Re: [ccp4bb] Fancy crystal, poor diffraction
Tiancen, I am not entirely clear - are you asking for techniques that can improve your *existing* material - or are you shopping for *any* technique that will eventually result in an improvement? If you're looking for the latter, and are willing to send me your protein sequence (I promise to keep it confidential, of course) - I can supply you with a list of changes you can do on the level of sequence modification, with fairly high likelihood of success (tested on a number of bad proteins to-date, with very high success ratio). I cannot disclose the algorithms used to calculate these mutations but they're not the same as Derewenda's surface entropy method (which also works) or some of the existing servers dedicated to the same purpose. If it helps, you can look at my recent short blurb for other ideas :) http://www.xtals.org/pdfs/rescue_crystals.pdf Artem > Dear all, > > Sorry for the non-CCP4 question. I think this is an old story but our > knowledge to deal with it is very limited. So any suggestions will be > greatly appreciated. > > We have crystallized a 21KD protein with 2 disulfide bonds grown for one > month in 0.1M tri-sodium citrate pH 5.6, 0.5M (NH4)2SO4 and 1M Li2SO4. The > crystals look big (~0.4mm x 0.4mm x 0.3mm) and pretty (sharp edge, clean > surface) but diffracted to only 4A in-house. The spots are quite strong > and isotropic at low resolution but decay sharply beyond 5-6A. The crystal > belongs to P4 pointgroup (P422 is also possible) with cell parameters of > 127.6, 127.6, 162.5, 90, 90, 90. The solutions we can think of to elevate > its diffraction ability are as follows: > > 1)Try synchrotron radiation > 2)Try a lot of similar crystals and hope one of them diffracts better > than others > 3)Let the crystals grow for a longer time and hope it could pack more > ¡°orderly¡± > 4)Additive screen based on the original condition > 5)Check the original plates for other crystallizing conditions > (unfortunately until now this is the only one out of ~300) > 6)Screen with other forms of the protein, i.e., N/C-terminus truncated > ones, complexed with its ligands etc. > > I believe many protein crystallographers have encountered similar > problems, are there any successful stories from these fancy poor crystals? > Any suggestions or references will be highly appreciated. > > Thanks in advance! > > Tiancen Hu > Shanghai Institute of Materia Medica > Rm. 2107, #555, ZuChongzhi Rd. > Shanghai 201203 > P.R. China > Tel: +86-21-50806600 ext 2107 > Email: [EMAIL PROTECTED] > >
Re: [ccp4bb] AKTA prime
Hi, Don't forget system losses (friction in the tubing). An empty AKTA prime typically eats up about 0.25 MPa so your maximum is closer to 0.7MPa loss on column. Artem > > > Dear Frank, > AKTA prime delivers a max. pressure of 1 MPa. The 24 ml SEC columns can > be run at pressures > up to 1.5 (S200) or even 3 MPa (superose 12). If your column is in pretty > good shape, expect to > reach 0.9 MPa at 0.4 ml/min for S200. So you can use an AKTA prime, but > ... > Best > Clemens > > > At 11:16 PM 2/13/2007, you wrote: > > Dear all, > > I need to decide between buying an AKTA prime and an AKTA FPLC from GE > health care. I understand AKTA prime is a low-pressure system, but > because it is too much cheaper than AKTA FPLC, it is still very > attractive to me. > > I will mainly use it for Nickel columns and gel filtration columns, and I > am worried about the latter. Is it true that using AKTA prime, you can > only run the 24 ml Superdex 200 column at 0.1 or 0.2 ml/min? > > Could anyone who has used AKTA prime give me some feedbacks? I would > appreciate it. > > Best, > Frank > > > Need Mail bonding? > Go to the Yahoo! Mail Q&A for great tips from Yahoo! Answers users. > Jun.-Prof. Dr. Clemens Steegborn > Ruhr-University Bochum > Dept. Physiol. Chemistry, MA 2/141 > Universitaetsstr. 150 > 44801 Bochum, Germany > > phone: 0049 234 32 27041 > fax: 0049 234 32 14193 > email: [EMAIL PROTECTED]
Re: [ccp4bb] Bio-rad DuoFlow
Hi folks, I've been an AKTA user for probably close to ten years, and in this time I've also used BioRad and several other systems. I'd like to make a couple of comments since everyone seems to be enjoying this lively discussion :) 0. Choose the device for what *you* need. Some applications are equally easy on whatever system you choose, whereas others are very difficult on some of the systems. Having said this, I would argue that I never had any trouble programming the AKTA systems (Explorer and purifier that is, NOT the Prime or the FPLC) for any of the crazy multistep methods I've used. 1. For some reason, BioRad software was and is never really designed with protein purification in mind. Not to say that you cannot use it - with some training it's OK but the Unicorn software is intuitive from the get-go. 2. Unicorn has two faces. For the beginner, or for people who only use the system sporadically, there is the pre-made 'wizard' interface that is quite nice and robust. In my small opinion, the wizards lack elegance. For the pro, there is the scripting interface which is exceptionally powerful. Either way, programming an AKTA from scratch takes a couple of minutes. 3. I love AKTAS. But AKTA Prime is a waste of money. You can assemble a component-based system for much less money that would do the same sort of stuff. 4. AKTA Purifiers can be configured for about the same cost as competitors. I think the DuoFlow is still cheaper in the end, but its default tubing set needs to be changed to much smaller diameter to minimize the itnernal volume of the system. AKTA's come with thin tubes to begin with. 5. The first thing I do on an AKTA system is take out the internal filter. It serves almost no purpose except to annoy the user. I've passed honey-think whole cell extracts through AKTA with no filters anywhere in the system (important - no column filters either - they would clog up as well!) with no ill consequences whatsoever. Naturally, I wouldn't put a lysate like this on my $7000 MonoQ column - that'd be silly (I've seen people do this, and the result isn't pretty). Filter your solutions and everything would be OK :) Cheers! Artem
[ccp4bb] journals with on-hold policy
Dear CCP4ers, This is a new question for me, and I would appreciate the forum's suggestions! I would like to find out which journals (that publish structural work) are OK with placing structures on hold for the full year permitted by the PDB. Yes, I could go site-by-site and look this stuff up, but I'd rather ask since many of us had to do this before. Before admoniting me for placing structures on hold, please note that I do not enjoy doing so - but this is the only way we get even a chance to publish this particular paper. Since the choice is not to publish versus publish and place a hold on the structure - it's not really a choice at all. Up until now, all my structures are always released *immediately* (yes, before publication) - so please try to avoid negative comments! Many thanks, Artem
[ccp4bb] on-hold policy - reasons why
Cheers, Many thanks to all of you who sent in the (mostly positive) helpful replies. A lot of folks asked why do we have to place the structure on hold - since this is a pretty typical example of how industrial crystallographers have to operate sometimes, I will attempt to explain. We have a choice at the moment - not to publish at all, versus (maybe) to publish but keep the structure on hold for a year. Since a whole lot of us are obliged to look for jobs (yours truly included), publications are important. Hopefully this explains things a bit better. Best regards, Artem
Re: [ccp4bb] human cDNA clones?
It depends on scale. With some synthetic DNA companies if you order many at once, you can get a huge discout, down to maybe 35-45 cents per base. Alternatively, if you have free labor (students) then you can multiparallel your PCR, but $100 per clone is going to be hard to beat given the fact that you still have to buy primers, use PCR reagents and cleanup/cloning kits, etc. etc. and at the end also would want to sequence the products. How many genes are you trying to get? Artem > Sorry a little offtopic... > > We'r trying to clones a number of putative human proteins for > crystallization. Besides IMAGE clones from OpenBiosystem, is there any > the cheaper way of obtaining human cDNA clones? OpenBiosystem > is okish, ~$100Cdn for each clone, but does get abit expensive when you > start getting alot. >
Re: [ccp4bb] Cannot running NTA to purify the protein having His-tag?
Dear TriNgo, Firstly, the fact that you can see your His-tag via Western blot does not mean that it's attached to the majority of the protein molecules in solution - strong Western signals may be observed for as little as 1% of the protein that is *supposed* to be His-tagged. So one of the causes may be that your protein is chewing itself up or is being chewed up by something that causes most of the molecules to lose the tag (but for a few percent to remain tagged and thus give you a Western signal). The other alternative is that your protein is misfolded and/or aggregated. I've seen several cases of aggregation resulting in His-tag being 'hidden' away from the resin. The way to check this is to take your lysate and add guanidine to 6-8M, then re-run the Ni-NTA column. If the protein suddenly binds - then you know what's up. Finally, your protein may be folded, but the tag may be 'concealed' in a suitable protein pocket and thus isn't exposed enough to the solvent and therefore to the resin. Common cases of such behavior typically involve oligomeric proteins. Again, denaturation may suggest an answer, alternatively just moving the tag may be the way to go. Note that IMAC works better at pH 8-8.5 so if you want, you could repeat your extraction at higher pH. Finally, it is not common to see insect cell media interfering with the binding of proteins to IMAC. If you have large amounts of medium in your cell paste, you may want to either wash the cells (gently!) during the initial pelleting process, or try adding ~5 mM MgCl2 which has been known to help (presumably by complexing away the chelators naturally present in insect cell medium). This may or may not work. You may also be able to do primary separation of some sort (such as ammonium sulfate or PEG precipitation, or perhaps bulk ion exchange etc.) prior to your IMAC step. Best regards, Artem > Dear CCP4 users, > > I'm purifying a kind of protease having His-tag. The protein is expressed > in > insect cells and broken by sonication. > I used NTA resin to purify this protein. > Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B is 50mM > phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole. > However, all proteins cannot bind to NTA resin. My protein is eluted in > Flow-through. I also check the NTA resin with the control His-tag. The > western blot also shows that my protein has His-tag. > > Do you have any ideas about my problem? I'm really appreciate all of your > advices how to solve this. Thank you very much! > > My best regards, > TriNgo > Sungkyunkwan University >
Re: [ccp4bb] Cannot running NTA to purify the protein having His-tag?
Indeed Strep-II (please note there is also a Strep-I which is not as good!) tag is a good tag to try if you can. There are a few caveats that you may want to consider when trying it: 0. if things work out, Strep-II affinity purification can yield essentially pure protein in one step. The question of whether this protein is homogenous or not still needs to be addressed though (via sizing columns, light scattering, etc.) We have had some very mystifying cases where the Strep-II tag just did not work, for no apparent reason - no matter what we tried. (So, in the end we blamed it on the 'hidden' tag scenario). 1. biotin in medium is a potential problem (not as bad for insect cells, but can be bad for yeast) 2. you may want to incubate your lysate with small amounts of avidin, to capture away all the biotinylated eukariotic proteins (Strep-II tag doesn't really bind avidin, but Strep-tactin binds biotin). 3. the resin is pricey and only binds roughly 1 mg/ml of an 'average' (300 aa) protein. It may be reused (very carefully) about 10 times if you handle the column like it's a 2500-year old mummy. Typically 5 or more re-uses result in significant loss of capacity. 4. the tag only works in native conditions as it is structured and binds to a protein that is structured as well. Passing 8M guanidine through a $6000 column only to find out that it's dead afterwards can cause much sadness. 5. the column can degrade very quickly if you have unusually high protease load in your lysate Caveat emptor :) Artem > Hi, > > > > We got some good experiences using the IBA streptag for baculo-expressed > proteins. You'll have to redo the cloning, but it will be worth your while > when you see the first purification step, > > > > cheers > > Joost > > > > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Alex > Berndt > Sent: Wednesday, 28 February, 2007 20:21 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Cannot running NTA to purify the protein having > His-tag? > > > > sometimes the insect cell medium intereferes (for whatever reasons) with > nta purifications when they ar employed as a first step in the > purification scheme. i experienced that occasionally. this can easily be > circumvented by doing an ion exchange step beforehand! > > alternatively you might want to introduce a linker between your protein > and the his-tag or create a 8xhis or 10xhis tag to enhance bing to the nta > matrix. make sure you wash your cells from residual medium before you > freeze your pellets. > > > > alex > > > > On 28 Feb 2007, at 19:18, Juergen Bosch wrote: > > > > > > Ngo Duc Tri wrote: > > > > Dear CCP4 users, > > > > I'm purifying a kind of protease having His-tag. The protein is > expressed > in insect cells and broken by sonication. > > I used NTA resin to purify this protein. > > Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B is > 50mM > phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole. > > However, all proteins cannot bind to NTA resin. My protein is eluted in > Flow-through. I also check the NTA resin with the control His-tag. The > western blot also shows that my protein has His-tag. > > > > Do you have any ideas about my problem? I'm really appreciate all of > your > advices how to solve this. Thank you very much! > > > > My best regards, > > TriNgo > > Sungkyunkwan University > > > > You His tag is most likely inaccessible, can you easily change the tag > from e.g the N-terminus to the C-terminus ? Or if you have a structural > homolog you could add the His tag into a loop, which is exposed. > > > > Alternatively you can purify your protein under denaturing conditions > using 8 M urea and refold it if you dare :-) > > > > Juergen > > > > -- > > Jürgen Bosch > > University of Washington > > Dept. of Biochemistry, K-426 > > 1705 NE Pacific Street > > Seattle, WA 98195 > > Box 357742 > > Phone: +1-206-616-4510 > > FAX:+1-206-685-7002 > > > > --- > > Alex Berndt > > MRC Laboratory of Molecular Biology > > Hills Road > > Cambridge CB2 2QH > > UK > > > > mail : [EMAIL PROTECTED] > > phone : +44 (0)1223 402113 > > --- > > > > > > > > > > > This message, including attachments, is confidential and may be > privileged. If you are not an intended recipient, please notify the sender > then delete and destroy the original message and all copies. You should > not copy, forward and/or disclose this message, in whole or in part, > without permission of the sender. >
Re: [ccp4bb] protease cleavage sites
Rene, There are many proteases that are suitable for digestion of an N-terminal tag. Interestingly, there are NOT many (any?) proteases suitable for digestion of thr C-terminal tag (except for the dual-enzyme His-tag digest system that sometimes work and sometimes does not). I personally like TVMV, TEV, and Thrombin. With the first two enzymes, you have no worries whatsoever about non-specific digestion, whereas Thrombin is pretty gentle but has been reported to eat things up sometimes. Cheers, Artem > Hi, > > A non-ccp4 Q. Sorry. > > I would like to use a cleavable purification tag at the N-terminus/ > extracellular end of my membrane protein for purification. Before I > start, I wonder if someone could recommend a particular protease site > that I can engineer between the tag and my protein? How about a > proprietary cleavage system such as the PreScission protease (GE > Healthcare)? I would be grateful to hear success and horror stories > in this area. > > Best wishes, > > Rene > > > Dr R.A.W. Frank, PhD > Royal Commission for the Exhibition of 1851 Research Fellow > > Prof Seth Grant Lab / Genes to Cognition > Wellcome Trust Sanger Institute > Hinxton > Cambridge CB10 1SA > > Work Tel: 0044 (0)1223 834244 ext. 7318 > Cell No.: 0044 (0)7870 208280 > === > > > >
Re: [ccp4bb] protease cleavage sites
The important bit about Thrombin is to find the right source of it. Some
suppliers are *much* better than others. I've had no problems with this
protease in the recent year, but had trouble earlier.
Note that Thrombin leaves 2 amino acids whereas TVMV, TEV, and 3C leave
one amino acid (or even none if you have the right N-terminus).
Artem
> I'd like to third TeV & second 3C ("PreScission").
>
> Both have high specificity, good processivity and I have had a lot of
> success with 3C.
>
> I have _never_ got Thrombin to cut cleanly - but I guess I could have been
> unlucky...
>
> Dave
>
> On 05/03/07, Cynthia Kinsland <[EMAIL PROTECTED]> wrote:
>>
>> Hi,
>> I'll second the TEV protease suggestion. We use it routinely because it
>> is highly specific and easy to make ourselves (and, therefore, cheap).
>> We
>> have never seen it cut non-specifically and, since it is cheap, we just
>> chuck in a bunch and let it go.
>>
>> The Prescission protease is also very specific and also available for
>> home
>> preparation (it is the 3C protease...prescission is a marketing name).
>> I
>> don't have as much experience with it, but it has behaved for me so far
>> and
>> I know that a number of people use it routinely with great success.
>>
>> Another nice thing about having the clones around to make your own is
>> that
>> you can make the protease with the same tag that you intend to cut off
>> (say,
>> His or GST or whatever your favorite is). Then, you can remove cleaved
>> tag,
>> uncleaved fusion protein and the protease all in one post-cleavage step.
>>
>> In our case, we almost always have a HisTag (often as part of some
>> larger
>> fusion) so our TEV is His-tagged. We have some of the GE pGEX vector
>> for
>> Prescission protease, so our 3C clone has GST on it.
>>
>> Best of luck,
>>
>> Cynthia
>>
>> On Mar 2, 2007, at 5:01 AM, Rene Frank wrote:
>>
>> Hi,
>>
>> A non-ccp4 Q. Sorry.
>>
>> I would like to use a cleavable purification tag at the
>> N-terminus/extracellular end of my membrane protein for purification.
>> Before
>> I start, I wonder if someone could recommend a particular protease site
>> that
>> I can engineer between the tag and my protein? How about a proprietary
>> cleavage system such as the PreScission protease (GE Healthcare)? I
>> would
>> be grateful to hear success and horror stories in this area.
>>
>> Best wishes,
>>
>> Rene
>>
>>
>> Dr R.A.W. Frank, PhD
>> Royal Commission for the Exhibition of 1851 Research Fellow
>>
>> Prof Seth Grant Lab / Genes to Cognition
>> Wellcome Trust Sanger Institute
>> Hinxton
>> Cambridge CB10 1SA
>>
>> Work Tel: 0044 (0)1223 834244 ext. 7318
>> Cell No.: 0044 (0)7870 208280
>> ===
>>
>>
>>
>>
>>
>> Cynthia Kinsland, Ph.D.
>> Cornell University
>> Protein Facility Director
>> 607-255-8844
>>
>>
>>
>>
>
>
> --
> ---
> David Briggs, PhD.
> Father & Crystallographer
> www.dbriggs.talktalk.net
> iChat AIM ID: DBassophile
> ---
> Anyone who is capable of getting themselves made President should on no
> account be allowed to do the job. - Douglas Adams
>
Re: [ccp4bb] protease cleavage sites
Since people asked:
http://www.usbweb.com/category.asp?cat=118&id=22293
is my preferred source. Please note that I don't have any relation to USB
whatsoever (but large amounts of money sent to my unnamed Swiss bank
account are always appreciated. The password for the account is BACON).
Artem
> The important bit about Thrombin is to find the right source of it. Some
> suppliers are *much* better than others. I've had no problems with this
> protease in the recent year, but had trouble earlier.
>
> Note that Thrombin leaves 2 amino acids whereas TVMV, TEV, and 3C leave
> one amino acid (or even none if you have the right N-terminus).
>
> Artem
>
>> I'd like to third TeV & second 3C ("PreScission").
>>
>> Both have high specificity, good processivity and I have had a lot of
>> success with 3C.
>>
>> I have _never_ got Thrombin to cut cleanly - but I guess I could have
>> been
>> unlucky...
>>
>> Dave
>>
>> On 05/03/07, Cynthia Kinsland <[EMAIL PROTECTED]> wrote:
>>>
>>> Hi,
>>> I'll second the TEV protease suggestion. We use it routinely because
>>> it
>>> is highly specific and easy to make ourselves (and, therefore, cheap).
