[ccp4bb] SeMet protein behaves much different with native protein..
Hi, I'm now facing a problem about selenomethionine-labelled protein crystallization.. Native protein crystallized very well and diffracted to 2.5A. However, SeMet-protein expression level decreased to only 1/10, and its crystals have different shape with before(native protein crystals have beautiful rhombohedral shape, while SeMet-protein crystals have a shuttle shape). For another crystalllization condition the SeMet protein don't crystallize at all while the native protein got crystals with even higher resolution. I don't know whether these "shuttle shaped" crystals are worthy to be used or not? But there are only 13 Met in my protein (43KD), why they have so much difference? Is this common, I mean, the huge difference between SeMet and native protein crystallization? And, any suggestions about what I can try to solve this Se-Met problem? (What I'm doing now is to screen crystallization kit with SeMet protein.) Thank you very much for your help! best, Sarah
[ccp4bb] anyone scrapping an Raxis-IV?
Is anyone about to "re-cycle" a Rigaku RAXIS-IV? One of the inverters that power the erase lamps in ours is smoking, and Rigaku have not been able to source a replacement for us, hence the message to the BB. Our machine was made in November 1996 and uses the type of inverter connected with four pairs of wires (Rigaku can source the more recent 3-pair types). I can give more detail, pictures etc. but would not want to clutter up your inboxes. This is not the first of these inverters to fail, so it would also be nice to find a source so we can keep going with it a while longer. By "we" of course I mean "I", as is the machine I play with whilst more serious people collect data on our more modern system, I clarify this just in case anyone should think my colleagues here are also stuck in the 20th century Thanks, Peter Peter Moody Henry Wellcome Laboratories University of Leicester Lancaster Road Leicester LE1 9HN UK tel. +44 (0)116 229 7097 http://www2.le.ac.uk/departments/biochemistry/staff/moody
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Dear Allister Crow, in cases like these, I would recommend to apply the Babinet bulk solvent correction instead of the mask bulk solvent correction as a control (Refmac5 -> Scaling -> Use Babinet scaling; uncheck "Calculcate the contribution from the solvent region"). The Babinet bulk solvent correction only uses two overall scaling factors and is usually simple, robust and, in my experience, does not show any local difference density artefacts. The mask bulk solvent correction is more powerful, but, depending on the project and the various mask radii for generating and shrinking the mask, could produce false positive or negative difference density. To exclude these cases, you can always calculate the Babinet bulk solvent correction as a control. Best regards, Dirk. Am 16.04.12 12:37, schrieb Allister Crow: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] SeMet protein behaves much different with native protein..
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sarah, it is very common that SeMet preps behave differently from the native crystal w.r.t. the crystallisation conditions, and 13Mets for a 43kDa protein is a lot! Don't judge a book by its cover: The only way to find out whether your differently shaped crystals diffract is to test them on an X-ray machine. Until you have done so you do not even know whether you have a "SeMet-Problem". Have you tried micro-seeding from the native crystals into a drop with SeMet-protein? Tim On 04/17/12 09:58, Qian Sarah wrote: > Hi, I'm now facing a problem about selenomethionine-labelled > protein crystallization.. Native protein crystallized very well and > diffracted to 2.5A. However, SeMet-protein expression level > decreased to only 1/10, and its crystals have different shape with > before(native protein crystals have beautiful rhombohedral shape, > while SeMet-protein crystals have a shuttle shape). For another > crystalllization condition the SeMet protein don't crystallize at > all while the native protein got crystals with even higher > resolution. I don't know whether these "shuttle shaped" crystals > are worthy to be used or not? But there are only 13 Met in my > protein (43KD), why they have so much difference? Is this common, I > mean, the huge difference between SeMet and native protein > crystallization? And, any suggestions about what I can try to > solve this Se-Met problem? (What I'm doing now is to screen > crystallization kit with SeMet protein.) > > Thank you very much for your help! best, Sarah > - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPjTTGUxlJ7aRr7hoRAoW+AJ9AquRYnXbaXfGl7MiRVMQ0KRaomQCfZ3NW 8GUTky+3a5/CS795RbqGDHI= =tgAe -END PGP SIGNATURE-
Re: [ccp4bb] SeMet protein behaves much different with native protein..
Hi sarah I believe, you might have used reducing agent in your SeMet-labeled protein sample. if avoiding reducing agent is not a problem to your protein (ignoring Selenium), try to purify/crystallize your protein in the absence of reducing agent. if u want to try this use all degassed buffers. With regards uday
Re: [ccp4bb] bulk solvent treatment inside protein cavities
On Tue, 2012-04-17 at 11:08 +0200, Dirk Kostrewa wrote: > The mask bulk solvent correction is more powerful Just to note that sometimes Babinet solvent correction returns lower Rfree and thus may be preferred to mask (assuming that the Rfree is the only thing that matters). Beginning with 5.6.0078, you can also optimize the bulk solvent mask parameters http://www.ysbl.york.ac.uk/~garib/refmac/data/refmac_news.html#Bulk_solvent This, imho, is not likely to remove the density artifacts, but is worth a try. -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
[ccp4bb] Checking in dry shippers?
Hi, what's the latest on flying with dry shippers? Until about 2009, I used to fly with dry shippers all the time: I just tossed them (dry!) on the check-in belt, and the airlines didn't mind. But that was only London-Zurich, using BA or SwissAir. Anybody know if this still works, especially flying to the US? Or have the securocrats now secured total victory? Any airlines / airports to avoid? phx.
[ccp4bb] CALL FOR PROPOSALS FOR ESRF BEAM TIME WITH ONLINE MICROSPEC
*CALL FOR PROPOSALS FOR ESRF BEAM TIME WITH ONLINE MICROSPEC* Proposal Deadline **1st May 2012** There will be beam time available at the ESRF for MX data collectionwith a setup that allows online monitoring of UV/VIS absorbance orfluorescence spectral changes of the crystal during the X-raydiffraction experiment. Users who are interested in using this beam time(including those who are members of BAG Groups) should use the followingmechanism:_http://www.esrf.fr/UsersAndScience/UserGuide/Applying/ProposalGuidelines/MXnon-BAGproposal_and *it must be clearly indicated in the title of the proposal form that**the online monitoring of spectral changes is necessary for the project*. A brief description of the device is given below however users areencouraged to consult the web pages for detailed information:_http://www.esrf.fr/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/Run_Your_Experiment/Microspectrophotometer_User_Guide_The device is also described in: McGeehan, J., Ravelli, R.B., Murray,J.W., Owen, R.L., Cipriani, F., McSweeney, S., Weik, M. and Garman, E.F.(2009) Colouring cryo-cooled crystals: online microspectrophotometry. JSynchrotron Radiat., 16, 163-172. As this is not a standard set-up, it might take a significant amount oftime to train users, align the device, and analyze the data in order toderive relevant data collection schemes. *We will therefore schedule 24**hours for each project*. The *deadline *for this specific application is*Tuesday 1st**May 2012*. It is strongly recommended to, beforehand, record an absorption(fluorescence) spectrum of the crystal on a home microspectrophotometersuch as the 4dx one, or at an off-line facility such as the ESRFCryobench, and to provide it in the application form. Such a spectrumwould greatly help to determine the feasibility of the experiment. Foroptimal experimental conditions, crystals should be frozen in minimalamounts of cryosolution, especially when the crystals are small.Finally, please note that *the ESRF sample changer cannot be operated at**the same time as the on-line microspec*.The use of specific LASER is possible if the device is compliant withthe beam line safety system (interlock on device power). Dates of beam-time: *13th June - 17th June 2012* Storage Ring: Uniform (200mA) Beamline: ID14-1 Energy: 13.27 keV (not tunable) Specifications: UV/VIS-range: 250-1100 nm Light source: Mikropack DH-2000-BAL (Deuterium/Halogen) Fluorescence/Actinic excitation wavelength: 405, 440, 473, 532, 561, 671 nm ODmax for UV-vis absorbance spectra: 2-2.5 Monitoring light size: 0.03 (min) - 0.15mm(max) Sampling freq (to disk): 10Hz or lower -- Dr David FLOT Beam-Line Operation Manager Tel : (+33) 4 76 88 17 63 Structural Biology GroupFax : (+33) 4 76 88 26 24 ESRF B.P. 220, 6 rue Jules Horowitz e-mail : david.f...@esrf.fr F-38043 GRENOBLE CEDEX http://www.esrf.eu
Re: [ccp4bb] SeMet protein behaves much different with native protein..