>>> We
>>> have never seen it cut non-specifically and, since it is cheap, we just
>>> chuck in a bunch and let it go.
>>>
>>> The Prescission protease is also very specific and also available for
>>> home
>>> preparation (it is the 3C protease...prescission is a marketing name).
>>> I
>>> don't have as much experience with it, but it has behaved for me so far
>>> and
>>> I know that a number of people use it routinely with great success.
>>>
>>> Another nice thing about having the clones around to make your own is
>>> that
>>> you can make the protease with the same tag that you intend to cut off
>>> (say,
>>> His or GST or whatever your favorite is). Then, you can remove cleaved
>>> tag,
>>> uncleaved fusion protein and the protease all in one post-cleavage
>>> step.
>>>
>>> In our case, we almost always have a HisTag (often as part of some
>>> larger
>>> fusion) so our TEV is His-tagged. We have some of the GE pGEX vector
>>> for
>>> Prescission protease, so our 3C clone has GST on it.
>>>
>>> Best of luck,
>>>
>>> Cynthia
>>>
>>> On Mar 2, 2007, at 5:01 AM, Rene Frank wrote:
>>>
>>> Hi,
>>>
>>> A non-ccp4 Q. Sorry.
>>>
>>> I would like to use a cleavable purification tag at the
>>> N-terminus/extracellular end of my membrane protein for purification.
>>> Before
>>> I start, I wonder if someone could recommend a particular protease site
>>> that
>>> I can engineer between the tag and my protein? How about a proprietary
>>> cleavage system such as the PreScission protease (GE Healthcare)? I
>>> would
>>> be grateful to hear success and horror stories in this area.
>>>
>>> Best wishes,
>>>
>>> Rene
>>>
>>>
>>> Dr R.A.W. Frank, PhD
>>> Royal Commission for the Exhibition of 1851 Research Fellow
>>>
>>> Prof Seth Grant Lab / Genes to Cognition
>>> Wellcome Trust Sanger Institute
>>> Hinxton
>>> Cambridge CB10 1SA
>>>
>>> Work Tel: 0044 (0)1223 834244 ext. 7318
>>> Cell No.: 0044 (0)7870 208280
>>> ===
>>>
>>>
>>>
>>>
>>>
>>> Cynthia Kinsland, Ph.D.
>>> Cornell University
>>> Protein Facility Director
>>> 607-255-8844
>>>
>>>
>>>
>>>
>>
>>
>> --
>> ---
>> David Briggs, PhD.
>> Father & Crystallographer
>> www.dbriggs.talktalk.net
>> iChat AIM ID: DBassophile
>> ---
>> Anyone who is capable of getting themselves made President should on no
>> account be allowed to do the job. - Douglas Adams
>>
>
Re: [ccp4bb] Nature policy update regarding source code
Hi, >From a very narrow point of view of a crystallographer/protein expressionist, this is an overkill. Obviously if you're dealing with articles in materials science, physics, etc. - not everything is as simple as getting some DNA. Generally speaking, if someone wants to reproduce nearly any crystallographic results all they need is the source DNA in whatever form. An obvious exception to that would be the case where the protein is purified from a natural source. Special cases excluded; cloning, expression, purification and so forth should be reproducible from source DNA onward. In fact, with the advent and proliferation of synthetic DNA even that requirement is not really all that important - all you need is the sequence, provided that the 'materials and methods' section of the work is well written and no significant details are omitted. The latter is (in my experience) the more common problem than the availability of sarting materials. It is very hard to include absolutely everything into the Materials and Methods. At least half of the published protocols we've reproduced required very significant investment of effort to get right (sometimes even changing expression systems from what's reported). The end point is clear - the crystal structures DO reproduce quite faithfully even if extra work has to be done to get there. For many industrial scientists the following sentence is basically the end of the road as far as publication is concerned: "One preferred form of disclosure is a link from the methods section to a copy of the relevant Material Transfer Agreement (MTA) form, which is hosted as Supplementary Information on the journal's web site." Pharma/biotech companies are typically very reluctant to make blanket provisions of this kind. Are the journals trying to exclude industrial submissions? Artem
Re: [ccp4bb] Multiple nucleation
Not a contradiction at all :) If the protein supply is adequate - this is a perfectly good approach to explore. I simply forgot to mention it. I've also not mentioned engineering the protein's surface (works really well for us because we have secret ways to do this, learned from ancient and mysterious Shaolin monks) or derivatizing the protein with chemicals - these are excellent but require more work on the researcher's behalf. If the methods that people suggested so far do not result in success (which is unfortunate but not uncommon) - you can always change the protein itself! Good luck :) Artem > Hi Jobi > > Sounds like you need to explore your protein vs PEG concentration, and my > guess (directly contradicting Artem's) is that chances are you need to > pump > [protein] way up and drop [PEG] way down. > > Like in [protein]=>30-40mg/ml, [PEG]<=2-4%. Often people don't go into > that > range when setting up optimizations, because the original condition was, > say, 25% PEG. But looking at the solubility curve, this is the regime > where > few nuclei form and then have lots of protein to grow with. > > phx.
Re: [ccp4bb] Question about cryoprotectant
Dear Yang Li, Cryoprotection of crystals is not an exact predictive science - you have to try a number of things, and in the worst case scenario, none of the stuff you try may work out. Assuming that your crystals are OK to start with (capillary mounted room temp. pattern would confirm that), you can explore a whole wide range of cryoprotectants. I am slightly surprised that you need one at all - at 20% isopropanol together with 20% PEG you have 40% organic content which is usually enough for glassification. You may want to explore the actual cryo-mounting method - there are several options for flash freezing and they're not all the same in terms of results. Assuming that you do really need cryoprotectant, you may want to experiment with addition of EG or glycerol, etc. right into the mother liquor when you set things up - this way, if the crystals grow they're already protected and ready to go. If you can somehow get rid of isopropanol, your life would be easier as well because this alcohol is very volatile which can cause no end of trouble when working with crystals in the open air (among other things, they tend to 'zoom' around the drop as the surface of the drop is pulled around by fluxes of evaporating iPrOH. There are numerous other things you can try, including the one mentioned here: http://www.xtals.org/ under 'stuff'. Regards, Artem > Hi, > l have a crystal grow at condition screen l 40: > 0.1M tri-sodium citrate dihydrate pH5.6 isoproponal 20%PEG4k 20% > and the crystal need a cryoprotectant, we have used the 30% glycerol but > it > is not good, the mosaicity of > the diffraction pattern is a little high, so anyone knows which is the > best > cryoprotectant for this crystal? > Thanks! >
Re: [ccp4bb] bigger size - > better diffraction?
Hello Jenny, 0.2 Rmerge may be an indication of incorrectly assigned SG or perhaps a misindexing by one, etc. Check what happens if you reprocess in P4. For a 10kDa protein, your unit cell is kind of large - depending of course on the symmetry, the higher obviusly the better. I would bet that you have pretty high solvent content - nothing scientific about this prediction, merely personal experience with several small proteins that crystallized in high-symmetry SG and had >80% solvent. What you may want to do is to very carefully study your cryo. When we were working on a structure of FliS-FliC complex(http://www.xtals.org/pdfs/FliC_FliS.pdf), the only way to freeze the crystals correctly was to increase the concentration of sulfate ion in the cryoprotectant. It later turned out that we had a sulfate ion bridging a symmetry threefold, and in low sulfate the ion would leave, resultuing in disorder in the crystals. Ultimately, if you've already tried all the 'standard' tricks, such as additives, glycerol/eg in the condition, replacing NaCl with an exotic salt such as KCl, CsCl, RbCl, and so forth - perhaps you may want to consider adjusting the surface of the protein. I would be glad to help you with that. Artem
Re: [ccp4bb] bigger size - > better diffraction?
A small comment: crystals that do not diffract well at R.T. can still diffract well when frozen. There are several reasons for this, including: * poor stability of crystal once the drop is open to the air * crystal has low tolerance for handling (capillary mount can be challenging and cause more physical damage, especially if the experimenter is rusty with capillary mounts) * the effect of freezing - it has been known to cause transitions between space groups etc. * the effect of cryoprotectant (dehydration, and so forth) So - even if your stuff doesn't diffract well in a capillary - it still can be saved sometimes :) Or not. Artem > diffracts in general. It is true it may be a cryo problem, but if > the non cryo protected crystals do not diffract then why would one > expect the cryo protected one to.
Re: [ccp4bb] oxidised cys
Hi Stefano, How certain are you that this link is truly what you think it is? If I understand what you're saying - you want to create a (thioperoxythio) link - this chemistry should be hideously unstable. Can you explain this using disorder, or perhaps the residual density is a symmetry artifact? Regards, Artem > Dear all > in my structure I think I can see an oxidised Cys in cys-SO. Refining > cys-SO > I observe a residual density between the oxigen of one oxidised cys and > the > one of the other molecule in AU. > I'd like to try to refine it as cys-SO-OS-cys. I didn't find an example of > it in the pdb database. Could anyone tell me whether there are other > cases? > I guess I just didn't find them. > Second question: > How could I "explain" to refmac that there is the OO bond? > I tried to write a line similar to the one for SSBOND in the pdb header > OOBOND 1 CEA A 42CEA D 42 > but refmac couldn't care less... > > thanks in advance > > Stefano > > _ > Express yourself instantly with MSN Messenger! Download today it's FREE! > http://messenger.msn.click-url.com/go/onm00200471ave/direct/01/ >
Re: [ccp4bb] Additive screens
Hi, We use additives extensively. Pretty much all available kits, as well as some of our own design. My favorite is Nextal (Qiagen) kit named the Salt Optimizer. Artem > Hello everyone, > > Does anyone have suggestions or comments on the various additive > screens that are available? > > I was looking at the kit from Hampton (HR2-138 Additive Screen HT) in > 96-well format. It appears that it should be straight forward to > array out our current protein/mother liquor condition into a 96-plate > and then add in the additives from the deep-well block provided by > the company. The additives come 10x concentrated in a 96-well deep- > well block for dilution into the arrayed crystallization condition. > > Does anyone do this routinely for the crystals they grow? It would > be interesting to know how often people find a systematic approach > using such a kit actually improves the quality/size etc. of their > crystals. > > Cheers, > > Brian > > --- > Brian L. Mark, MSc, PhD > Department of Microbiology > Room 418, Buller Building > University of Manitoba > Winnipeg, Manitoba > CANADA R3T 2N2 > > Phone (204) 480-1430 > Fax (204) 474-7603 > Web: http://www.umanitoba.ca/science/microbiology/staff/mark/
Re: [ccp4bb] Unusual Difference Fourier near Methionines
This appears to be a symmetry threefold axis. These areas are notoriously prone to have density artefacts. Also it looks like your terminal carbons are pointing in the opposite way, and the entire end of Met is slightly shifted as a result. You may never be able to fit this area to your satisfaction - due to the symmetry axis. I would definitely experiment with allowing partial overlap - remove steric restraints on this residue so they don't bump into one another then see how the density looks - if things return to normal - perhaps you're forcing a close collision - maybe there is a small error in unit cell parameters for instance, etc. Artem > > Dear all > > I am doing one structure at 2.6A resolution where I found unusual density > near methionines. Actually three methionines come close together nearby > and I get difference even at 5sigma cutoff. I don't understand nature of > the density. The amino acid sequnce is same I mean met by chemical > sequncing so there is no change amino acid residues. I am sending the > snapshot of the difference fourier observed at methionines. The crystal > belong to R32 system with one molecule and biological trimer. In snapshot > two of the methionines are symmetry related. > > Details of the data collection. > Data collected at synchrotron > > Overall Rsym = 6 % and I/sigma = 2.1. Completeness = 99% > > Can anyone suggestion what could be chemical nature for the difference > fourier. > > Thanks in advance > > Ethayathulla > > ### > A.S.Ethayathulla,Ph.D. > Department of Biophysics > All India Institute of Medical Sciences > Ansari Nagar > New Delhi-110029 > India. > ### >
Re: [ccp4bb] Differentiating bound Mn & Ca.
Dave, Others have given many excellent suggestions :) I'd like to mention that to a protein biochemist such as myself, another obvious solution would be to take an aliquot of the protein for a metal analysis (via ICP or AA). ICP is more sensitive. Both Mn and Ca will light up the spectrum like new year's tree lights. Artem > The obvious solution is go to a synchrotron and scan around the Mn edge > and > see what happens, however, whilst waiting for beam time, is there any way
Re: [ccp4bb] Selenomethionine reduction v.s. disulfide bond
Hello Tiancen, The pundits often suggest keeping the selenium reduced (and indeed, it's not a bad idea) however if you're worried about disulphides - I would say that they take precedence over the selenium. If you work reasonably fast you should be able to have the best of both worlds - have the selenium safe and the disulphides oxidized. Not to mention that there always are the good old heavy atom derivatives to fall back on if the selenium data does not pan out :) Artem
Re: [ccp4bb] salt or protein?
Multiple overlapping salt lattices can sometimes look like protein diffraction, as long as you're looking in only two dimensions. However, if you can find the dominant rings, you should be able to discriminate since the c-spacing of salt would nearly always be pretty small. Consider powder patterns, and you should see what I mean. Generally speaking, if your crystals visually appear to be single, and give huge salt peaks - then they're probably salt. Exceptions - large, non-diffracting protein crystals that have small salt crystals stuck to them. Ultimately, you can use the 'stick a fork in it' method: stick a needle in your rod-like crystals and push. If you hear a crack, and see sharp clean edges on the break - it's salt. If you feel the crystal 'give' and see bending or pitting - it's probably protein. Good luck! Artem > Hi All! > > I have been trying to screen for my protein crystals, from the > crystals grown in 0.5 M Ammonium Sulphate, 1.0M Lithium Sulphate > Monohydrate in 0.1 M TriSodium Citrate Buffer dihydrate Buffer at pH 5.6. > Two different kinds of crystals observed: rod shaped and thin platy > ones. Whenever I am trying to collect data from the rod shaped ones I am > getting dominantly salt patterns but also spots at 15 A resolution bin > repeatedly, where some of the spots very much look like from protein! > what can be the reason for this? if anyone has come across similar > situation please help. > > with regards, > Sreeram Mahesh > > > Research Student > Prof S Ramakumar's lab > PHYSICS department > IISc Bangalore-560 012. > ph:080-2293 2718. > mobile: 9241145183. > > > -- > This message has been scanned for viruses and > dangerous content by MailScanner, and is > believed to be clean. >
Re: [ccp4bb] phosphorylation of protein is troublesome or not for crystallization
Rongjin, Yes, there most definitely are numerous examples of phosphorylated, glycosylated, lipidilated, etc. etc. proteins crystallized. Since you are asking specifically about phosphorylation here are my two cents: a. over one half of all heterogenously expressed kinases are phosphorylated b. about half of those phosphorylate themselves, sometimes to a great degree. The most common example of the latter is PKA which under the right expression conditions can be phosphorlyated on over 20 sites c. there are several major techniques people have employed to crystallize phosphorylated proteins. Nearly all of them focus on obtaining homogenous species. d. some proteins can crystallize even when heterogenously phosphorylated, although the crystals are not always any good. Yet others absolutely require homogeneity. Several important notes: hyperphosphorylating kinases can be very toxic to their expression hosts. Depending on the host, some kinases may be super-active, silent, or somewhere in between. Some kinases become insoluble when they're not phosphorylated, the opposite also can happen. Methods to control phosphorylation include: expression host choice coexpression with other kinase(s) coexpression with phosphatase(s) in vitro enzymatic treatment (phosphorylation or dephosphorylation) mutagenesis of key residues - either of the phosphorlyation sites or of the active site coexpression with regulatory proteins expression in the presence of inhibitors modulation of growth conditions (which is how PKA was done) etc. Artem > Dear all, > > Below is a question my friend asked me, but I have never worked on > phosphorylated proteins. > Has anyone worked on crystallizing phosphorylated proteins and can you > comment on it? > > Thanks > > Rongjin Guan > -- > I would like to ask how feasible it is to crystalize protein containing > post-translational modification, such as phosphorylation. To my limited > knowledge, I think the heterogenicity of modified and unmodified proteins > causes the major obstacle in the crystal structure. Is there any specific > method to crystalized modified proteins (like phosphorylated proteins ) > these days? >
[ccp4bb]
Hi, Without knowing exactly what you did, it's hard to guess however here are a few suggestions, in addition to the ideas already given by others: a. try iodination. Simply place an iodine crystal near the drop (glue it to the cover slip using a tiny bit of grease) and let the vapors penetrate the drop gradually. Also you can add 0.1 mM of KI to the solution then add a speck of iodine - the KI will slowly transition the iodine via the KI3 complex to the protein. You have to experiment with timing - what worked for me was 1 day exposure. The crystals may or may not turn yellow - depends on your protein and on conditions. b. I am surprised that mercury derivatives did not work. Have you tried increasing amounts of ethyl mercury phosphate? c. Have you tried my favorite platinum derivative - KPt(NO2)3? You can add a LOT of this stuff - it's very mild on the protein. Artem > > > > Hey there, > > I have crystallised a protein in 30% MPD and it it gives nice native > data. However, it seems impossible to get heavy metal derivatives and > I read that MPD does chelate heavy metals. Did any of you make > similar experiences with xtals in MPD conditions? If so did you find > a good solution (introduction of selmet makes the protein ustable) > to make heavy metal derivatives? > > Best Regards > > Daniel
Re: [ccp4bb] disorder, solvent content, and diffraction limit
Mike, This is a complicated question :) The floppy bits can contribute to packing in several non-obvious ways. I may be able to help you get a better crystal form, if you're willing to share the specifics of the protein (which will be kept confidential of course). I've developed new procedures for rational protein crystal engineering and now need more Guinea pigs to test them. Artem > Greetings, > > I've taken a protein (structure known) with some disordered termini and > created a new truncated construct that lacks these disordered bits > (approx. > 5% by mass of the total protein). > > To my delight, the new construct crystallizes much more readily than the > full-length one (more hits in screening, nicer looking crystals, etc.) > However, while the crystallization is more robust and the crystals look > much > "nicer", they don't diffract as well as the full-length crystals (go from > around 2.7 angs for full-length to 3.2 or worse for truncated construct). > > While the truncated form gives many more crystallization hits than > full-length, optimization always gives the same crystal form (same space > group and unit cell dimensions), no matter which conditions are used. > > Given that I've removed floppy bits from my protein, I would have expected > improved diffraction. But since I keep getting the same unit cell, I > wonder > if I've just increased the solvent content in my truncated construct > crystals (less mass per same unit volume). > > Does this rationale make sense? Is it possible that the disordered parts > I > removed were in some way restraining the ordered parts of the structure? > Should I keep searching for a new crystal form with improved lattice > packing? btw, I have tried several different approaches to dehydrate the > crystals and none has successfully improved diffraction. > > Thanks for your help. > > -Mike >