If SeMet oxidation is an issue then EDTA is your friend. This is because Met, SeMet and even Cys-Cys are not oxidized directly by O2, but rather via a trace transition metal intermediate. So, keeping enough chelator around to soak up any trace metals will dramatically slow down the oxidation. I can't remember where I first read about this, but once upon a time when I was working out how to make fmoc-SeMet (before you could buy it) and assaying the product by HPLC I found that I could easily see both the single- and double-oxidized forms on the chromatograms. Adding H2O2 was a good way to make the oxidized species (positive control), but a solution of SeMet in water at neutral pH and ~0.5 mM EDTA was stable in air for weeks. I discovered quite by accident that using a steel probe as a scraper was not a good idea. Lost a whole batch that way. The singly-oxidized SeMet can be converted back to SeMet with DTT, but the double-oxidized form cannot. -James Holton MAD Scientist On 4/17/2012 4:03 AM, Uday Kumar wrote: Hi sarah I believe, you might have used reducing agent in your SeMet-labeled protein sample. if avoiding reducing agent is not a problem to your protein (ignoring Selenium), try to purify/crystallize your protein in the absence of reducing agent. if u want to try this use all degassed buffers. With regards uday
Re: [ccp4bb] SeMet protein behaves much different with native protein..
just out of curiosity, was one Se enough in this case to solve the structure of your 0.2 kD protein by MAD? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 17 Apr 2012, at 18:13, James Holton wrote: > If SeMet oxidation is an issue then EDTA is your friend. This is because > Met, SeMet and even Cys-Cys are not oxidized directly by O2, but rather via > a trace transition metal intermediate. So, keeping enough chelator around to > soak up any trace metals will dramatically slow down the oxidation. I can't > remember where I first read about this, but once upon a time when I was > working out how to make fmoc-SeMet (before you could buy it) and assaying the > product by HPLC I found that I could easily see both the single- and > double-oxidized forms on the chromatograms. Adding H2O2 was a good way to > make the oxidized species (positive control), but a solution of SeMet in > water at neutral pH and ~0.5 mM EDTA was stable in air for weeks. I > discovered quite by accident that using a steel probe as a scraper was not a > good idea. Lost a whole batch that way. The singly-oxidized SeMet can be > converted back to SeMet with DTT, but the double-oxidized form cannot. > > -James Holton > MAD Scientist > > On 4/17/2012 4:03 AM, Uday Kumar wrote: >> Hi sarah >> >> I believe, you might have used reducing agent in your SeMet-labeled protein >> sample. >> >> if avoiding reducing agent is not a problem to your protein (ignoring >> Selenium), try to purify/crystallize your protein in the absence of reducing >> agent. >> >> if u want to try this use all degassed buffers. >> >> With regards >> uday
Re: [ccp4bb] Checking in dry shippers?
Hi, thanks for all responses. Most people suggested avoiding the scenario altogether, which was cute but not the question. Answers below the original question: On 17/04/2012 15:59, Frank von Delft wrote: Hi, what's the latest on flying with dry shippers? Until about 2009, I used to fly with dry shippers all the time: I just tossed them (dry!) on the check-in belt, and the airlines didn't mind. But that was only London-Zurich, using BA or SwissAir. Anybody know if this still works, especially flying to the US? Or have the securocrats now secured total victory? Any airlines / airports to avoid? phx. Harry: for a trouble free journey with your dry shipper, I'd say avoid any airlines that are flying to the States, and avoid any airports in the States Herb: We ship by FedEx ahead of time rather than try to bring on commercial passenger flight. It may be possible to do so but it's just not worth the hassle of running into an unexpected problem. Liz: For what its worth I always use a courier because i didnt think you could fly with them as luggage. THats between here and Berlin. Marko: no idea about flying to US, but Easyjet has been ok still to Lyon etc. We have designated contact at the airline, she approves the dewar, although the final decision is with guy in the uniform (pilot). Stanstead is ok as well, we simply call the security at airport, they as for a form by fax and ask to speak to supervisor when going through security. Doubt they let the dewar inside though, and hence the latter deal with airport security - I carry the dewar to the plane myself, so it is upright at least until it leaves the plane at the other end. Kris: If you are returning and the dewar has no samples, it is just a box. It will get X-rayed and forwarded to the cargo area. I generally keep it closed with zip ties so that no one can place anything in it without my permission. Also, remove all hazardous, flamable, or cryogenic stickers.
Re: [ccp4bb] Checking in dry shippers?
On Tue, 2012-04-17 at 20:03 +0100, Frank von Delft wrote: > Hi, thanks for all responses. Most people suggested avoiding the > scenario altogether, which was cute but not the question. As far as US is concerned, the FAA instructions to air carriers http://lmgtfy.com/?q=faa+liquid+nitrogen&l=1 clearly state that dry shippers are OK as long as there is no residual liquid. I assume that if you are flying from Europe to the US, similar lack of restrictions should be in place in the country of origin and all stops along the way. Naturally, the TSA personnel may be not so friendly to the scary you-know-what looking like item http://xkcd.com/651/ so perhaps printing out FAA advisory and some dry shipper disclaimer from the manufacturer could be helpful. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
[ccp4bb] Molecular Replacement
Hello, I have a question about molecular replacement. I use "Phaser" or "AutoMR" to generate models of my target protein. Input .mtz is from X-ray diffraction. Template is from a known structure. I also set up seq file using my target protein. The sequence identity between template and my target protein is quite high, over 90%. When I exam the ouputs, I found the sequence of the output .pdb is exactly same as the template. Is this normal for Molecular replacement? In order to have my target .pdb, I need to mutate the residues using coot? Thank you for advice Ros
[ccp4bb] Off-topic: Site-directed mutagenesis
Dear all I would like to get some opinions on site-directed mutagenesis. What are the current methods available? I know the Quick Change, are there others that work better? Thank you.