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, Assuming that you need the minimal medium for Se, have you tried the regular heavy atom derivatives? You already have diffracting crystals... M9 medium can be slightly difficult. However, no one says that you *have* to use minimal medium such as M9 (again, assuming that you're doing Se-Met). There are numbers of defined media compositions out there, many with very good growth characteristics. Finally, you can spike the M9 with a small quantity of yeast extract. This will give your culture a decent initial boost and the amount of normal methionine in the extract should be relatively small so it will all get eaten up while the cells are still in early log phase. Regards, Artem > Hello everybody, > > Sorry for an offtopic question. I am trying to express a protein in M9 > minimal media for Selenomet incorporation. When grown in LB this protein > expressed very well and got good crystals. Diffraction was upto 2 A. I am > having a hard time expressing the same protein in Minimal media. It took > nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in > minimal media and eventually got no protein expression. It looks like the > cells are not growing or taking very long to grow. The cell line I am > using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). > > It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use > RosettaBlue (DE3). It worked very well in LB, but having a hard time > while expresing the same in minimal media using Rosetta Blue. Has anybody > tried expression in minimal media using Rosetta Blue cell line? I am > planning to try overnight induction. > > Any suggestions would be greatly appreciated. > > Thanks, > > Manish > > > > > > > > > * > Manish B. Shah, PhD. > Postdoctoral Fellow > Hauptman-Woodward Medical Research Institute > 700 Ellicott Street > Buffalo, NY 14203. > * >
Re: [ccp4bb] recommendation for heavy atoms used in lithium sulfate and ammonium sulfate
Tiancen If you have enough crystals to experiment with, just do a normal series of heavy atom derivatives, starting with good old favorites such as mercury and platinum. As an added touch of class you can try tungstate - it's like sulphate, but has fat W so it can be used probably just like a halide soak... Let me know if you need more detailed help :) Artem > Dear all, > > Could any one recommend some heavy atoms used for crystals grown in 0.1M > tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate? I > read from Hampton user guide of heavy atom kit that "high salt > concentrations are not the ideal medium for heavy atom reactions with > macromolecules", so is there any type of heavy atom we should avoid using? > We do not have any experience in preparing heavy atom derivative, so any > suggestions, experience or references will be greatly appreciated. > > Thanks in advance! > > Tiancen Hu > Shanghai Institute of Materia Medica >
Re: [ccp4bb] Superdex 200 PC columns
It greatly depends on the state of your instrument - how well you took care of the optical elements, etc. It also depends on the initial concentration of the sample (and therefore the peak volume at detection time) and on the optical activity of the sample and the buffer. If the stars align and everything works, you should be able to easily detect 5 ug of protein in a *reasonable* peak volume. This would require a true injection of perhaps 15 ul assuming a dilution down to about 50 ul in the peak. Oviously if you have multiple smeary peaks than you won't get any signal at all :) Artem > Sorry for perhaps off-topic question but I am writing to ask if anyone has > experience in using Amersham Superdex 200 PC 3.2/30 columns in conjuction > with > the Precision Column holder on a standard (not SMART system) AKTA FPLC. > At the > moment using a Superdex 200 10/300 column on a standard AKTA I can get a > reasonable signal from down to about 20 ug protein - I am wondering how > much > less protein I will be able to use with the narrower Superdex 200 PC > 3.2/30 > column when I hook it up to the same FPLC via the Precision Column holder. >
Re: [ccp4bb] S-tagged protein purification
Hello, This tag is very hard to release, as it forms an integral part of the RNAse A. It maybe released with 2M NaSCN but this often results in denaturation of the carrier protein. This is why the manufacturer strongly recommends having cleavage site(s) engineered in between the tag and the carrier. The low capacity of the column is also not surprising since the affinity element is the entire RNAseS protein. While it is not a large protein, it's still considerably larger than the tiny NTA, IDA, or other metal-chelating group used to make IMAC resins. I've used S-tag to purify milligram quantities of proteins but inevitably engineered these constructs away from the tag in the end, for convenience reasons. If you know that your protein cannot be expressed with other tags, then your best bet would be to engineer a cleavage site between the S-tag and the protein of interest (although even this can potentially screw up expression). If all else fails, you can try using mutagenized S-tag - there is a report out there describing rational mutagenesis of the peptide sequence to reduce the affinity of the S-peptide for the S-protein. Thus, you may be able to elute with lower concntrations of chaotropes. Good luck, Artem > Hi, > > I have a membrane protein with a C-terminal S-tag, and there is no > cleavage > site for the S-tag (novagen, e.g, thrombin, EK). I wonder whether I still > can use this for purification without cleavage of the S-tag. Here are my > questions: > > 1) How to release the protein bound to S-tag resin? I noticed they suggest > 3M NaSCN, 3M MgCl2 or 0.5 M Citrate pH2, but these sound crazy for a > membrane protein. Does anyone have experience with this? > > 2) It seems that S-tag resin has lower binding capacity than does Ni > resin. > Did anyone ever use S-tag resin for purification of at least 10 mg > protein? > > I would probably put a his tag on my protein for purification later. > > Many thanks for any suggestions. > > Yong >
Re: [ccp4bb] Disordered active sites
This is very protein-specific, for some proteins it is better to co-crystallize with an inhibitor, then countersoak with a different inhibitor, yet for others it is better to co-crystallize with an inhibitor of interest directly. For all it's worth, I personally am a proponent of the second approach, since soaking can and does generate scary artifacts. Artem > Hi, all, > I met some crystal structures with disordered active sites. Soaking > common ligands can not make it become ordered. I am wondering what > people generally do in such situation. > > Thanks, > > Nian Huang >
Re: [ccp4bb] Survey on computer usage in crystallography
To steal the words from one of my favorite cartoon shows, to me stereo is just a headache with pictures. Artem (the show is Futurama) > I was just curious to see how popular stereo graphics is for > crystallographers. I personally think its a wonderful teaching tool which > is > currently under-utilized. > > Paul
Re: [ccp4bb] CCP4 Wiki
Hey, When was the announcement made? I can't claim to read all the CCP4 mail very carefully, so I probably missed the it... The wiki I've set up is essentially a sandlot - something to play with and something to give people ideas (in other terms, not intended as a final product), but I'll gladly remove it so as not to cause confusion. Artem > Yes, this is how a wiki page for CCP4 might look like. However, to make a > wiki successful we have to make sure it is secure and that there is a > dedicated core team to maintain it. Kevin was asked by the CCP4 to lead > the wiki project and while he is setting things up, it would be > counterproductive to start setting up independent "CCP4 wiki" sites. > > Tadeusz > > > > > > > > "Artem Evdokimov" <[EMAIL PROTECTED]> > Sent by: "CCP4 bulletin board" > 23-Jul-2007 00:29 > Please respond to "Artem Evdokimov" <[EMAIL PROTECTED]> > > > To > CCP4BB@JISCMAIL.AC.UK > cc > > Subject > [ccp4bb] CCP4 Wiki > > > > > > > Hi, > > Would something like this work? > > http://www.xtals.org/wiki/ > > I've only set it up today, but it seems to work just fine. Feel free to > play > with it. > > Artem > > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of > Lucas > Bleicher > Sent: Sunday, July 22, 2007 11:02 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Suggestion: Wiki -- was:Re: [ccp4bb] need > help--Rfree > is not decreasing > > That would be a great idea. In fact, I keep on my > mailbox dozens of great postings (most of them > summaries) in CCP4 which would be very useful to > everybody if there's an online resource, with > information organized in topics. I would gladly copy > them to this wiki. > > Lucas > > --- Kay Diederichs <[EMAIL PROTECTED]> > escreveu: > >> So - rather than repeat things that are obvious to >> some people, would it >> not be good to have a crystallography-FAQ that one >> could point people >> to? This should be part of a Wiki where "we >> crystallographers" could >> collect our wisdom. This would be much more >> systematic, and less >> volatile, than the postings of this mailing list >> (which to me _is_ a >> very valuable ressource). >> >> A Wiki is not difficult to set up. Maybe it could be >> part of the CCP4 >> pages? We set up a Wiki for our lab at the beginning >> of the year, and it >> was a great success, in particular because it works >> the same way as >> Wikipedia - anybody can contribute. There should be >> some means of >> controlling "write access", but that could simply be >> granted to people >> who are subscribed to the CCP4 mailing list. >> >> I'd at least volunteer in helping to get a Wiki >> started. And one way to >> get it filled with articles would be that those >> people who used to write >> a "summary" of responses would simply compose a new >> Wiki article, and >> report to the mailing list that this article exists, >> which could then be >> expanded by others. >> >> best, >> >> Kay >> >> >> Anastassis Perrakis schrieb: >> > Sorry for the cliche, but *the goal of refinement >> is not to reduce R >> > factors, but to produce a good model.* >> > >> > ARP/wARP uses the 'WEIGHT AUTO' option of REFMAC5 >> to get a good geometry. >> > You should set the weight to a value that produces >> 1-2 and 1-3 distances >> > rms deviations similar to the ARP/wARP job, >> > to be able to compare. The fact that weight is 0.3 >> says nothing. >> > >> > The correct weight can vary wildly from 0.02 to >> 0.5, in my experience. >> > for 2.0 data 0.3 sound loose, 0.15-0.2 is what I >> am used to, >> > depending on dataset. But, The only way to tell >> what is right is >> > inspecting the geometry and aim for a 'reasonable' >> rms 1-2 distances >> > deviation. >> > >> > What is 'reasonable', can cause yet another long >> discussion, but my >> > personal favorite for 1-2 distances rms deviation >> is between 0.015-0.020. >> > In Refmac these also give the lowest R factors, in >> my hands. >> > >> > The invisible side chains is yet another long >> discussion that you can >> > retrieve from the ccp4bb archives. >> > Again, my personal preference is to leave them in &g
Re: [ccp4bb] CCP4 Wiki
Hi, I personally am not concerned as much (as I should be?) with the security - Wiki's tend to get attacked but as you point out the databases contain all the old articles and can be backed up on a regular basis. There has been enough concern expressed to me by different folks that I took down the xtals.org/wiki, in order to avoid confusion. I would guess the biggest advantage of a truly commercial solution for this wiki is the fact that it would be managed and backed up by someone who is paid for their services. For all it's worth, I generally agree with the proponents of the more "open" approach to wiki - such as articles could be written by anyone who has a 'real name registration'. Any kind of rigorous procedure designed to select 'worthy' contributors goes against the spirit of the whole enterprise, in my opinion :) Artem > Kevin Cowtan schrieb: >> Yes, that is our intention. CCP4 would probably be at the third level of >> a hierarchy including: >> >> Macromolecular crystallographic techniques >> Structure solution, analysis and visualisation software >> The CCP4 suite > > Kevin, > > why does/will/should the CCP4 Wiki have a hierarchy at all? I thought > that the structure of the Wiki emerges from the links between the > articles. > As far as I know the MediaWiki software does not even support a > hierarchy (but of course I may be wrong, and other software might > support it). > > And why are you concerned about security? In what sense? Breakins into > the computer that runs the software, or vandalism of articles? I believe > the first point is best dealt with by setting up a virtual machine > dedicated to the Wiki, and running an OS that gets automatic security > updates for a number of years. The "vandalism" thing is probably less of > an issue than it is in Wikipedia - but even at the Wikipedia scale it > can be handled well, it seems. > > One other thing that I thought about has to do with permissions. Let me > explain. The easiest way to come up with useful articles would probably > be to just write summaries citing people's emails to the CPP4BB. Citing > Eleanor's postings alone would certainly produce a great Wiki! But - is > it appropriate/allowed to cite people's old emails to the CCP4BB on the > Wiki, without their consent? I for one would certainly give permission > to cite my earlier CCP4BB postings, provided they are cited in the > proper context. > > And finally: why is there a concern by some people about the "premature" > setup of a Wiki, like the one that was set up by Artem? If the articles > in a Wiki are good they can just be copied over to the "CCP4 Wiki" when > it exists. At least that is my understanding concerning Wikipedia > articles - you can just use them under the GNU Free Documentation License. > > thanks, > > Kay > >> >> So there would be room for other software packages at the same level as >> CCP4, and things like purification and crystallisation off of the top >> level. >> >> James Stroud wrote: >>> Would a "CCP4 wiki" be different from a general crystallography wiki? >>> Would it reflect, for instance, the breadth of topics on the CCP4BB? >>> >>> >>> On Monday 23 July 2007 09:57, [EMAIL PROTECTED] wrote: >>>> Contributions from volunteers to establish and maintain the CCP4 wiki >>>> will >>>> be definitely appreciated. >>> >>> > >
Re: [ccp4bb] Dnase activity in E. coli expressed his tag proteins
Hi, This is an intriguing question :) It's quite hard to test for 'stray' nucleases when you potentially have mg/ml amounts of 'real' nuclease. Typically, the DNAse contamination of proteins purified from E. coli is not massive - especially after several purification steps. Otherwise, the companies who make recombinant site-specific nucleases would be in huge trouble :) In fact, when we did test a few things for nuclease contamination a while back, we found RNAse contamination (not surprising) but not DNAse contamination to any reasonable degree. Some of the things that come to mind immediately include: a) trying the cleavage experiment in presence of DNAse inhibitors, EDTA, and so forth. b) testing whether the protein of interest binds to DNA somehow (start by looking at pI - if the protein has a pI of 10 then Bob's your uncle) c) check if this protein works on non-methylated as well as methylated DNA There are enzymes out there that can perform DNA hydrolysis without themselves being 'classical' DNAses (for instance, under some condition polymerases, topoisomerases, or helicases can exhibit DNAse activity). In addition you may have a chemical effect in place (irradiation of certain metal complexes with visible light can generate radicals, causing breakdown of nucleic acids to which these complexes bind - see e.g. DNA and RNA footprinting). It is not a huge stretch of imagination to say that a strongly positively charged enzyme with an appropriate metal center *could* also degrade DNA in a process that is completely unrelated to the normal function of the enzyme. Could you describe in more detail what do you see when you refer to DNA being cleaved as well as with a commercial nuclease? What products are formed (by gel)? Does this happen in darkness as well as under light? Can you repeat this in an oxygen-free environment? Does this work with linear DNA or synthetic oligos? Can you cleave single-stranded DNA (i.e. just one oligo without any self-complementarity). Artem
[ccp4bb] J. Struct. Biology EndNote style
Dear CCP4ers, My turn to ask an unrelated question :) Sorry! Does anyone here have an EndNote style for the Journal of Structural Biology? I am a lazy slob, and would like to check before making my own. The EndNote online collection does not seem to have one. Alternatively, if you can suggest an existing style that is identical to the one I look, I would very much appreciate this! Regards, Artem
Re: [ccp4bb] speeding crystallisation
Hi, Temperature is one obvious factor that comes to mind. If you have access to MS, you could check the protein from the crystallization drops - find out if there's something 'chemical' going on such as proteolysis, modification of some sort, etc. - this has happened to me once before, the protein was undergoing very slow proteolysis, causing the resulting fragment to crystallize. In the absence of MS access you could check by SDSPAGE but not all modifications will show. Likewise, check the pH of the solution in the reservoir of the drops that gae you crystals (if you have a microelectrode, you can try checking the drop as well). Sometimes it gradually changes (e.g. MES buffers exposed to light, acetate buffers leaking acetic acid through the plastic) which can cause your final pH to be way off what you think it is. Likewise with salinity (conductivity). Finally, did you try seeding? If seeding does not work then you should take a really close look at the above :) Good luck! Artem > hi all, > I am working on a protein for which we get nicely diffracting > crystals but the problem is that the crystals grow from anywhere between > 4-6 months. Does anyone has any general suggestions as to what things > can be changed or tried to speed up the process. Any help or suggestions > would be welcome as i am crawling towards getting enough crystals to > make a synchroton visit:):) > > thanks > Neeraj > > -- > Neeraj Kapoor > TPCB Graduate Fellow > Sakmar Lab/ Molecular Biology & Biochemistry > The Rockefeller University > 1230 York Avenue, RRB 510 > New York, NY 10021 > lab.1.212.327.8284:fax.7904 > mobile: 917.535.2030 > http://www.rockefeller.edu/labheads/sakmar/sakmar-lab.html >
[ccp4bb] endnote style file found
Thank you all who sent in the link to the correct endnote library - somehow I failed to find it on the endnote website, which proves that not only I am a lazy slob, but a blind one as well :) Thank you, Artem
Re: [ccp4bb] highly soluble proteins
Yvonne, Several 'old' proteins have been crystallized from insanely high concentraitons - concanavalin A for instance can be grown from 100-250 mg/ml solutions by means of 'salting in' using microdialysis. This is of course highly labor-intensive and also expensive on the protein side. Look at the distribution of charged residues on the protein (especially helpful if you have a model of some sort). I would recommend mutating some of them to non-charged or even nonpolar residues in order to lower solubility. Alternatively, you could try preparing chemical derivatives of the protein using e.g. heavy atoms or amine-modifying reagents like NHS. If you block enough amines, your solubility should go down and your pI will change as well. Likewise you could try modifying exposed acidic residues etc. Mutagenesis can in the end be easier to do because chemical modification tends to produce complex mixtures of products, unless you do it with a huge excess of the reagent and allow the reaction to proceed to exhaustion. Artem > Our lab is trying to crystallize a highly soluble (100+ mg/ml) protein > with a molecular weight of 35 kd. > > The protein was screened against 1536 conditions at 20 mg/mL. Most drops > were either clear or produced "bubbles" (often oily looking). The few > that had precipitate contained high concentrations of K3PO4, cobalt, or > zinc. We have tried repeating some of the bubble conditions at 100+ > mg/mL and are still getting clear drops or bubbles. > > Is there something about highly soluble proteins and/or secreted > proteins and/or proteins with unusual portions of their sequence that > needs to be considered in order to successfully crystallize it? > > I am considering trying "salting out" using dialysis, and also adding > ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer. > > I welcome thoughts and suggestions on crystallization ideas, > publications, etc. > > Thank you > > Yvonne >
Re: [ccp4bb] Question regards to binding affinity of the protein complex?
Dear TriNgo, The Kd is only 1/2 of the picture. You also should contemplate the consequences of the on and off rate for the complex formation. If the off rate is in the range of minutes then you can have successful purification over size exclusion even though the Kd may be high micromolar. Other considerations include the ratio between the plate volume of the sizing column (a theoretical number but useful nevertheless) and the volume of the loaded complex, as well as the concentration of the latter and the rate of diffusion in the column. This is a sticky question, and ultimately the proof is in the pudding - do the experiment on a small scale and check whether the complex comes out 1:1 or you get individual components as well as some ill-defined ratio in the higher m.w. range. As for the second question - yes, many people have co-crystallized complex components by simply mixing them in the (approximately) right ratio. Keep in mind that this ratio isn't necessarily 1:1 - some people only had success with great excess of one component over the other, whereas others found that 1:1 worked best. Therefore in my opinion your best chance to find the right solution is to try a few ratios in a simple screen (say, one 96-well screen over 6 or 9 ratios) to see how things develop from there. Good luck, Artem > Dear CCP4 Users, > I'd like to solve the structure of the protein-protein complex. I > intend to purify and incubate the complex then run gel-filtration > before setting crystal. > I'd like to know your experience about the Kd value of the interaction > in order to get the complex after running the gel-filtration. In other > words, what is the lowest Kd that we can purify the protein complex > using gel-filtration? > And if the binding affinity is too low to purify the complex, is it > possible to get the crystal of the complex if I just incubate and set > crystal of this mixture? > > Thank you very much for all of your advice! > My best regards, > TriNgo > Sungkyunkwan University >
Re: [ccp4bb] Has anyone seen such density?
Hello, This density is the 'evil sausage-like blobule' that tends to crop up whenever something ill-defined (but sausage-like) binds weakly to the surface of the protein. It could be almost anything - something from the polyacrylic acid, something from Triton (it has lots of sausage-like shapes), or perhaps a chain of poorly ordered and incompletely occupied waters. I would leave this density unfilled and mention it briefly in the description of the structure. Alternatively you could model this as disordered water and again, briefly mention this treatment in the "results". Good luck, Artem > Hi all, > > I have a 1.8A structure for which I am in the final stages of refinement. > In a critical hinge region there are two unknown molecules bound to my > protein, which appear to induce the formation of a domain swap. I am > totally out of ideas as to what this molecule could be. I have attached a > a jpg of the offending region. The total length of the unknown molecule is > ~4.4A. The environment is polar and solvent exposed. > > The crystallisation condition was: 0.1 M HEPES, 18% polacrylic acid, > MgCl2, pH 7.0. Cryoprotectant was 20% glycerol. I wish it hadn't been, but > triton-X was used during cell lysis as were Roche Complete Mini Protease > Inhibitor Cocktail Tablets. > > Glycerol is of the correct length but after one round of refinement a > strong negative difference density appears over the middle -OH group and R > factors increase. In addition, any molecule which is alkane-based has > serious clashes with the protein backbone (eg. glycerol or ethylene > glycol, C2H6O2). > > Has anyone seen similarly shaped density, or have any ideas what it could > be? > > Thanks a lot for your help guys, I'm very grateful for any suggestions. > All the best, > Miriam > > PS. Also thanks to everyone who writes in publicly to the bb - it has been > a great learning resource! > >
Re: [ccp4bb] "Quick soak" method
Hi, I do not use their method as such - however, I love heavy atom soaks and do them any time I can, so I've got very similar experiences in the past. Heavy atoms can bind very quickly even from quite dilute solutions - the quickest I've ever soaked (and got useful data) was sodium chloroplatinate in under ten minutes at ~ 1mM concentration. The next quickest was K2Pt(NO2)4 which tends to take between 10 minutes and 30 minutes, almost regardless of the concentration. This is by far my favorite HAD reagent, incidentally. In general my soaks are all less than one hour with a few exceptions, such as iodine [not iodide!] soak to iodinate tyrosines which is typically better done during a day or so with very low amount of I2, via vapor phase. Artem > Hi, > > I'd like to find out how successful the "quick soak" method for heavy > atom derivatisation proposed by Radaev and Sun: > > Sun PD, Radaev S, Kattah M. Generating isomorphous heavy-atom > derivatives by a quick-soak method. Part I: test cases. Acta Cryst. > 2002. D58:1092-1098. > > has been in comparison to the "classical" method of longer soaks at > low concentrations of heavy atom compound. The method was quite > successful in our hands a few years ago but (fortunately?) it's > becoming increasingly rare that we use heavy atoms. I understand that > evidence will necessarily be anecdotal, but let's not let that stop us. > > Derek > -- > Derek Logan tel: +46 46 222 1443 > Associate professor fax: +46 46 222 4692 > Molecular Biophysicsmob: +46 76 8585 707 > Lund University > Box 124, Lund, Sweden > > >
Re: [ccp4bb] Ketone inhibitors
Here's a Cathepsin K family of inhibitors with a straight ketone: Bioorganic & Medicinal Chemistry Letters Volume 17, Issue 1, 1 January 2007, Pages 22-27 and here's a structure paper (covalent adduct with the ketone) www.jbc.org/cgi/reprint/266/26/17695.pdf Here's a couple of Cruzain structures with ketone inhibitors. Look for ligands in the pdb called ZRA or ZYA http://www.rcsb.org/pdb/ligand/ligandsummary.do?hetId=ZYA http://www.rcsb.org/pdb/ligand/ligandsummary.do?hetId=ZRA Bioorg Med Chem. 2003 Jan 2;11(1):21-9. BACE (memapsin) and related enzymes are some of the most studied peptidase targets. I believe there were a few ketone inhibitors pulished for these enzymes, but I am honestly not sure if any of the structures are in the public domain. Here's an alcohol version of a peptidomimetic: http://www.jbc.org/cgi/content/full/277/7/4687 Here's a difluoro ketone one: Moore CL, Leatherwood DD, Diehl TS, Selkoe DJ, Wolfe MS. Difluoro Ketone Peptidomimetics Suggest a Large S1 Pocket for Alzheimer's gamma-Secretase: Implications for Inhibitor Design. J Med Chem. 2000 Sep 7;43(18):3434-3442. Michael adduct traps (alpha-unsaturated ketones) have been proposed for kinases with a Cys in the ATP site: http://www.freshpatents.com/Specific-kinase-inhibitors-dt20060413ptan20060079494.php Good luck, Artem > Hi all > > I have searched the PDB without much luck and wondered whether anyone out > there could confirm: > > I'm looking for co-crystal structures of enzymes with inhibitors that bind > to the catalytic group via a ketone moiety. Our case is a metalloprotease > with a ketomethylene peptide analogue but any kind will do. > > As far as I can tell, the PDB has a few examples with an aldehyde at one > of the termini of the inhibitor (e.g. 1LAN, 1CBX), and some with modified > ketones (e.g fluoro-, 1OD1, 1NJS) but none with an unmodified internal > ketone. > > So far, I've found some carboxypeptidase A papers from the 80's and > nothing since. Have I missed something? > > Sincerely > Jean Watermeyer > > > Jean Watermeyer > Division of Medical Biochemistry > IIDMM > University of Cape Town Health Sciences > Anzio Rd Observatory > 7925 > South Africa > > Tel. 021 4066832 > > > PLEASE UPDATE: my email address has changed: [EMAIL PROTECTED] > > Jean Watermeyer > Division of Medical Biochemistry > IIDMM > University of Cape Town Health Sciences > Anzio Rd Observatory > 7925 > South Africa > > Tel. 021 4066832 >
Re: [ccp4bb] artificial dimerization modules
C-terminal 'clasp' domain of any bacterial gyrA or parC - very small, pretty tight dimers. Artem > This is not strictly a crystallography question, but I imagine (and > hope) that some of you would be able to advise me. In short, I would > like to add to t7he N-terminal part of my construct a sequence that will > promote dimerization of the molecule. > > Background: I am working on a large protein that is built from multiple > domains. I am interested in the structure of some of the more C-terminal > domains. Obviously, I do not have well diffracting crystals at this > point (and hence this post). N-terminal to the domains of interest is a > dimerization domain. I would like to mimic the dimerization induced by > this domain by adding as short as possible a sequence. Moreover, I would > like to try both a parallel and an anti-parallel dimerization motif. > > Would you know of an appropriate short sequence (e.g. one that makes a > helix which dimerizes), or of a very small domain that I could add to my > construct? > > Thanks. > > Arnon >
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
This is not entirely uncommon. Did you try removing just one of the two sites (sometimes it helps) - combined with enzymatic deglycosylation this may give you good enough protein to work with. If you try other hosts, I would definitely consider Schisosaccharomyces pombe - its glycosylation patterns are much simpler. Other likely candidates include regular bakers' yeast and Hansenula polymorpha. I would try both the native and the mutant proteins in those hosts. There are tricks one can play with Pichia to try and make it express the deglycosylated protein but it almost sounds like you've hit on something very essential since you're going from good expression down to no expression. Lastly, you could try insect cells :) Artem > > Dear all, > > Our lab is new to working with Pichia pastoris, also new to working with > glycosylated proteins. We have a construct for a secreted protein that > expresses pretty well in Picha, but upon mutation of the 2 N-linked > glycosylation sites to Ala, we get no expression at all, nada. The > nucleic acid sequence appears to be correct, i.e. we have not introduced > any unintentional frame shifts, stop codons, or anything like that. Is > this a common phenomenon? Are there any tricks to get the Pichia to do > its thing? Any chance that alternative substitutions will work when Ala > does not? Or are we better off (a) trying to deglycosylate > enzymatically, or (b) trying a different expression host? All opinions > and anecdotes welcome. > > Thanks! > Evette > > Evette S. Radisky, Ph.D. > Assistant Professor and Associate Consultant II > Mayo Clinic Cancer Center > Griffin Cancer Research Building, Rm 310 > 4500 San Pablo Road > Jacksonville, FL 32224 > (904) 953-6372 (office) > (904) 953-0046 (lab) > >
Re: [ccp4bb] intersection curve of cone with cylinder
In topology, long time ago, I was taught that these are indeed 'intersection curves of surfaces of the second order'. Emphasis on surface being second order, not the curve. This was in Russian, which means that the English terms may be different. You might also encounter 'intersection curves of quadric surfaces'. There are seventeen types of quadric surfaces (including real and imaginary ones) and they're a right pain in the neck to calculate intersections of. Artem > Dear All - > > if memory serves me right, the intersection curve of > a cone with a cylinder (like in a powder diffraction camera) is > not an ellipse, but a "Schnittfigur zweiter Ordnung", > loosly translated as 'intersection curve of second order'. > > Is there a correct English term for that type of curve? > > Thx, br > - > Bernhard Rupp > 001 (925) 209-7429 > +43 (676) 571-0536 > [EMAIL PROTECTED] > [EMAIL PROTECTED] > http://www.ruppweb.org/ > - > The hard part about playing chicken > is to know when to flinch > - >
Re: [ccp4bb] Econo column with AKTA FPLC
What you need is a tubing and connector kit. Every decent lab has one, and you also usually can order these in part or whole from FPLC/HPLC manufacturers. The two types of female fittings are then attached to a short length of tubing and presto. Artem > Dear All, > > I have econo column from Biorad and AKTA FPLC system. how shall i connect > the econo column to AKTA FPLC system i.e what are the different > connections / tubings to be used since akta system the tubing are all > small > sized and econo column tubing are large capacity. > > does anyone have any experience with the above and help me out with this. > > thanks in anticipation. > > > exec >
Re: [ccp4bb] Sephadex resins
Any basic chromatography (for proteins) text will have those. It's generally not hard - if you have dry sephadex just resuspend it in 10-20% alcohol, stir well (but gently!) and let sit overnight, then pack the column, wash, and go. Artem > does anyone have protocols / method of sephadex resin. about swelling the > resin use of buffers, elution of sample from the resin. i have referred to > the GE healthcare website, it gives details of other resins well, but > sephadex resin not much detail is given. > > kindly guide >
Re: [ccp4bb] histidine modification
Hi, 1. the same image in jpg or png format occupies a mere 180 - 300 kB. It's nice to be considerate of other people's bandwidth especially since you're asking for help 2. how do you know it's a covalent modification? Anything on MS to confirm that? 3. this reminds me of http://www.jbc.org/cgi/content/full/278/3/2008/F1 4. in theory this could be a particularly stable calcium complex (what's the distance between the center of the fat blob in the middle and the N of His?) Cheers, Artem > Hi -- I am close to finishing up a protein structure of an aldo-keto > reductase but for a modification of a histidine residue shown here > (one in each molecule of the AU). Note the following electron density > in which this residue in both molecules of the AU was mutated to an > Ala, Refmac'd for 5 cycles and then 2fofc and fofc maps are shown. > Resolution goes to 1.6 A, and the Rwork/Rfree is 16/18.9%. However > this modification flumoxes me and I was hoping someone (someone!!) has > seen something like this before. This crystal was shot at LS-CAT but > I see something similar with a slightly lower resolution crystal I > shot on our homesourse Raxis4. Crystallization conditions are 0.2M Ca > Acetate, 9% PEG 8K,0.1M Na Cacodylate pH6.5, Cryo- 25%Et.Glycol. > Other people have suggested this may be a modification of the > cacodylate, however the cacodylate modifications I've seen in the > lit/pdb seem to refer to modifs of cysteine in which the arsenic > contains two oxygens attached to the arsenic atom which is in turn > bound to the cysteine sulfur. However in this case, there we seem to > have at least 4 atoms shown coming off the Nepsilon of the his residue. > > Thank you for your time. > > Alex Singer >
Re: [ccp4bb] Spooky, moving crystals
We had exactly the same - and there were two culprits: 1. frozen gas trapped between the jacket of the pre-mounted Hampton loop and the loop itself 2. ice on the base of the pin. When the ice slowly melted (on contact with the goniometer head at room temp.) the base shifted by infinitesmally small amount - enough to drive the displacement at the end appreciably. Depending on the amount of ice you may experience angular, lateral, or vertical movement. Drying the pins before use eliminated most of the issues. Artem > Dear all, > > in a recent synchrotron trip we had a problem with our crystals moving > after mounting them onto the goniometer, in some cases they moved out > of the beam and even out of the zoomed camera picture - it seemed the > pins, upon equilibrating to room temperature, extended. It happened > with pre-mounted litho-loops only, not with pre-mounted mitegen loops > on the same trip, so one possible cause is different metal allows used > in the pins, somehow the mitegen ones being more suitable. > > We used two-component glue to stick the pins into the metal bases > (Spine), so that might be another possible culprit. Perhaps we did not > allow sufficient time for the glue to react before freezing into > liquid N2 and it continued its reaction upon thawing, somehow pushing > the pin a bit out of the base. In this case the difference between > litholoops and mitegen loops may have been the thickness of the pins, > the latter somehow allowing expansion of the glue along the sides, the > former not. > > In any case, I am wondering if any of you has seen this before, so we > know how to avoid it in the future. > In some cases, it took 10-20 min. for the crystal to stop moving, > which, with the current data collection speed and robotic mounting, is > significant. Fortunately, it did not affect our trip too much, as we > has sufficient time in the end. > > Greetings, > > Mark > > Mark J. van Raaij > Dpto de Bioquímica, Facultad de Farmacia > Universidad de Santiago > 15782 Santiago de Compostela > Spain > http://web.usc.es/~vanraaij/ >
Re: [ccp4bb] Running Resolve after Sharp?
Yes, I did that recently and it worked although I found SOLOMON maps to be (subjectively) better. You should probably use the 'centroid' phases from the eden.mtz (SHARP gurus might want to correct me :)) Artem > Hi > > Sorry for a mixed up question about two great software. > > Has anybody tried to use Resolve after sharp / autosharp? i.e., > escaping the Solomon step other than handedness determination and > doing the density modification (& maybe NCS / building) directly with > the sharp output? If so, I would appreciate any input.. I am confused > with the eden file.. > > Cheers, Partha > > > > > > -- > MRC National Institute for Medical Research > Division of Molecular Structure > The Ridgeway, NW7 1AA, UK > Email: [EMAIL PROTECTED] > Phone: + 44 208 816 2515 >
Re: [ccp4bb] Off-topic: Native gel electrophoresis of basic proteins
You could try Coomassie Blue Native gel. It's a very neat technique and it worked for me on a couple of occasions. In one unfortunate case, it resulted in dissociation of a heterotetrameric complex, though. Artem > Dear CCP4 community, > > Sorry for the off-topic subject, but I would really appreciate some > suggestions and/or protocols relating to native gel electrophoresis of > basic proteins. I have used a general acidic PAGE protocol for my protein, > which has a PI of 9.5. Briefly, the protein was loaded onto a native gel > (I have tried both the pre-made Biorad gels (7.5% and a gradient gel: > 4-15%) and freshly prepared Tris native gels adjusted to pH 6.8) and run > in a 1X acetic acid/b-alanine pH 4.5 running buffer. The electrodes were > reversed and the gel run on ice for ~ 2hrs at 100V. In all cases, the > native protein was unable to enter the gel. Some protein samples incubated > with heavy atoms were able to enter the gel (possibly indicating binding) > but these samples too had problems entering the gel as the bands were at > or just a little bit below the edge of the well. Any suggestions and > comments would be most welcome! > > Thank you so much in advance for your help, > Sincerely, > Ming Lye >
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Hello Meg, Since your protein is quite acidic, your next step could be e.g. anion exchange - provided that you are able to get the protein into a suitable buffer w/o losing it (since you will pass through the pI). If you can, the simplest way to do so is to add TRIS to the pH4.5 buffer until you get the desired pH (7-8) and dilute the sample a bit with pure water. An alternative, or a follow-up step could be hydrophobic column - these are very useful for removing aggregates, partially folded stuff and similar entities. Again, nothing is completely safe - HIC may cause problems since you are starting at fairly high salt (1-2 M NaCl or KCl typically). But at least you won't have to pass the pI. Cheers, Artem > Dear All, > > This is with reference to the purification of our recombinant protein > sample > expressed in E.coli as inclusion bodies. After Solubilization refolding we > perform > the cation exchange chromatography of our protein sample using SP > sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF > results of the collected fractions. > > In addition to our protein of interest we are also getting high molecular > weigh > contaminants, which we cannot get rid of in IEX. Can anyone please guide > me > on a technique to get rid of these bands as even after gel filtration of > samples > few high mol wt contaminant bands are not separated from main proteins and > sample gets diluted too. > > In cation IEX procedure is > Column Sp Sepharose Fast flow packed in fineline 35 column packed bed > volume 100 ml > System AKTA FPLC > Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein > conc], > washing to remove unbound materials 2 C.V. step elution 0-35% gradient 1 > C.V., 35 80% gradient 10 C.V. and 60 100% gradient 1 C.V. > Protein elutes at 40-50% gradient. > Protein details: Our protein is stable at acidic pH and has a pI of 5.8 > 6.3 and > buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer > containing 0.4 M NaCl. > > We get only one peak on AKTA but on running SDS page we get so many > bands even IEF shows 1-2 bands at the most. > > > How can we modify the method or what can be done to get rid of extra high > mol wt bands. > > Any help will be deeply appreciated. > >
Re: [ccp4bb] foam dewar usage ?
Yes, we use them all the time and they're great. Stuff does not slip on the bottom like it does in the glass Dewars and the formation of ice is greatly reduced. Plus, they're much easier to dry. Artem > Does anyone in the biocrystallogaphy community use foam dewars for > handly liquid nitrogen and freezing/manipulating frozen protein > crystals ?