Re: [ccp4bb] Off-topic: Site-directed mutagenesis
Hi, Theresa, Our lab uses Phusion high-fidelity DNA polymerase. It might not be better though. Regards, Xun Sent from my iPad On Apr 17, 2012, at 5:53 PM, Theresa Hsu wrote: > Dear all > > I would like to get some opinions on site-directed mutagenesis. What are the > current methods available? I know the Quick Change, are there others that > work better? > > Thank you.
Re: [ccp4bb] Molecular Replacement
I would say yes. In any case you need to examine each mutated residue to be sure the correct conformation is chosen, so you might as well do the mutagenesis in coot or O. The ccp4 "chainsaw" program is sometimes used to prepare models for MR, but it truncates the mutated residues to variable extent rather than mutating- the purpose is to improve chances of success, not to arrive at a model with the correct sequence. If your resolution is high (2.2 A or better?) and you have ARP/wARP installed, run A/W in the mode "to improve an existing model", giving it your current model and the correct sequence. Not only will it build most of the mutated residues correctly, but in its role as a "model bias remover" it will fix or remove incorrect parts of the structure that may not be obvious in the initial maps. Uma Ratu wrote: Hello, I have a question about molecular replacement. I use "Phaser" or "AutoMR" to generate models of my target protein. Input .mtz is from X-ray diffraction. Template is from a known structure. I also set up seq file using my target protein. The sequence identity between template and my target protein is quite high, over 90%. When I exam the ouputs, I found the sequence of the output .pdb is exactly same as the template. Is this normal for Molecular replacement? In order to have my target .pdb, I need to mutate the residues using coot? Thank you for advice Ros
Re: [ccp4bb] Molecular Replacement
Thank you very much for your inputs and comments. I am getting understand what is going on now. > If your resolution is high (2.2 A or better?) and you have ARP/wARP Yes, the resolution is about 2A. I have ARP/wARP. Will give a try. One more question about Molecular Replacement. With Phaser, I got tetramer conformation. With autoMR, it gave me dimer conformation. Each molecuale was trucated into two chains. The scores from both methods were high, as expected from 90% sequence identity between template and target. Thank you for advice Ros On 4/18/12, Edward A. Berry wrote: > I would say yes. In any case you need to examine each mutated > residue to be sure the correct conformation is chosen, so you might > as well do the mutagenesis in coot or O. The ccp4 "chainsaw" program > is sometimes used to prepare models for MR, but it truncates the mutated > residues to variable extent rather than mutating- the purpose is to improve > chances of success, not to arrive at a model with the correct sequence. > > If your resolution is high (2.2 A or better?) and you have ARP/wARP > installed, run A/W in the mode "to improve an existing model", giving it > your current model and the correct sequence. Not only will it build > most of the mutated residues correctly, but in its role as a "model bias > remover" it will fix or remove incorrect parts of the structure that may > not be obvious in the initial maps. > > Uma Ratu wrote: >> Hello, >> I have a question about molecular replacement. >> I use "Phaser" or "AutoMR" to generate models of my target protein. >> Input .mtz is from X-ray diffraction. Template is from a known >> structure. I also set up seq file using my target protein. The sequence >> identity between template and my target protein is quite high, over 90%. >> When I exam the ouputs, I found the sequence of the output .pdb is >> exactly same as the template. >> Is this normal for Molecular replacement? >> In order to have my target .pdb, I need to mutate the residues using coot? >> Thank you for advice >> Ros > >
Re: [ccp4bb] Molecular Replacement
> Does that mean a different number of molecules in the asymmetric was found, With Phaser, 4 monomers. With AutoMR, 2 monomers. >did you divide the molecule and find each part separately? Each monomer (by AutoMR) is composed of two chains. One chain is part of my target. The other chain matchs to the rest of my target. The original template .pdb is a tetramer with four monomers. I used one of the monomer as the template for the molecualr replacement in both Phaser and AutoMR. The seq file is the whole of my target. >Provide more details like space group Space group is P21. > whether the tetramer is crystallograhic or all in the asymmetric unit, The tetramer is crystallograhic Thank you Ros On 4/18/12, Edward A. Berry wrote: > > One more question about Molecular Replacement. With Phaser, I got > > tetramer conformation. With autoMR, it gave me dimer