Re: [ccp4bb] Potential N-terminal cyclization - 2nd attempt with formula attached
Hi, Not sure what you mean by 'other way around' - the formula you drew is what I was thinking about :) Mesomeric states in normal amidines are averaged and should result in planarity - since this case does not seem to have a planar geometry, there's a chance that the amidine never formed and the transient Schiff base was reduced by something. There is also an off chance that the Histidine side chain was attacked on the *carbon* rather than the NH. This does happen and has been observed in studies dealing with heat- and aldehyde-driven crosslinking of proteins. However the image of the e.d. that I saw does not support this. Artem > Dear Colleagues, > > The amidine originating from the condensation reaction between the > Met N-formyl group and the His side chain should be the other way > around! This would also be roughly consistent with the reported > geometry. In fact, there should be two mesomeric states, provided > there is N protonation. > > Arne Skerra > > > > At 5:56 Uhr -0400 10.10.2008, Artem Evdokimov wrote: >>If you're 100% sure that this is only one atom then amination comes to >> mind. >>I have no clue what conditions would favor such reactivity but it is >>possible that the formyl group on the Met was aminated with the cyclic N >> of >>the histidine, resulting in either a substituted bis-amine (requires >>reduction, may not be stable) or in a cyclic amidine (the latter requires >>four atoms - N1-C=N-Ca - to be in the same plane). This amidine should >> have >>some pretty interesting properties (probably more like a Schiff base than >> an >>amidine). >> >>Could you tell us a bit more about the system you're working with? >> >>Artem >> >>P.S. The bond lengths you describe are not typical for C-N, however at >> 1.9A >>resolution it is not very likely that the values you measured actually >>correspond to the bond lengths (hopefully, because otherwise you have >> >>-Original Message- >>From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of >>gerwald jogl >>Sent: Thursday, October 09, 2008 4:24 PM >>To: CCP4BB@JISCMAIL.AC.UK >>Subject: [ccp4bb] Potential N-terminal cyclization >> >>Hi All, >> >>I am looking for some input regarding an unusual feature in one of our >>structures. Maybe someone has come across something similar or has some >>thoughts about it... >> >>We have a 1.9 A crystal structure with well-defined density in the >>N-terminal region. The side chain of His3 is oriented towards the >>N-terminal amino group of Met1 and there is a nice difference density >>'ball' right in between the His and the amino group suggesting that >>there is an additional atom that is covalently linked. There are two >>molecules in the asu and both show the same feature (no ncs refinement). >>My problem is that I cannot come up with a reaction that would result in >>such a linkage and there is not much to be found in the literature. >> >>If I place a hypothetical atom in the difference density peak, I can >>measure distances and angles. Here are some numbers: Distance from atom >>X to the N-terminal nitrogen 1.72 (1.64 in chain B). Distance from atom >>X to His NE2 1.58 (1.45 in chain B; the temperature factors of the His >>side chain are slightly more consistent with this ring orientation >>compared to the 180 degree flip that would bring CE1 towards atom X). >>Atom X is coplanar with the His ring. The angle between NE2 - X - N1 is >>95 deg (106 in chain B). The angle between X - N1 - Calpha1 is 111 >>degrees in both chains. As the N-terminal methionine is still present, >>it is possible that the methionine formyl-group was present before the >>hypothetical reaction. However, there is only one 'atom' in the >>difference density. >> >>Any comments or suggestions would be highly welcome. >>Gerwald > > > -- > > > Prof. Dr. Arne Skerra [EMAIL PROTECTED] > Lehrstuhl f. Biologische Chemie Phone: +49 (0)8161 71-4351 > Technische Universitaet MuenchenFax: -4352 > 85350 Freising-Weihenstephan > Germany http://www.wzw.tum.de/bc >
Re: [ccp4bb] warning message will NOT go away
To quote Terry Pratchett: +++ Divide By Cucumber Error. Please Reinstall Universe And Reboot +++ Couldn't resist :) Artem > You have an error in your mtz file.. > > OK- so I am sorry! But the Warning will not disappear.. > Eleanor >
Re: [ccp4bb] tricoordinated ion?
May be something that tagged along from purification or one of the impurities in the chemicals... nitrate, carbonate, etc. Incidentally, a 3.9 M BMP file was a nasty surprise for my mail box. A JPEG or PNG file of similar quality would have taken less than 100K... Artem > > Hi all, > I wanted to ask you what would you model in the density in which I > have at the moment modelled 4 water molecules, which are however too > close to be waters, I guess (see attached image). > My crystallisation conditions contain NaCl, MgCl2, Peg400, TrisHCl, TCEP, > glycerol. > I can't think at a tricoordinated ion like that... > thanks in advance for the hints, > ciao > S > > -- > Sebastiano Pasqualato, PhD > IFOM-IEO Campus > Dipartimento di Oncologia Sperimentale > Istituto Europeo di Oncologia > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5094 > fax +39 02 574 303 310
Re: [ccp4bb] getting weak diffracting crystals
Dear Deb, We've seen the detrimental effects of local disorder time and tme again. So - yes it is very likely that the putative disordered loops detrimentally affect the quality of your crystals. You can try to engineer your protein to be better - it usually takes a number of internally engineered constructs to get things right (about 10 per 350aa protein, in my experience). This is different from terminal truncations - which also can have a huge effect on crystallization. You might also try some of the methods summarized here: http://www.xtals.org/pdfs/rescue_crystals.pdf Cheers, Artem > > Dear Members, > > I am getting crystals of my protein. The secondary structure prediction > implies that it has N-terminal with high degree of loop regions. I also > get some mountable crystals yielding weak diffraction pattern(10 A). The > quality of the crystals can also be assumed from its texture for it has > tortuous surface. The big crystals are achieved at very high concentration > in hanging drop method (44mg/ml) whereas the initial hit (small crystals) > is at lower concentration in sitting drop (~ 5mg/ml). > > I have some queries about it. Does the N terminal loop regions are having > any effect of the crystal quality. Any suggestions of its quality > improvement are welcome. I will be highly benefited with your generous > replies. > > Sincerely > Deb >
Re: [ccp4bb] Off topic: 182 kDa protein is too large to crystallize
Hello Marek, 182 kDa protein is nothing special - unless it has huge areas of disorder, membrane-association domains, coiled coils, or something like that. Much larger proteins and protein complexes have been successfully crystallized. With respect to purification - this is where you may want to diversify. There are numerous avenues open to you: you can explore other forms of distributive chromatography (HIC, IE, etc.); you can opt to use other affinity matrices (dye resins, etc.) or you can try sizing using a non-carbohydrate matrix. Not to mention that there are numerous options for IMAC - Ni-NTA is just one of many. I would be glad to help you further off the main list if you want to share more details. Cheers, Artem
Re: [ccp4bb] unknown density for a small molecule
Hi, It could be all sorts of things, but the one that for some reason is stuck in my mind is isopentenyl phosphate (phosphate, not pyrophosphate!). Of course w/o seeing the density in 3D this is just a guess. Artem > Dear All, > > When I was refining my structure, I found some unmodeled blobs, shown as > attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor > 0.18, refined to 1.9 angstrom ). > > The protein was expressed in E.coli and purified by nickel column and gel > filtration, both in tris buffer. The crystallization condition has 2 M > ammonium sulfate and 0.1 M sodium acetate. > > The lower part looks like a sulfate group, which is held by one Arg, one > his, one lys and one asn. The latter three residues are from another > asymmetric unit. > > The other end of the small molecule is stunk by the rings of Tyr and Phe. > It > also interacts with the OH group of another tyr and one water molecule. > > Is there a program can build small molecule models according to the > densities? Or could anyone tell what it might be from the density? > > Thanks a lot! Any suggestions will be appreciated! > > Mengxiao >
Re: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space
Sure, it's not always 'disastrously bad' to have EDTA (hence my use of the word 'bad' rather than a more categorical statement. Donuts are bad for me yet I can't stop eating them :) Yes, you can take a risk. However since periplasmic isolation is already a PITA, why add an extra concern? Artem > Artem, > > Artem Evdokimov wrote: >> Please note that osmotic shock extraction typically employs EDTA which >> is >> obviously bad for IMAC. >> > This is not entirely correct. > I have used extracts with 5 mM EDTA for IMAC in the past. > If your IMAC column volume is large enough, only the top 1-2 mm will be > depleted of Me2+ (easily seen with Cu2+). > Moreover, it is always possible to add some Me2+ to your extract prior > to IMAC. > All this was published long ago (Biochemistry. 31: 2690-2702, 1992). > > Nadir > > -- > > Pr. Nadir T. Mrabet > Cellular & Molecular Biochemistry > INSERM U-724 > Nancy University, School of Medicine > 9, Avenue de la Foret de Haye, BP 184 > 54505 Vandoeuvre-les-Nancy Cedex > France > Phone: +33 (0)3.83.68.32.73 > Fax: +33 (0)3.83.68.32.79 > E-mail: nadir.mra...@medecine.uhp-nancy.fr > > > >
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi, The question you have to ask yourself first is - does my gene actually *have* the rare codons that you're trying to avoid? Experience shows that usually it's not single codons that are a problem but pairs or triplets of rare ones. If your gene does not have obviously bad codon combinations you still may derive a benefit from codon optimization and especially from mRNA structure shuffling. There are articles out there that attempt to present statistically significant evidence and I tend to agree with them - the practice of optimizing codon usage AND mRNA structure is a good and useful one. >From what you describe, you're working with a kinase. It is not at all uncommon to have toxicity of kinases for E. coli. Additional issues include heterogenous phosphorylation if the kinase is normally active or can auto-activate in E.coli (classical example is PKA which can phosphorylate itself in up to 30 places given the right conditions). Toxicity isn't difficult to spot - classical signs include microcolonies, heterogenous colonies, slow growth, plasmid instability and so on. Even with a native gene you would likely notice these symptoms to some extent. Incidentally - toxicity usually equals folding, i.e. for a kinase to be toxic at least some of it has to be folded enough to work. This is actually *good news* because toxicity can be combated on a different level than lack of folded expression. For example, co-expression with phosphatases tends to work miracles for kinase-based toxicity. Finally, to answer your question directly - yes, i've seen several cases of proteins not expressing even with rare codon tRNA supplemented in trans, but expressing well from optimized DNA. Again - optimized for codons AND structure, as I've never separated the two processes. Synthetic DNA is cheap these days. If you can afford it - it's useful to try before taking the next step. In this case the obvious next step is attempt at expression in insect cells - kinases usually work out really well in IC. Artem
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi, Servers to check for rare codons definitely do exist. http://genomes.urv.es/OPTIMIZER/ http://www.doe-mbi.ucla.edu/~sumchan/caltor.html and several others As to comparing Rosetta and Codon+ - your best bet here is just to dig up product literature from the manufacturers. Rosetta uses pRARE: AGG, AGA, AUA, CUA, CCC, GGA Codonplus BL21-CodonPlus(DE3)-RIPL strain argU (AGA, AGG), ileY (AUA) , proL (CCC), leuW (CUA) BL21-CodonPlus-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA) BL21-CodonPlus(DE3)-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA) BL21-CodonPlus(DE3)-RIL-X strain argU (AGA, AGG), ileY (AUA), leuW (CUA) BL21-CodonPlus-RP strain argU (AGA, AGG), proL (CCC) BL21-CodonPlus(DE3)-RP strain argU (AGA, AGG), proL (CCC) BL21-CodonPlus(DE3)-RP-X strain argU (AGA, AGG), proL (CCC) Artem > Thanks for the reply. > > I've checked my sequence for rare codons; however, what would be useful to > a > pseudo-molecular biologist like me is a web server which will look at your > input DNA sequence and guesstimate the success of expression in E. coli > (i.e., consider codon frequency). Does one exist? Even better, a web > server > that offers the choice of different commercial forms of E. coli (i.e., > Rosetta and Codon+)? I tried Googling this a few days ago and didn't find > anything too useful? > > Cheers! > > On Tue, Feb 24, 2009 at 11:00 AM, wrote: > >> Hi, >> >> The question you have to ask yourself first is - does my gene actually >> *have* the rare codons that you're trying to avoid? Experience shows >> that >> usually it's not single codons that are a problem but pairs or triplets >> of >> rare ones. If your gene does not have obviously bad codon combinations >> you >> still may derive a benefit from codon optimization and especially from >> mRNA structure shuffling. There are articles out there that attempt to >> present statistically significant evidence and I tend to agree with them >> - >> the practice of optimizing codon usage AND mRNA structure is a good and >> useful one. >> >> From what you describe, you're working with a kinase. It is not at all >> uncommon to have toxicity of kinases for E. coli. Additional issues >> include heterogenous phosphorylation if the kinase is normally active or >> can auto-activate in E.coli (classical example is PKA which can >> phosphorylate itself in up to 30 places given the right conditions). >> >> Toxicity isn't difficult to spot - classical signs include >> microcolonies, >> heterogenous colonies, slow growth, plasmid instability and so on. Even >> with a native gene you would likely notice these symptoms to some >> extent. >> >> Incidentally - toxicity usually equals folding, i.e. for a kinase to be >> toxic at least some of it has to be folded enough to work. This is >> actually *good news* because toxicity can be combated on a different >> level >> than lack of folded expression. For example, co-expression with >> phosphatases tends to work miracles for kinase-based toxicity. >> >> Finally, to answer your question directly - yes, i've seen several cases >> of proteins not expressing even with rare codon tRNA supplemented in >> trans, but expressing well from optimized DNA. Again - optimized for >> codons AND structure, as I've never separated the two processes. >> >> Synthetic DNA is cheap these days. If you can afford it - it's useful to >> try before taking the next step. In this case the obvious next step is >> attempt at expression in insect cells - kinases usually work out really >> well in IC. >> >> Artem >> >
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi, I respectfully disagree with the doom&gloom feelings regarding fusion proteins. In my not very limited experience, fusion proteins *can* fix expression issues. Do they always work - of course not :) But there are very few things in this field that work most of the time. Is it better to try a fusion protein or to go into a higher-order expression system? If you can afford it, usually higher order systems tend to work better. But what if you cannot afford it? Regarding precipitation upon cleavage - consider the example of PTPbeta catalytic domain: this protein expresses very poorly on its own, however it expresses extremely well with a His-MBP N-terminal fusion, and the activity of the fusion protein is very high. If you cleave the protein in 'just buffer' then PTPbeta rapidly precipitates. Bad news, right? However if you cleave the fusion in the presence of 0.1% BOG the protein stays perfectly soluble and monomeric, concentrates to 15 mg/ml and produces marvellous crystals (about six structures in the PDB). So - do not be too quick to dismiss fusion proteins as a way to try and salvage your desperate cases, especially if going to a different expression system is hard for some reason. Regarding SUMO - I have personally tested it on about 30-35 proteins. It only worked for *one* - but it made the protein nice and soluble, and it stayed soluble after cleavage (note - we do not use the SUMO-protease, just regular protease sites). Is ratio like that worht the trouble? You decide :) Artem > Some thoughts about SUMO tags and fusion tags in general. > > Fusion tags also follow the "Garbage In, Garbage Out" philosophy. > Yes, if for many of the reasons already hashed out extensively on > CCP4BB, one is dealing with lack of expression or miniscule > expression, often tagging the protein with a fusion/cleavable tag > does indeed bump up the expression and lead to 'improved solubility'. > Sometimes, it's very important to ask: improved solubility of what > though? > > Everything that Phoebe describes, namely the chaperone contamination, > precipitation after cutting off tag etc., reeks of an intrinsically > misfolded/unstable/unhappy protein. My experience-- and those of many > others-- is that the fusion tag and fusion tag alone can only fix > little in cases: 1) when one observes lots of degradation of the > untagged protein, 2) where the untagged protein is made as an > intrinsically misfolded/unstable protein. In these cases, the carrier > protein then notoriously comes along for the ride in the soluble > fraction with the fusion/cleavable tag, initially giving the > impression of improved expression and improved solubility. Even then, > one might even see multiple degradation products with the tagged > expression product. Next, cleave the tag off in such a case and lo > and behold! all protein precipitates and you are back to square one. > > I am not trying to discourage anyone from using fusion tags -- to > improve expression, solubility, crystallization etc. We all know of > many examples where fusion tags have worked wonders. I only caution > that if your favourite protein is intrinsically misfolded in a > particular expression system and then you have tried tagging a fusion/ > cleavable tag onto the protein in the same expression system and you > observe all that Phoebe describes, perhaps it is time to bang your > head against a different wall now. In many difficult cases, I am > unaware that a fusion tag actually aids in the proper folding of a > carrier protein. I will not rule out this possibility but I do not > know that this is the general rule. > > I have worked quite a bit with SUMO tags. As far as GST and SUMO tags > are concerned, I banged my head against the GST-tag and SUMO- tag > wall for my target protein for a frustrating while. I tried a His > tag, then a GST tag, then a SUMO tag. All had exactly the same > symptoms. In my case, clearly the problem lay with the carrier > problem but I was never allowed to conclude so. > > Just my two cents, the worth of which will already have diminished by > the time you have read this email. > > Raji > > > > > > > On Feb 26, 2009, at 11:30 AM, Phoebe Rice wrote: > >> We haven't tried SUMO, but had some frustrating results with >> GST fusions. They did improve expression and solubility - BUT >> in one case the target protein precipitated immediately when >> the tag was cleaved off, and resisted all attempts to bring it >> back to life. In another case, the fusion protein dragged >> chaperones into the prep that were nearly impossible to get >> rid of completely, thus ruining our ATPase assays. >> >> Is SUMO, being smaller, less likely to drag such crud along >> with it? >> >
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi, In a case like the one Raji outlined below - after all the attempts - I would have most cerainly switched to insect cells as the next step :) If you suspect that protein of interest has large disordered regions, expression in a higher order system by itself may not be enough (still good chances of proteolysis). I am guessing here, but co-expression with a partner is probably necessary (as per your point #4). All of this assumes that you need the entire protein, of course (as Raji said) - because otherwise the fairly obvious next step could be to chop that beast into manageable chunks :) Is this 'normal' and 'expected'? I would say that yes, if you are working with a large human protein that has potentially disordered regions and is normally a part of a stable complex with other stuff - yes you can expect problems exactly like these (or worse). Artem > BUT...I recently had a similar problem to what Mo outlines -- human > protein in E. coli, 'zero induction', wondering if the protein is > toxic based on colony morphology and effect on cell growth, and all > the good stuff that Mo describes. In my case, for my human protein: > 1. Disorder-prediction and secondary-structure-prediction programs > indicate stretches of disordered protein > 2. After the usual tricks of bacterial expression with a His-tagged > version (temp, IPTG variation, autoinduction, cells), I got 'zero > induction'. Then I switched to larger fusion tags like those both of > you mention and then I could see expressed protein, though there was > only a small fraction of full-length protein and copious amounts of > proteolysis and/or truncated products (mass spec, Western blot etc. > etc.). > 3. When one tag didn't work, I switched to another. After switching a > bunch of tags, I consistently faced the same kinds of problematic > issues like persistent proteolytic degradation, persistent > aggregation even when fusion tag was left intact, and hard-to-remove > chaperone contamination during purification. I was unable to recover > purified full-length protein and these issues could not be mitigated > by buffer, pH optimization etc. during purification etc. > 4. Literature indicates that the protein might function in a complex > > In my case, I used all of the above information collectively to > hypothesize that my target protein, under the given conditions, was > likely poorly folded on its own. In this specific case, even before I > determined whether to move to a different expression system or not, I > did still have at least a few choices that I can think of: > a. To attempt co-expression with a protein binding partner > b. To attempt co-expression in a chaperone-aided expression system > c. To resort to a synthetic gene > (Chopping up the protein and going after individual domains was not a > real option in my case.) > > Is my example the norm or the exception? I leave that open. > > Even if my case is extremely rare, my original intention was to > caution that there may come a time with a beast of a protein-- after > a LOT has been attempted and tried-- when one has to start to quickly > distinguish between a promising approach and a futile time-sink. > > To me, it seems we concur and there is not much scope for a healthy > debate :)! > Raji > > > > > On Feb 26, 2009, at 10:07 PM, Artem Evdokimov wrote: > >> I personally think that debate is healthy. >> >> Having said this I do not advertise one fusion partner over >> another and I did not intend to overweight the MBP success in my >> previous message, it was just a familiar example. As I mentioned, >> SUMO also worked for us perhaps not as frequently as MBP but >> there is no statistical significance in this because the number of >> studied cases was way under a thousand (which intuitively sounds >> like a useful number, but in fact even a thousand is probably too >> small to be meaningful). >> >> Unlike choosing one brand of OS over another, the choice of fusion >> partner (or indeed whether to use or not to use one) is not an >> expensive one since cloning is generally very cheap and expression >> in E. coli is also extremely affordable. Therefore it is perfectly >> easy to try as many expression tricks as one desires. Having said >> this I also should mention that enough trials in E. coli eventually >> out-price a few trials in insect cells or other higher order >> system so everyone should apply their own judgement as to when the >> aggregate cost advantage disappears. I dont think that anyone >> would disagree that chances of successful expression of a >> eukaryotic protein in e.g. insect cells are considerably higher >> compared to the sa