conformation. > > Each molecuale was trucated into two chains. The scores from both > > Does that mean a different number of molecules in the asymmetric > was found, or just that they were arranged differently? If the latter, > try generating symmetry-mates around one dimer and see if the tetramer > is created. Ideally if the solutions are right they should be the > same, within an arbitrary choice of asymmetric unit and origin. > > What does it mean, each molecule was . . two chains? did you > divide the molecule and find each part separately? Is you > dimer a heterodimer? Provide more details like space group and > whether the tetramer is crystallograhic or all in the asymmetric > unit, and some expert may be able to provide suggestions. > > > Uma Ratu wrote: >> Thank you very much for your inputs and comments. >> >> I am getting understand what is going on now. >> >>> If your resolution is high (2.2 A or better?) and you have ARP/wARP >> >> Yes, the resolution is about 2A. I have ARP/wARP. Will give a try. >> >> One more question about Molecular Replacement. With Phaser, I got >> tetramer conformation. With autoMR, it gave me dimer conformation. >> Each molecuale was trucated into two chains. The scores from both >> methods were high, as expected from 90% sequence identity between >> template and target. >> >> Thank you for advice >> >> Ros >> >> >> On 4/18/12, Edward A. Berry wrote: >>> I would say yes. In any case you need to examine each mutated >>> residue to be sure the correct conformation is chosen, so you might >>> as well do the mutagenesis in coot or O. The ccp4 "chainsaw" program >>> is sometimes used to prepare models for MR, but it truncates the mutated >>> residues to variable extent rather than mutating- the purpose is to >>> improve >>> chances of success, not to arrive at a model with the correct sequence. >>> >>> If your resolution is high (2.2 A or better?) and you have ARP/wARP >>> installed, run A/W in the mode "to improve an existing model", giving it >>> your current model and the correct sequence. Not only will it build >>> most of the mutated residues correctly, but in its role as a "model bias >>> remover" it will fix or remove incorrect parts of the structure that may >>> not be obvious in the initial maps. >>> >>> Uma Ratu wrote: Hello, I have a question about molecular replacement. I use "Phaser" or "AutoMR" to generate models of my target protein. Input .mtz is from X-ray diffraction. Template is from a known structure. I also set up seq file using my target protein. The sequence identity between template and my target protein is quite high, over 90%. When I exam the ouputs, I found the sequence of the output .pdb is exactly same as the template. Is this normal for Molecular replacement? In order to have my target .pdb, I need to mutate the residues using coot? Thank you for advice Ros >>> >>> >> > >
[ccp4bb] Post-Doc position at ICR, London
We seek to appoint a post-doctoral training fellow to the Division of Structural Biology in the new team led by Dr Alessandro Vannini to undertake crystallographic, single particle electron microscopy analysis and biochemical analysis of large macromolecular complexes that assemble at RNA Polymerase III loci across the eukaryotic genome, in order to mechanistically understand their role in cancer and other fundamental cellular processes (www.icr.ac.uk/alessandrovannini). The Division of Structural Biology has managed facilities for protein crystallography (Bruker Microstar and CCD detector and crystallisation robots), cryo-electron microscopy (FEI Tecnai F20 and T12), and protein production with expertise in multi-subunit expression (insect cell, yeast and bacterial expression, including a 60 L fermentor). The Division is also well equipped with equipment for biophysical analysis (e.g. ITC, fluorescence, multi-angle light scattering). Applicants should possess a PhD (or equivalent) in biochemistry or molecular biology with a sound knowledge of protein crystallography an/or single particle electron microscopy. Experience in the biochemical and biophysical characterization of multi-subunit and protein-nuclei acids complexes is highly desirable. Experience in protein production in insect cells (MultiBac) or yeast genetics would be an advantage. Appointment will be on Fixed Term Contract for 3 years in the first instance, with a starting salary in the range of £27,536 to £33,852 p.a. inclusive (based on previous post-doctoral experience). For further particulars and details of how to apply, please check the current vacancies in the division of Structural Biology on our online application website (https://www.icr.ac.uk/jobs/index.shtml) and refer to this vacancy (n. 1228522). Closing date: 16th May 2012 Please direct informal inquiries to Dr. Alessandro Vannini at alessandro.vann...@icr.ac.uk