Re: [ccp4bb] Structural biology inside the cell
Hello, 1. As long as all proteins have seventy amino-acids or less and express in E. coli in mM concentrations - we're in business. 2. As for the question below - my favorite answer is 'It will take a week and ten million dollars in unmarked bills. We begin as soon as the money arrives.'. Artem > As another example, more than once I have been asked by someone if > they give me the sequence or name of a protein (even membrane > proteins), how many days would it take us to provide them the crystal > structure. > > Mark > > Mark J. van Raaij > Dpto de Bioquímica, Facultad de Farmacia > Universidad de Santiago > 15782 Santiago de Compostela > Spain > http://web.usc.es/~vanraaij/ > > > > > > > > On 12 Mar 2009, at 21:57, Felix Frolow wrote: > >> Dear Mark >> Stay calm >> Buzz-words come and very frequently do not stay, they go away with >> the artifacts they advocate... >> >> Dr Felix Frolow >> Professor of Structural Biology and Biotechnology >> Department of Molecular Microbiology >> and Biotechnology >> Tel Aviv University 69978, Israel >> >> Acta Crystallographica D, co-editor >> >> e-mail: mbfro...@post.tau.ac.il >> Tel: ++972 3640 8723 >> Fax: ++972 3640 9407 >> Cellular: ++972 547 459 608 >> >> On Mar 12, 2009, at 6:14 PM, Mark J. van Raaij wrote: >> >>> Dear All, >>> >>> a News & Views article in Nature 458, pages 37-38 of 5 March 2009 >>> (link below) states: >>> "The development of structural biology WAS historically based on >>> the principle of divide and conquer individual proteins were >>> purified to homogeneity and their atomic structures were solved in >>> vitro by using either X-ray crystallography or nuclear magnetic >>> resonance (NMR) spectroscopy. This approach WAS tremendously >>> successful, and led to the creation of a protein-structure databank >>> that currently contains more than 50,000 structures." >>> >>> I find the past tense here too much... >>> >>> Greetings, >>> >>> Mark >>> >>> Mark J. van Raaij >>> Dpto de Bioquímica, Facultad de Farmacia >>> Universidad de Santiago >>> 15782 Santiago de Compostela >>> Spain >>> http://web.usc.es/~vanraaij/ >>> >>> http://www.nature.com/nature/journal/v458/n7234/full/458037a.html >>> Structural biology: Inside the living cell >> >
Re: [ccp4bb] fake images
Steganographic encoding of a PGP-encoded graphical segment should not be difficult to incorporate... The problem of course is that the delicate signal of such a 'hidden' message would be subject to deliberate erasure unless the encoding is actually done on the level of reflections themselves (i.e. something clever like arranging background pixels differing by 1 bit in a specific pattern around specific reflections). The alternative is to ask for ubiquitous incorporation of such watermarks into all detector-generated images. The absence of watermarks would indicate either 1) an older detector which cannot be software-updated to produce the codes or 2) fake image. Returning to real world - even if images are faked and fake structures are produced, and all sorts of N-S-C papers are written - experience shows that fraud does not survive for too long, especially when the subject matter is 'hot' and interesting, and doubly so if the results are unexpected or remarkable in some other way. I doubt that people would go to the trouble of faking routine structures, since those generally do not make their authors any money/fame/recognition. Artem > After consultation with Ton Spek, I should correct my last email. > It turns out that my 'watermark' was not clever enough, because > PLATON - his program used to make the picture that I had randomly > picked as an example - can emulate the XP watermark (the way of > shading the ellipsoids which I intended to be different from the > 'genuine ORTEP') rather well, and had indeed done so in > the picture in question. However it is technically an 'ORTEP > clone' (like XP), not an 'XP clone'. I am using 'clone' to mean > that one has intentionally created the 'same look and feel' as > an existing program, not to imply that the same code is used. > > This shows how difficult it will be for James to include an > unforgeable 'watermark' in his calculated frames, though I would > still encourage him to try. Being able to emulate the experiment > so well is wonderful for debugging the programs used to process > the frames, but it will surely make fraud more difficult to > detect! > > George > > Prof. George M. Sheldrick FRS > Dept. Structural Chemistry, > University of Goettingen, > Tammannstr. 4, > D37077 Goettingen, Germany > Tel. +49-551-39-3021 or -3068 > Fax. +49-551-39-22582 > > > On Fri, 20 Mar 2009, George M. Sheldrick wrote: > >> Dear James, >> >> About 30 years ago I wrote a clone of the program ORTEP; the clone >> was called XP, but unfortunately Microsoft later stole the name >> and brought it into disrepute. So that I would always be able to >> see at a glance which plots were 'genuine ORTEP' and which were >> my clone, I built in a sort of watermark (a slightly different way >> of shading the ellipsoids). I hope that you have done something >> similar in mlfsom! But please don't reveal what it is to ccp4bb, >> I suggest that you deposit the information in encripted form with >> the PDB. Otherwise you may suffer the same fate as I did: the >> 'ORTEP' plot on the cover of the February 2009 Acta C appears to >> have been made by a clone of my clone! >> >> The information that data produced by mlfsom give Rcryst ~ Rsym >> is very interesting and makes it even more likely that the >> typical R-values of 19.9% for proteins have something to do with >> the ca. 50% of the structure that is rather poorly modeled - the >> 'solvent' region. >> >> Best wishes, George >> >> Prof. George M. Sheldrick FRS >> Dept. Structural Chemistry, >> University of Goettingen, >> Tammannstr. 4, >> D37077 Goettingen, Germany >> Tel. +49-551-39-3021 or -3068 >> Fax. +49-551-39-22582 >> >> >> On Fri, 20 Mar 2009, James Holton wrote: >> >> > Yes, Harry, indeed there is a program for simulating diffraction >> patterns. >> > You can get a development snapshot of it here: >> > http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz >> > >> > MLFSOM (mosflm in reverse) is not the only program of its kind in >> existence >> > and I don't think it is a good idea to keep the fact that they exist a >> secret >> > in any way. In fact, MLFSOM has been extremely instructive in >> establishing >> > how important different sources of error are to the structure >> determination >> > process, and where the "threshold of solvability" is in real-world >> units. I >> > am writing this up now and I have given several talks about it >> recently but
Re: [ccp4bb] low insect cell expression
Hello Chen, You did not mention perhaps the most important element that can make or break the secretion process - namely the secretion signal. Which signal(s) have you already tried? Have you attempted to quantify the protein made in the total IC? In other terms, is the bottleneck in the protein synthesis/folding or in secretion itself. More details would be awesome! Artem > Hi, > > We are trying to express a protein in insect cells as secreted. However, > the yield is VERY low using either pFastBAC or pAcGP67A. We have tried > many different constructs and this is the only one that at least > expresses. I wonder whether anyone knows how to improve. Many thanks. > > Best, > Chen > > >
Re: [ccp4bb] low insect cell expression
Hi, Which secretion signal(s) did you use? The choice of secretion signal can make or break the entire process. Have you checked total yield of your protein in the cytoplasm and in the medium? Can you make assumptions regarding the bottleneck - is it protein synthesis/folding or is it the secretion process itself? More details would be great to have! Artem > Hi, > > We are trying to express a protein in insect cells as secreted. However, > the yield is VERY low using either pFastBAC or pAcGP67A. We have tried > many different constructs and this is the only one that at least > expresses. I wonder whether anyone knows how to improve. Many thanks. > > Best, > Chen > > >
Re: [ccp4bb] Reagent Grade from Hampton
Hi, Phosphate + calcium - not an easy combination :) Fluka brand PEGs used to be the way to go. I would carefully test the pH of the actual solution in the kit, and compare it with the pH of the solutions you're making yourself. There are several reasons as to why - but these values aren't always the same which (in my experience) is responsible for 40-50% of cases when screening conditions do not reproduce. Old PEG solutions can go very acidic, for instance - see previous CCP4bb exchanges on this subject. Good luck! Artem > Hello, > > I am trying to reproduce two hits obtained from the PEG/Ion screen of > Hampton which showed up with 20% PEG3350 and 0.2M K2HPO4/0.2M CaCl2. I > tried optimizations making up my own solutions with the two salts from > Sigma > (The PEG is from Hampton). To my surprise, the hits do not translate > during > optimizations. I am suspecting the salts. My question is, what grade of > Sigma > chmicals would be equivalent to the ones sold by Hampton? > > Any other suggestions would be more than welcome. > > Regards > Hari >
Re: [ccp4bb] Design Constructs
Hi, There are several semi-rational ways to design constructs on the basis of existing structures. It is important to remember that we only partially understand the protein expression and folding - which means that sometimes (way more often then I'd like) all the design considerations have to go out of the window and one has to painstakingly scan the termini in small increments. If you're interested in further discussion, we should probably take this out of the list. Feel free to drop me a note. Artem > Hello > > I am designing a protein construct for structural biology. It is a protein > kinase > which has not been crystallized earlier. I was comparing the kinase > domains of > other closely related family members characterized biochemically vs > crystallization constructs. For crystallography constructs, there are > different > stretches of amino acid residues particularly at the N-terminus (some > contain > extra 2-5 residues while others have 15-20 residues. > > My question: Is there a rational way of designing exact constructs one > would propose to make, eg., by a sequence alignment showing nearest > homology neighbors that guided construct design etc.. > > > Sincerely > Hari > > Haridasan V. M. Namboodiri, PhD > Scientist-Structural Biology > Locus Pharmaceuticals, Inc. > Four Valley Square > 512 Township Line Road > Blue Bell, PA 19422 > email: vnambood...@locuspharma.com > Ph: 215-358-2012 > Fax: 215-358-2020 >
Re: [ccp4bb] Halide soaking
I'd like to remind folks here that if one's crystals are sturdy enough to survive halide soaking, they're *probably* also sturdy enough to live through covalent iodination. Iodination is easy to set up and if it does work out - one gets awesome quality derivatives with multiple sites (as long as there are enough Tyrosines in the protein). The drawback is that iodination can and often does kill the crystal - which is the reason for my earlier comment regarding crystal machismo/resilience. If you want to try it, I would be happy to supply an easy protocol that worked for me several times :) Artem > You always get the entry of Bromide into crystal by quick soaking, > because it does not require the incorporation of Bromide into the > protein. But whether the signal is good enough for phasing is another > story. You have to collect the full data set to know the answer. > > Nian > > 2009/3/31 tat cheung cheng : >> Hi all >> >> I am now trying to do bromide soaking, but i am not really sure does the >> bromide atom enter my crystal. So is there any signs that indicate the >> entry of bromide atom? e.g. does the space group, cell dimension change? >> or just nothing change, and the bromide atom just get in? >> Thanks very much. >> >> T.C. Cheng >> >> >> Yahoo!»´ä´£¨Ñºô¤W¦w¥þ§ð²¤¡A±Ð§A¦p¦ó¨¾½d¶Â«È! ½Ð«e©¹ >> http://hk.promo.yahoo.com/security/ ¤F¸Ñ§ó¦h! >> >
Re: [ccp4bb] Halide soaking
The conditions in the drop play a huge role in the success of iodination. If you see iodinated histidines, this means that you had high pH - higher than 8 at least, as histidines are much harder to iodinate than tyrosines (which will work even at pH 5, and definitely at 6). Paul's classic experiment is very nice :) If you're into even more classic experiments, check out: INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN ROGER M. HERRIOTT, 1936 http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2141501&blobtype=pdf The heavily modified method that I will be posting shortly does not involve adding iodine directly to the crystals, which is its sole (but I think significant) benefit :) Artem > Hi everyone, > the in situ iodination reaction described in the following classic paper > by > the late Paul Sigler works quite well. > > Iodination of a single tyrosine in crystals of alpha-chymotrypsin. > Sigler PB. > Biochemistry. 1970 Sep 1;9(18):3609-17. > > The primary purpose of my experiment (which took place 11 years ago > according to my notebook) was indeed to iodinate tyrosines, but difference > fourier analysis using calculated phases from the final refined MIR > structure to reveal the complete iodination model (out of curiosity), > showed > that in addition to iodination of two tyrosines, two histidines had also > been iodinated! In retrospect, I had actually run across those peaks in my > cross-difference fourier maps but thought that they were too 'secondary' > to > be included in the heavy atom model. > > Best wishes > Savas > > > Savvas Savvides > L-ProBE, Unit for Structural Biology > Ghent University > K.L. Ledeganckstraat 35 > 9000 Ghent, BELGIUM > office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 > Email: savvas.savvi...@ugent.be > http://www.lprobe.ugent.be/xray.html > > > > -Original Message- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > Alessandro Vannini > Sent: Tuesday, March 31, 2009 9:11 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Halide soaking > > Hi, > it worked very nice for me in 1 out of 1 case where I tried it :-). > Very well diffracting crystals (1.8 Ang), rather small protein 20 > kDa, 50 %solvent content, 1 mol/ASU. 20-30 s soak in 0.5 M NaBr > resulted in 6 nice ordered sites. > It was crucial for us to collect a 3 wavelength MAD data set. A SAD > data set (using just the peak, even if with high redundancy ) was not > enough to obtain traceable electron density map, even-though > one could distinguish clearly protein boundaries and solvent channels. > > Good luck > > Ale > > On 31 Mar 2009, at 18:19, tat cheung cheng wrote: > >> Hi all >> >> I am now trying to do bromide soaking, but i am not really sure does >> the bromide atom enter my crystal. So is there any signs that >> indicate the entry of bromide atom? e.g. does the space group, cell >> dimension change? or just nothing change, and the bromide atom just >> get in? >> Thanks very much. >> >> T.C. Cheng >> >> >> Yahoo!Ïã¸ÛÌṩ¾WÉÏ°²È«¹¥ÂÔ£¬½ÌÄãÈçºÎ·À¹ ºÚ¿Í! ÕÇ°Íù http://hk.pro >> mo.yahoo.com/security/ Á˽â¸ü¶à! > > > > > > E-mail message checked by Spyware Doctor (6.0.0.386) > Database version: 5.12080 > http://www.pctools.com/en/spyware-doctor-antivirus/ >
Re: [ccp4bb] [ANNOUNCE] Journal of Failed Crystallization Experiments
Hey, This is *the* place to report my experiments with Uranium (IV) Astatide (UAt4) and Radon for phasing! Artem > Dear Crystallography Community: > ... good stuff ... > Sehl Oediter > Chief Guy in Charge > Journal of Failed Crystallization Trials > Sell Press > Boston, MA >
Re: [ccp4bb] New human genome policy - please read.
In a powerful counter-proposal I recommend that human genes be named after actual people. I think many of us have heard of the star catalogue - for fifty bucks you can have a star named after you (not sure if they still do this). So, the same deal applies - only a bit more expensive because there's considerably fewer human proteins than theer are stars. Imagine weary crystallographer coming home: Darling, we finally solved the structure of Edgar Allan Poe! What, full-length? No, unfortunately we had to chop the head off - it was wobbling all over the place. Artem > H... just saw this. !!!?!??! > > > > The International Human Genome Organization (HUGO) has announced its > intention to commercialize the naming of human genes. They put it out for > public consultation, and are asking for responses on their web site ( > http://www.internationalhugo.org/). If you have strong opinions on this, > please do respond. > >
Re: [ccp4bb] [ANNOUNCE] Journal of Failed Crystallization Experiments
The impact factor of UAt4 has recently been upgraded from 'huge' to 'all over the place' after the unfortunate accident at the Miscatonic university labs which left half the building as glowing multi-colored glass puddle. Artem > What is the impact factor for that? 100 or 1000? > > leo > > ar...@xtals.org wrote: >> Hey, >> >> This is *the* place to report my experiments with Uranium (IV) Astatide >> (UAt4) and Radon for phasing! >> >> Artem >> >> >>> Dear Crystallography Community: >>> >>> >> ... good stuff ... >> >>> Sehl Oediter >>> Chief Guy in Charge >>> Journal of Failed Crystallization Trials >>> Sell Press >>> Boston, MA >>> >>> >> >> >
Re: [ccp4bb] Link two proteins into one polypeptide
We often have good luck using protease cleavage sites as linkers. They're evolved to have certain flexibility to them and they usually have a healthy mixture of hydrophobic and hydrophilic amino acids. As a delightful bonus they also offer the option to separate the two partners at will. Artem > I recently had some success in this area. My approach was to get lucky > when I guessed the linker. In this case, my linker became ordered > which seemed to help my fusion construct settle down enough to exhibit > density. My linker sequence came was (approximately) alternating > hydrophilic and hydrophobic residues. It ordered as part of a sheet > that mediated inter-protein contacts. I purposely did not use a low- > complexity motif for reasons I'll explain below. > > While staring at this structure and allowing my imagination to wander, > I came up with the idea of an "anything goes" linker motif. Find one > that can form an alpha helix or an extended conformation with equal > (in a subjective sense) regularity. The idea is that you want a > segment that can "adapt" to arbitrary crystal packing environments. I > think many natural segments that display multiple conformations have > been observed. Check out 1owr for a natural motif that can be alpha > helical or extended/disordered. (1owr is not the chimeric construct I > refer to above which is unpublished.) I think the natural linker in > 1owr would make an ideal linker in a chimeric construct. > > Although I base this advice on nothing but a hunch, stay away from > poly-gly or other absurdly simple motifs. The idea is purely > mathematical: think of choosing a linker as a guessing game, and you > want to guess the right linker. Let's pretend you want a 7 aa linker. > What are the chances that the optimal linker is going to be any random > sequence of residues? 20**7. So poly-gly, from a purely combinatorial > perspective, has a 1 in 20**7 chance to be the optimal sequence. But > the chances are probably lower than 1/20**7, because we know that poly- > gly is going to be disordered and the optimal sequence will probably > be ordered. In other words, common protein structure sense and > mathematics suggest that you will be shooting yourself in the foot if > you choose a poly-gly motif as a linker. > > An exception to the low complexity "rule" is the poly-A motif used in > fusion constructs with MBP. The poly-A is usually ordered and alpha > helical in these constructs. > > James > > On Apr 2, 2009, at 1:11 PM, Raji Edayathumangalam wrote: > >> Hi People, >> >> Could anyone point me to successful examples for two unrelated >> proteins that have been stitched together into one single >> polypeptide chain with flexible amino acids to create a functional >> chimera that was subsequently crystallized. I've looked up a few. >> >> I am particularly interested in understanding all the important >> considerations while designing a flexible linker even though many of >> these factors might be case dependent might be variable, obvious and >> commonsensical. Either way, I'd like to hear what folks have to say. >> >> Thanks very much. >> Raji >
Re: [ccp4bb] unknown density
Theoretically, citric acid can decompose into beta-ketoglutaric acid, and the latter may decarboxylate into malonic acid... Not sure if this happens in aqueous solutions, but if your citrate had malonate contamination and your enzyme prefers malonate to anything else - that's where it may have come from. Pure speculation, of course! Artem > Dear all, > i am working on a structure of M.tb, solved at 2.18 A. In my structure > there > is a density near to the active site and we modelled it as a malonate (C3 > O4 > H2) molecule acorrding to the density, which was not used in any of our > buffer except we used 10 mM DTT, 1.6 M Ammonium sulphate, 100 mM citric > acid. > > Is their any possible way to check for unknown densities or can any one > suggest me for any possible compound which can be similar to the malonate > or > can be formed from above mentioned solutions (byproduct). > > Thanks > Rajan > > -- > > Current Address: > > Rajan Vyas > Research Scholar > Deptt. of Biotechnology > Panjab University Chandigarh, India > 160014Mob. +919417374197 > Fax: +91-172-2625254 > >
Re: [ccp4bb] how to purify protein in its native form
Hello Jerry, I would be glad to provide protocol suggestions, but it'd help to know a bit more regarding what you are working on. If you're planning to purify material from its native source (i.e. someone's chopped up sittin' muscle?) - this would be tremendously different from purifying protein from transfected CHO, HEK, etc. cells where you can expect reasonable abundance. Details are very important :) Artem > > Hi, All: > >Although it is off-topic, definitely I think I can get some help > here because we crystallographers are dealing with protein > purification almost every day. > > My protein was expressed in E.coli as a soluble octamer with or > without his-tag revealed by gel-filtration. For the his-tagged > protein, the final product is an octamer after Ni-column and > gel-fil. However, purification of the non-tagged protein results in > a tetramer because of a partially denatured condition. > > When I tested the enzymatic activity , it turns out the tetramer was > active although both of them can be crystallized. Now I am > wonderring whether its native form in human is an octamer or > tetramer. > > I am planning to purify a little protein from human cells to verify > its native form. > > Can anybody recommend some protocols? > > Thanks a million, > > Jerry McCully > > > _ > Rediscover Hotmail®: Now available on your iPhone or BlackBerry > http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Mobile1_042009
Re: [ccp4bb] how to purify protein in its native form
Yes,
However the even holier single method suitable for purification of
proteins from native sources is immuno-capture. If you have a good
antibody (i.e. an antibody that recognizes a native epitope) you can get
very high purity very quickly. If you don't have an antibody - you can get
polyclonals made very rapidly. Polyclonals are potentially even better for
immunocapture as some of them will recognize natively folded epitopes,
unlike monoclonals that may be raised against a peptide from deep inside
the protein.
Again, more details are needed :)
Artem
> The holy trinity of protein chromatography is ion exchange (IEX),
> hydrophobic interaction chromatography (HIC), and gel exclusion
> (GEC), assuming no affinity purification is possible. Depending on
> the abundance of protein, these three steps, perhaps in concert
> with additional chemical steps, including but not limited to salt
> precipitation and/or thermal denaturation, are often sufficient to
> purify proteins to homogeneity from natural sources. For
> minisculely abundant proteins, purification can be nightmarish.
> (Been there done that, got the dirty T-shirt.)
>
> The basis for any protein purification is an efficient assay for
> determining the presence of the protein in a mixture, even at low
> concentration. If you don't have a good assay (activity, antibody
> binding, spectroscopic ligand, etc., you are in dire straits indeed. But
> if you have an assay, you may not need to purify your protein at all to
> determine its oligomerization state in vivo. You may only need to pass a
> suitably concentrated crude extract containing your protein over a
> calibrated gel exclusion column. The emergence of the active fractions
> will provide an elution volume that will point you to the native MW, +/-
> 20% or so. The difference between a tetramer and an octamer (2X) should
> be cake using GEC. (Caveats apply: some proteins like to stick to GEC
> columns, but this can be suppressed with 100 mM NaCl, normally, but
> YMMV!)
>
> Cheers,
>
> --
> Roger S. Rowlett
> Professor
> Colgate University Presidential Scholar
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@mail.colgate.edu
>
> Jerry McCully wrote: .hmmessage P { margin:0px; padding:0px }
> body.hmmessage { font-size: 10pt; font-family:Verdana } Hi, All:
>
> Although it is off-topic, definitely I think I can get some help
> here because we crystallographers are dealing with protein
> purification almost every day.
>
>My protein was expressed in E.coli as a soluble octamer with or
> without his-tag revealed by gel-filtration. For the his-tagged
> protein, the final product is an octamer after Ni-column and
> gel-fil. However, purification of the non-tagged protein results in
> a tetramer because of a partially denatured condition.
>
>When I tested the enzymatic activity , it turns out the tetramer
> was active although both of them can be crystallized. Now I am
> wonderring whether its native form in human is an octamer or
> tetramer.
>
> I am planning to purify a little protein from human cells to verify
> its native form.
>
> Can anybody recommend some protocols?
>
> Thanks a million,
>
> Jerry McCully
>
>
>Rediscover Hotmail®: Now available on your iPhone or BlackBerry
> Check it out.
>
Re: [ccp4bb] problem in transformation of pqe 30 clone
Hi, You are using a pQE vector which has a T5 promoter. T5 is a native-like promoter, recognized by the E. coli RNA polymerase - and this means that even with lac operatorsupstream there is a huge amount of leakage in this system. If your protein is even moderately toxic then you have issues. Your solutions are 1) to use cells with higher levels of lac repressor (XL1-blue for example) 2) to re-clone this ORF under some tightly controlled promoter Artem > > i have cloned my gene successfully into qiagen vector into pqe30 but i do > transformation of this into BL21,pLys,Rosseta,C41, i dont get any > colonies,comp cells are good other clones give good no of colonies upon > transformation. > can someone help??? > thanks > atul > > -Original Message- > From: CCP4 bulletin board on behalf of herman.schreu...@sanofi-aventis.com > Sent: Mon 5/4/2009 3:49 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG molecule > crossing a two-fold crystallographic symmetry axis > > Dear Lidefeng, > > One "Tassos" afterthought; you are sure you traced the chain correctly? It > is not that both change make a crystal contact and then each go their own > (disordered) way? > > Best regards, > Herman > > -Original Message- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > lidefeng > Sent: Friday, May 01, 2009 2:48 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG molecule > crossing a two-fold crystallographic symmetry axis > > Dear Herman.Schreuder, > > Perhaps there is some misunderstanding about my question. There is > one molecule in the asymmetric unit (showed as symbos A). After the > 2nd crystallographic symmetric operation, another molecule appears > (symbol B). However, the density show that there is one peptide chain > cross the 2nd axis (symbol C). If we give chain C occupancy of 0.5, > only 50% of chain C belongs to molecules A and B, respectively. In > another word, , it looks like one half of chain C belongs to molecule > A and the other belongs to B. It means that one half of chain C > belongs to molecule A disappear, so do that belongs to molecule B. > > 2nd axis > | > AA | BB > AA | BB > AA | BB > \ | / > CCC|CCC | > | > | >Your sincerely > De-Feng Li > lidef...@moon.ibp.ac.cn > ??2009-05-01 > > Defeng Li, Dr., > Email: lidef...@moon.ibp.ac.cn > National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese > Academy of Sciences, > 15 Datun Road, Chaoyang District, > Beijing 100101, China > > > === 2009-04-30 14:52:00 You writed in your letter:=== > >>Dear Feng-Li, >> >>The other half occupancy peptide is generated by crystallographic >> symmetry (the twofold), you need only to build one. To check that >> everything fits properly, you should switch on the crystallographic >> symmetry in coot. (Draw -> Cell & Symmetry). >> >>Good luck! >>Herman >> >> >>-Original Message- >>From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of >>lidefeng >>Sent: Thursday, April 30, 2009 3:44 AM >>To: CCP4BB@JISCMAIL.AC.UK >>Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG >>molecule crossing a two-fold crystallographic symmetry axis >> >>Dear Tim Gruene, >> >>But how to illustrate the other one half occupancy of peptide? >> Disorder ? >> >> >> Your sincerely >>De-Feng Li >>lidef...@moon.ibp.ac.cn >>??2009-04-30 >> >>Defeng Li, Dr., >>Email: lidef...@moon.ibp.ac.cn >>National Laboratory of Biomacromolecules, Institute of Biophysics, >>Chinese Academy of Sciences, >>15 Datun Road, Chaoyang District, >>Beijing 100101, China >> >> >>=== 2009-04-29 10:55:00 You writed in your letter:=== >> >>>Hello De-Feng Li, >>> >>>first of all sorry for changing the subject: I think starting a new >>>thread from an existing one may hamper people who are going to search >>>the archives in the future, therefore it is good practice to give it >>>its separate subject line, even though it certainly is be very closely >>>related. >>> >>>In your case you can refine two peptides each with an occupancy of >>>0.5, one for
Re: [ccp4bb] problem in transformation of pqe 30 clone
XL1-Blue is a strain of E. coli. Whether it is or isn't an expression host depends on the definition, and I am not going to argue semantics. The T5 promoter works with regular garden variety RNApol of E. coli. Therefore ANY E. coli strain is an 'expression host' for vectors that contain this promoter. I've expressed many proteins in XL1-Blue and I see no reason why you can't express yours, either. Artem > xl1-blue is not an expression host,since i have cloned it successfully,i > need to transform into expression host, i am able to transform it into dh5 > alfa,but not in any of expression host > > > > From: ar...@xtals.org [mailto:ar...@xtals.org] > Sent: Mon 5/4/2009 6:32 PM > To: atul kumar > Cc: ccp4bb@jiscmail.ac.uk > Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone > > > > Hi, > > You are using a pQE vector which has a T5 promoter. T5 is a native-like > promoter, recognized by the E. coli RNA polymerase - and this means that > even with lac operatorsupstream there is a huge amount of leakage in this > system. If your protein is even moderately toxic then you have issues. > > Your solutions are > 1) to use cells with higher levels of lac repressor (XL1-blue for example) > 2) to re-clone this ORF under some tightly controlled promoter > > Artem > >> >> i have cloned my gene successfully into qiagen vector into pqe30 but i >> do >> transformation of this into BL21,pLys,Rosseta,C41, i dont get any >> colonies,comp cells are good other clones give good no of colonies upon >> transformation. >> can someone help??? >> thanks >> atul >> >> -Original Message- >> From: CCP4 bulletin board on behalf of >> herman.schreu...@sanofi-aventis.com >> Sent: Mon 5/4/2009 3:49 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG >> molecule >> crossing a two-fold crystallographic symmetry axis >> >> Dear Lidefeng, >> >> One "Tassos" afterthought; you are sure you traced the chain correctly? >> It >> is not that both change make a crystal contact and then each go their >> own >> (disordered) way? >> >> Best regards, >> Herman >> >> -Original Message- >> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of >> lidefeng >> Sent: Friday, May 01, 2009 2:48 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG >> molecule >> crossing a two-fold crystallographic symmetry axis >> >> Dear Herman.Schreuder, >> >> Perhaps there is some misunderstanding about my question. >> There is >> one molecule in the asymmetric unit (showed as symbos A). After the >> 2nd crystallographic symmetric operation, another molecule appears >> (symbol B). However, the density show that there is one peptide chain >> cross the 2nd axis (symbol C). If we give chain C occupancy of 0.5, >> only 50% of chain C belongs to molecules A and B, respectively. In >> another word, , it looks like one half of chain C belongs to molecule >> A and the other belongs to B. It means that one half of chain C >> belongs to molecule A disappear, so do that belongs to molecule B. >> >> 2nd axis >> | >> AA | BB >> AA | BB >> AA | BB >> \ | / >> CCC|CCC | >> | >> | >>Your sincerely >> De-Feng Li >> lidef...@moon.ibp.ac.cn >> ??2009-05-01 >> >> Defeng Li, Dr., >> Email: lidef...@moon.ibp.ac.cn >> National Laboratory of Biomacromolecules, Institute of Biophysics, >> Chinese >> Academy of Sciences, >> 15 Datun Road, Chaoyang District, >> Beijing 100101, China >> >> >> === 2009-04-30 14:52:00 You writed in your letter:=== >> >>>Dear Feng-Li, >>> >>>The other half occupancy peptide is generated by crystallographic >>> symmetry (the twofold), you need only to build one. To check that >>> everything fits properly, you should switch on the crystallographic >>> symmetry in coot. (Draw -> Cell & Symmetry). >>> >>>Good luck! >>>Herman >>> >>> >>>-Original Message- >>>From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of >>>lidefeng >>>
Re: [ccp4bb] problem in transformation of pqe 30 clone
To clarify: I am not implying that I've worked with many proteins that express better in XL1-blue cells than they would express in BL21(DE3) or such if cloned into a pET vector or similar. In fact I can probably recall only one or two that expressed *better* in XL1 cells. However in the good old days pQE series of vectors was quite commonly used and I had things in that were inherited from others - these 'things' were fairly simple to express in XL1-blue whereas they gave me loads of trouble in other strains and I was too busy/lazy to re-clone them. Artem > Hello Artem, > > We express almost all our proteins in BL21 derivatives. It sounds > like you've worked with many proteins that express/behave better in > XL1-Blue? > > > ho > UC Berkeley > > - > XL1-Blue is a strain of E. coli. Whether it is or isn't an expression host > depends on the definition, and I am not going to argue semantics. > > The T5 promoter works with regular garden variety RNApol of E. coli. > Therefore ANY E. coli strain is an 'expression host' for vectors that > contain this promoter. > > I've expressed many proteins in XL1-Blue and I see no reason why you can't > express yours, either. > > Artem >
Re: [ccp4bb] Refolding of Denatured Protein
I hate to be the Negative Nancy here - but removal of detergent can be a significantly harder task than removal of urea. Here's why: Urea is a small molecule which diffuses freely and does not normally stick to proteins - so removal of urea is no harder than removal of salt (we ignore the considerations of protein stability here). On the other hand, detergents can be easy or very hard to get rid of, depending on which particular detergent and what particular protein are brought together. Some of the nastier polymeric detergents are quite different to remove 'completely' because they stick to proteins, stick to plastic and/or resin beads, form micelles (thus refusing to dialyze) and so on and so forth. There are numerous acceptable ways to remove sticky detergents, and some of them include treatment with hydrophobic matrices that 'suck up' detergent molecules, exchange with a 'milder' detergent, and so forth. Good luck! Artem > > dear sanjiv > i dont think that the removal of detergent is more difficult than > urea,since u are purifying ur protein over Ni-NTA column,so after a few > wash over there u can remove the detergent completely. > > best > atul kumar > > -Original Message- > From: CCP4 bulletin board on behalf of David Cobessi > Sent: Tue 5/5/2009 6:18 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Refolding of Denatured Protein > > Sanjiv Kumar wrote: >> I have not tried purifying this protein either with detergent or with >> /Guanidine Hydrochloride. I can try with both /Guanidine HCl and >> detergent. Since I got good purification with the 8M urea, I was >> thinking if this protein could be refolded and used further. Isn't >> removal of detergent more difficult then Urea? >> Sanjiv Kumar >> Lab. No. 411, >> Functional Genomics Unit, >> Institute of Genomics and Integrative Biology, >> New Delhi-110007 >> India > Is it a transmembrane protein or not? or just a protein associated to > the membrane (with a GPI anchor for example)? > If it is a transmembrane protein, you should try to use detergents for > extraction and during the purification. > David > > -- > David Cobessi > Institut de Biologie Structurale > 41, Rue Jules Horowitz > 38027 Grenoble Cedex-1, France > Tel:33(0)438789613 > 33(0)608164340 > Fax:33(0)438785122 > >
Re: [ccp4bb] problem in transformation of pqe 30 clone
Hello In order to know *exactly* what the reason for poor transformation outcome was one has to do all sorts of experiments. This is not likely to be your goal, right? Leaky expression, overload of DNA, somehow compromised cells, or even plain old user error - and numerous other reasons can be proposed (including some esoteric ones like incompatibility of your gene product with a specific strain, for reasons going beyond protocol errors and simple considerations). This can take a couple of years to sort out. If you're talking about using M15[pREP4] cells then the reason why you failed earlier is likely to be toxicity - pREP4 plasmid supplies lacI in-trans, which represses 'stray' transcription reasonably well. The cells that you used before and did not succeed with must have had low intrinsic levels of lacI and thus permitted leakage. To test this theory you could streak a couple of colonies of the M15[pREP4] cells on a plate that has a small amount of IPTG in the agar (say 0.1 mM) - if the colonies don't grow at all, or appear to be very tiny then you know it's your protein toxicity that's the issue here. Keep in mind that bacteria are extremely survival-oriented and therefore you will eventually generate 'safe' mutants and recombinants that will be able to grow even on IPTG. Likely those mutants will be useless to you from practical standpoint. Once you induce the M15[pREP4] cells - the toxicity will manifest itself again. You will likely observe total cessation of growth (for a long period of time) and possibly even lysis of the culture. Therefore you may have to adjust your fermentation protocol to take this into account. One of the common adjustments is deliberate induction at higher OD, another is to include 0.8% glucose in the growth medium; there are other options available to you. Hopefully once you induce transcription, the gene products do not shut down protein synthesis - which would be a disaster since it would likely shut down its own synthesis as well. Since I don't know what you're growing, this is just one of the possibilities :) Artem > > now i have transformed pqe 30 clone into the m15 host successfully, can > someone let me know exactly what was reason behind the problem into > transformation??is it leaky expression into the other host that is toxic > for the cells,if it so then will i would be able to get good expression > into this host??? > > atul > -Original Message- > From: CCP4 bulletin board on behalf of ar...@xtals.org > Sent: Tue 5/5/2009 6:24 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone > > To clarify: I am not implying that I've worked with many proteins that > express better in XL1-blue cells than they would express in BL21(DE3) or > such if cloned into a pET vector or similar. In fact I can probably recall > only one or two that expressed *better* in XL1 cells. However in the good > old days pQE series of vectors was quite commonly used and I had things in > that were inherited from others - these 'things' were fairly simple to > express in XL1-blue whereas they gave me loads of trouble in other strains > and I was too busy/lazy to re-clone them. > > Artem > >> Hello Artem, >> >> We express almost all our proteins in BL21 derivatives. It sounds >> like you've worked with many proteins that express/behave better in >> XL1-Blue? >> >> >> ho >> UC Berkeley >> >> - >> XL1-Blue is a strain of E. coli. Whether it is or isn't an expression >> host >> depends on the definition, and I am not going to argue semantics. >> >> The T5 promoter works with regular garden variety RNApol of E. coli. >> Therefore ANY E. coli strain is an 'expression host' for vectors that >> contain this promoter. >> >> I've expressed many proteins in XL1-Blue and I see no reason why you >> can't >> express yours, either. >> >> Artem >> > >
Re: [ccp4bb] Na/K Phosphate
The reason for the odd K/Na combination is solubility - mixed phosphate is more soluble than either of the individual ones. I try to avoid high phosphate conditions as the Plague. It's great for molecular biology but horrible for crystallization because phosphate + a variety of other ions = lovely salt crystals. It all usually ends up in tears. If you absolutely must make phosphate buffers - just open up any basic practical biochemistry book, there are standard ratios (both by weight and by volumes of molar solutions) that produce specified pH in a wide range. Artem > Dear all, > > Quite a few crystallisation conditions in the screens feature > 'sodium/potassium phosphate'. I'm curious to know why such a Na/K mix > is there. As the pH is mostly determined by the (H2PO4)- to (HPO4)2- > ratio, is there such a need to have both cations? If so, is the Na to > K ratio important? > > On the more practical side of things, if you want to explore an > initial hit based on sodium/potassium phosphate, how would you go > about it - e.g. what phosphate buffers would you make and at what > ratio would you mix them? > > Thanks in advance, > Geoffrey Kong >
Re: [ccp4bb] BioRad CFX96 for thermofluor/DSF?
Use the FRET channel - it's ideal for SYPRO orange. Artem > Sorry Charlie for the late reply. > I'm using the CFX96 5 channel with Sypro and other dyes. I tested the > machine against the Eppendorf product nd decided to go for the Biorad > version as running identical samples gave better results on the > Biorad. This could be a machine specific problem but on a 96 well > plate the detection level was ~1.5x better, you can use the HEX > channel for detection. > > Jürgen > > On 29 Apr 2009, at 03:06, Charlie Bond wrote: > >> Hi Folks, >> >> Has anyone here had any success running a thermofluor (differential >> scanning fluorimetry) experiment on a BioRad CFX96 with SYPRO orange >> as >> the fluorophore? >> >> Our local 'technical expert' tells us it should work and gave us some >> settings to test, but was very sketchy about any detail, and in a test >> we ran we got some strange results (every well that contains SYPRO >> orange - even those with no protein - have a peak in fluorescence at >> 58oC). >> >> Before we tinker, or go and persuade a friendly molbiol lab to let us >> play with the filters in their iq5, I thought I'd see if anyone here >> had >> any advice. >> >> I'll summarise any responses I get. >> >> Cheers, >> Charlie >> >> -- >> Charlie Bond >> Professorial Fellow >> University of Western Australia >> School of Biomedical, Biomolecular and Chemical Sciences >> M310 >> 35 Stirling Highway >> Crawley WA 6009 >> Australia >> charles.b...@uwa.edu.au >> +61 8 6488 4406 > > - > Jürgen Bosch > Johns Hopkins Bloomberg School of Public Health > Biochemistry and Molecular Biology, W8708 > 615 North Wolfe Street > Baltimore, MD 21205 > Phone: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-3655 > http://web.me.com/bosch_lab/ >
Re: [ccp4bb] Bubbles on crystals
Hi, If you're worried - instead of transfering the crystal, just flood the entire drop with excess of your cryo buffer. No exposure to air this way. I bet that the bubbles won't go away though :) Artem > Dear members, > I have protein crystals grown in a condition with Isopropanol (13%) and > citric acid (0.2 M). When I transfer the crystals to cryo-buffer (30% > glycerol or 25% ethyleneglycol with 0.25 M citric acid and 13%-15% > Isopropanol), small bubbles appear on the surface of the crystals. These > tiny bubbles sometimes disappear in few seconds and sometimes not. > Crystals > are exposed to air only for 2-3 seconds in the process of transfer. I am > worried if the formation of these bubbles is due to evaporation of > isopropanol form the crystal and if it affects the crystal lattice. These > crystals diffract X-rays upto 4 angstroms. Any ideas how to avoid this > problem? > Thank you > Kumar >
Re: [ccp4bb] Van der Waals contacts
Hello, Van der Waals Forces: A Handbook for Biologists, Chemists, Engineers, and Physicists by V. Adrian Parsegian Give it a try! Artem > Hi CCP4ers > > Perhaps I am hashing over old news.but > > We are having a discussion about Van Der Waals contacts and effective > contacts i.e. the "real distance" of a VDW bump between say a CH and a > CH group which sometimes is described as between a C and a C as i.e. 2x > 1.6A and ending about 4A but not including hydrogen. > > Some programs list contacts, to say a ligand, as far as 6A apart and > some of the simulation programs use that distance too for contacts for > protein protein interactions. > > Does anyone know of a good paper that discusses the effective distance > or has a comment on where a VDW force may begin and end or it's > effective distance - though some say it never truly ends just > approaches zero... > > > G > > > > Notice: This e-mail message, together with any attachments, contains > information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, > New Jersey, USA 08889), and/or its affiliates (which may be known > outside the United States as Merck Frosst, Merck Sharp & Dohme or > MSD and in Japan, as Banyu - direct contact information for affiliates is > available at http://www.merck.com/contact/contacts.html) that may be > confidential, proprietary copyrighted and/or legally privileged. It is > intended solely for the use of the individual or entity named on this > message. If you are not the intended recipient, and have received this > message in error, please notify us immediately by reply e-mail and > then delete it from your system. >
Re: [ccp4bb] heavy atom derivative choice
Hi, 1. KSCN will suck up a lot of the Hg and other metals -- can you switch to something else? 2. Hg typically does not enter the protein core. 3. It's generally not necessary to use a special tank, as long as you treat the resulting solutions as heavy-atom waste; but of course each lab's safety precautions are different. Artem > > Does mercury tends to get into the protein core to denature protein or > not? > > For gel-shift assay, do people normally use a special gel tank for heavy > metal work? > > thanks in advance > > Xiaoli Xiong (Alex) > > > --On 15 July 2009 14:33 +0200 Sebastiano Pasqualato > wrote: > >> Hi all, >> I've got crystals of a protein of ca 200 residues, with 2 free >> cysteines, 5 histidines, 2 methionines. >> We have nice diffraction for the native crystals, that grow in 150 mM >> KSCN, 17% PEG 3350, bis tris propane pH 8.8. >> We are crystallising the SeMet derivative, but I'm not completely sure >> I >> will be able to have nice crystals by Saturday, when we have tunable >> time at the ESRF. >> I was thinking of trying with some heavy atom soaks, but only have like >> 30 crystals, so limited trials allowed! >> Which compound would you advice as more likely to work, and thus worth >> testing? >> Thanks in advance for the suggestions, >> ciao >> s >> >> >> >> -- >> Sebastiano Pasqualato, PhD >> IFOM-IEO Campus >> Dipartimento di Oncologia Sperimentale >> Istituto Europeo di Oncologia >> via Adamello, 16 >> 20139 - Milano >> Italy >> >> tel +39 02 9437 5094 > > > > -- > Xiaoli Xiong > PhD Candidate > Department of Cellular and Molecular Medicine > School of Medical Sciences > University of Bristol > University Walk > Bristol BS8 1TD, UK > x.l.xi...@bristol.ac.uk >
[ccp4bb] centrifuge bottle/rotor liners [unrelated but fun topic]
Hi folks, Pfizer protein folks taught me a useful trick: the 1L centrifuge rotor liners from Beckman-Coutler (catalog number 369256 if you're curious). They are quite awesome in the sense that instead of scraping biomass out of the centrifuge tube one can instead heat-seal and freeze the lower portion of the liner directly. Saves a lot of space in storage, too - and the resulting flat pellet can be thawed really quickly. They can be used in the rotor directly, or placed inside a wide-mouth 1L centrifuge tube. Unfortunately, these liners aren't cheap ($900+ per 120 in the US, that's kind of high for disposables) and also they only come in 1000-ml capacity. I wonder if anyone on this list have seen/used something like that but a) cheaper and b) in a wider variety of volumes. Thank you, Artem
Re: [ccp4bb] MAD wavelength
I second Phil's opinion - it is better to scan and be sure - as long as the scan results are not hideously abnormal. If you cannot scan for whatever reason but are sure that the X-ray optical system is properly calibrated - then use Phil's numbers below :) Artem > Always take the scan results ahead of the typical values unless they are > obviously wrong. Only use the "predicted" values if the scan is broken > or too weak (e.g. very small crystals) and in that case I'd be tempted > to add 10-20 eV to the "typical" peak wavelength to make sure you > weren't actually collecting the inflection point since they are > typically very close in SeMet. > > In my NSLS X29-dominated data collections, I find I end up using > something like this for non-oxidized SeMet: > > Peak: 12664 eV, 0.9790 Angstrom (usually in range 12662-12664) > Infl: 12662 eV, 0.9792 Angstrom (usually in range 12660-12662) > I also typically use high energy remote: 12860 eV, 0.964 Angstrom > > give or take a few eV. This tends to translate well between the > relatively small number of beamlines that I personally end up using. > But I always prefer to take the results from the Chooch analysis of the > scan from the actual crystal. > > Cheers (and good luck) > Phil Jeffrey > Princeton > > > Jerry McCully wrote: >> Dear All: >> >> Next week we are going to try some seleno-Met labeled crystals. >> >> We checked the literature to try to find out the peak wavelength >> that has been used for SAD or MAD data collection. But they are slightly >> different ( may be 50 ev) in different papers. >> >> I guess this is due to the discrepancy between the fluorescence >> scanning and the theoretical vaules of f' and f''. >> >> When we collect the data, which wavelength should we use? Should >> we trust the scanning results? >
Re: [ccp4bb] Problem with different map when ligand refinement
Hi, Barring orientation mishaps - it almost sounds like you have an ethyl rather than a methyl ester... Could it be that your compound is an ester of ethanol rather than methanol? Artem > Dear ccp4 experts, > I am trying to solve the structure of one esterase with its substrate. The > structure was solved by MR to the resolution 2.2 A. In the active site, I > can see clearly the different density map of the substrate (S- methyl > hydroxy isobutyrate). After puting the substrate into that density map, I > use Refmac to run restrain refinement at 0.1 weighting term. > Even though the substrate fits to the different map very well, a big > different map appears at one end of the substrate after Refmac refinement. > I > attach the image so that it's easy to see the problem. > I tried to put water or Na (because the crystallization contains: 1M > sodium > citrate, 100mM Na Cacodylate, 20% glycerol) but it didn't work. > I tried to use CNS instead of Refmac, however the result is worse since > the > R-free is increased and the different map is still there. Because I am new > with CNS so I don't know which parameters I should adjust. > Do you have any suggestion to solve this problem? Thank you very much for > all of your helpful advice! > > My best regards, > TriNgo, > PhD student, > Sungkyunkwan University, Korea >
Re: [ccp4bb] Linux flavour and hard disks
I've just set up two home computers for crystallographic work. I decided to play around, and set one up with Ubuntu and the other one with Fedora core 11. Both did OK but I'd say that I had to perform marginally less tweaking with Fedora than with Ubuntu. As far as disk space - you can cram together almost any number of hard drives using Logical Volume Management. And then there are numerous RAID versions - both hardware and software (my two cents: don't get involved in nerdy discussions of which one is 'better' - just get the one that is easier for you). As long as you're backed up it really does not matter which hard drive configuration you choose. I recommend running updates with whatever you like for software - and also cloning your hard drives using CloneZilla or any other tool so that in the event of system drive failure you can just literally swap a cloned drive in and your computer won't know the difference! Artem > > > > Dear List, > > > > I'm planning to migrate soon from Red Hat Linux 7.0 on an HP xw6000 > workstation with dual Xeon processor. > > > > Please, any suggestion for the best Linux flavour to get the most out of > today's crystallographic software? I've seen that both Ubuntu and Fedora > are quite common. > > > > Also I'm in doubt about the following: will it be safer to use two mirror > hard disks (as I'm doing now) or to use one HD for the software and one > for the data? > > And, finally, please, what HD size is today most reasonable (big, but > still fast enough)? > > > > Thanks a lot, > > > > Claudia > > > > > > > > > > Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia > Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. > 0039 0382 986335/8/1 Facs 0039 0382 303673 > > > > _ > More than messagescheck out the rest of the Windows Live. > http://www.microsoft.com/windows/windowslive/
Re: [ccp4bb] How to improve the Se-Met crystal diffracted to 5.6A
My suggestion would be to attempt finding Se atoms in the MAD dataset and then use these coordinates to do sulphur phasing - assuming of course that you can get the native crystals to diffract at an appropriately low-energy X-ray source. Artem > > Dear all, > > > > I have a Se-Met crystal diffracted to 5.6A and a full data set of the same > native crystal diffracted to 2.8A. Since I can hardly improve the quality > of the Se-Met crystal, may I ask some suggestion from the community about > am I able to solve the structure based on the above resolution, and how? > > > > Zhen > > _ > Show them the way! Add maps and directions to your party invites. > http://www.microsoft.com/windows/windowslive/products/events.aspx
Re: [ccp4bb] Help with improving these crystals
Beyond improvement via 'chemical' means, I would heartily recommend mutagenesis for crystal packing improvement. I would be glad to help you with the latter if you're interested. Artem > Dear All: > We have been struggling to improve the crystals shown in the attachment. > These crystals form from a protein solution 8 mg/mL (before drop), drop > sizes are 2 microL of protein + 2 microL of well solution (0.1 MES > buffer, pH 6.5, 1.8 M Ammonium sulfate with 12 mM CoCl2). > > The protein was dissolved initially in 20 mM Tris.HCl, pH 8.0 containing > 1 mM EDTA. > > Low cobalt also gives smaller crystals. These diffract up to 8 A. > > Any suggestions to improve these crystals would be most welcome. > > Subbu > > PS: We have used many additives (Hampton Research - including detergent > additives) to alter the morphology but with little success. >
Re: [ccp4bb] lysozyme
pLysS contains a phage lysozyme. You can get it from any pLysS cells. Ditto pLysE Artem > Dear crystallographers, > > does anyone happen to have a plasmid containing a lysozyme gene (any > naturally occurring sequence) that would be suitable for use as a PCR > template? We're hoping to use the plasmid for our lunchtime projects club > for biology and chemistry A-level students. > > > > Many thanks in advance! > > > > Camille Shammas >
Re: [ccp4bb] Mounting needle-shaped crystals
Hi Charlie, The bending of needles is hard to avoid :) My advice is to investigate different areas of the needle using finely focused beam - in particular focus on the protruding end and on the area inside the loop. The protruding area is particularly good since it has the least solvent attached to it. One of those may work out :) There's an alternative to this and that's using a very large loop so the needle never touches the nylon. The disadvantage of this approach is obviously the large volume of solvent/oil present in the loop, resulting in large background scatter. As far as systematic absences lost due to special orientation - yes, although if you have a bent loop (similar to the cheater pins from the bygone days) that's not much of an issue. Additionally, precise selection of space group can be always made as the structure's solved :) Artem > Useful summary, Artem. > > Two comments spring to mind. > > - I have had trouble with slight bending of needles when frozen like > this, causing problems with mosaicity. (Presumably due to different > forces acting on the parts of the crystal within and without the loop.) > > - You can have lots of trouble measuring systematic absences in crystals > mounted like this as the unique axis in hexagonal or tetragonal is close > to the rotation axis. > > Cheers, > Charlie > > > Artem Evdokimov wrote: >> Sorry, hit send by accident. Here it is: >> >> http://www.xtals.org/pdfs/needles.pdf >> >> Artem >> >> -Original Message- >> From: Artem Evdokimov [mailto:ar...@xtals.org] >> Sent: Monday, October 05, 2009 9:05 PM >> To: 'Tanner, John J.'; 'CCP4BB@JISCMAIL.AC.UK' >> Subject: RE: [ccp4bb] Mounting needle-shaped crystals >> >> Hello felow MO crystallographer, >> >> For all it's worth, a while ago I've compiled a little document which >> describes mounting needles. I've converted it into PDF and posted it >> here: >> >> >> "Nothing is built on stone; all is built on sand, but we must build as >> if >> the sand were stone" >> Jorge Luis Borges >> >> >> -Original Message- >> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of >> Tanner, John J. >> Sent: Monday, October 05, 2009 5:52 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] Mounting needle-shaped crystals >> >> Dear CCP4, >> >> I'm looking for advice on mounting thin needles for low temperature data >> collection. Our needles are fairly long (100-200 microns) but only 20 >> microns or less thick. When I pick them up with Hampton loops (0.05-0.1 >> mm >> size), the crystals tend to break as they are moved out of the drop and >> through the liquid-air interface. >> >> I see that Mitegen sells MicroLoops E, which are advertised as working >> well >> for mounting needles. Can anyone recommend them? Can anyone recommend >> Mitegen MicroMeshes or another tool for mounting needles? >> >> Thanks, >> >> Jack Tanner >> >> > > -- > Charlie Bond > Professorial Fellow > University of Western Australia > School of Biomedical, Biomolecular and Chemical Sciences > M310 > 35 Stirling Highway > Crawley WA 6009 > Australia > charles.b...@uwa.edu.au > +61 8 6488 4406 >
Re: [ccp4bb] off topic, design of a self-cleaving tag
Good times :) When you patent this design, be sure to give royalties to all the contributors. Daddy needs a new Ferrari. I assume you've seen Inteins already, right? Cleavage induced by DTT or BME. Artem > Hi, > > We would like to design a self-cleaving tag. It will be similar to the one > Roger Tsien has developed but I hope to find one that works in the > opposite way. > > Lin, M.Z. et al. A drug-controllable tag for visualizing newly synthesized > proteins in cells and whole animals. Proc. Natl. Acad. Sci. USA 105, > 7744â7749 (2008). > > Tsien used the NS3 protease which is inhibited by a cell-permeant drug. > The tag will be cleaved in the absence of this drug. I would like to find > a small and monomeric protease which is inactive until a drug or a > co-factor is applied. Any suggestions will be highly appreciated. > > Best, > Daniel > > > >
Re: [ccp4bb] Duet vectors
Hi, This is not typical. Try different cell lines for propagation and also add more sugar and glycerol to your medium - this helps. Artem > Dear all, > > We are attempting to clone into pETDuet, pACYCDuet, pCola1Duet and > pCDFDuet > and we are encountering numerous difficulties. > > We are aware that most of them are low copy number, but even taking this > into account, we find it puzzling that whereas the first preps of the > vectors gave low, but significant amount of DNA, as time goes by the > quantities we get are constantly decreasing. And any subsequent step > (ligation, etc.) is a real challenge. > > Does anyone have any experience (succesful or unsuccessful) with these > vectors? > > Many thanks, > Silvia > > > Silvia Onesti > > Sincrotrone Trieste S.C.p.A. > SS14 - km 163,5 in AREA Science Park > 34149 Basovizza, Trieste ITALY > Tel:+39 040 3758451 > Mob: +39 366 6878001 > > Email: silvia.one...@elettra.trieste.it > http://www.elettra.trieste.it/PEOPLE/index.php?n=SilviaOnesti.HomePage > http://www.sissa.it/sbp/web_2008/C_Structural_Biology.html > >
Re: [ccp4bb] off topic: microscope in glove box
Short version: It should be OK, especially since the vacuum is transient and not particularly 'strong*' :) Long version: if this is an older microscope there may be further delamination of optically bonded components if air is already admitted between glass planes (i.e. the optical cement is worn and old). Also some of the fancier models may have pneumatic balance elements for gross motion of the optical column - those may experience pressure differentials above their maximum tolerances. Finally, and very unlikely you may have a situation where multiple optical elements are sealed together in a single tube with air trapped between them - if the 'vent' is blocked (by e.g. old grease or something) then these may pop. https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/oculars/ But the short version is right 99% of the time. Artem *"Professor Hubert Farnsworth <https://www.imdb.com/name/nm0921942/?ref_=tt_ch>: Well, it's a space ship, so I'd say anywhere between zero and one." https://www.imdb.com/title/tt0584455/characters/nm0921942 - Cosmic Cats approve of this message On Mon, Oct 21, 2019 at 10:00 AM Guenter Fritz < guenter.fritz.phenix.c...@gmail.com> wrote: > Dear all, > > I want to put one of our microscopes into the glove box. Does anybody > know whether some parts of the microscope optics do not like vacuum in > the air lock ? > > Thanks and best regards, Guenter > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
