Re: [ccp4bb] negative density in difference map [SEC=UNCLASSIFIED]
Delete (set occupancies to zero) the side chain back to CA. Do a few rounds of refinement and calculate Fo-Fc and examine. It is possible that the side chain is disordered, or ordered in multiple conformations. Compare the density for alternate confonformers against the density for CA. Alternatively, your side chain B-factor restaints might be too tight. Anthony From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina Edgooms [careinaedgo...@yahoo.com] Sent: Wednesday, 23 November 2011 6:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] negative density in difference map Good morning CCP4 members I have a question about a 2F0-Fc difference map that I calculated with Refmac. In some instances it gives me negative (red) density around part of a side chain and no positive density in sight. Furthermore the entire residue fits well into the blue density of the complete map, including the part with negative density. I am struggling to interpret this. Does the fact that it fits the blue density mean that the side chain is in the correct place or does the red blob on part of the side chain (eg on the sulphur in a Met residue) mean that something funky is happening with this side chain? Thanks for any assistance Careina
Re: [ccp4bb] crystal orientation during data collection
I would interpret "random" as aimed to be uniformly distributed, or highly diverse,... continuing this chain of definitions and having a precise kappa goniometer in hand, we can easily arrive to a "planned" strategy to follow. Certainly, if you can get only a few frames per xtal - Frank (vD), do you remember when we had a project like this together- you may not have the luxury of being able to afford to loose a complete frame(s) just to get to know the initial orientation of the crystal(s). In any case, I am happy to learn that our solution 'STAC' is useful for the community. Actually, we are organising an International Kappa Workgroup Meeting in Berlin on Nov 28-29. Registration is normally over, but if there is anyone interested in learning more about current developments for sample reorintation on synchrotron beamlines, please contact us directly and join us in Berlin. http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/kappa-meeting/index_en.html Sandor Jacob Keller wrote: Generally speaking, don't we agree that "planned" or "rational" is better than "random?" (Having trouble understanding the argument for randomness here...) Jacob On Fri, Nov 18, 2011 at 9:40 AM, Sanishvili, Ruslan wrote: Depending on the crystal shape, “random orientation” is not always random. Many crystals have tendencies of sitting themselves in one predominant posture in the mount. Compounding this, many experimenters have tendencies of rotating the mount into a specific orientation when centering. Then crystal orientation ends up being not random at all, so understanding it’s true orientation as my neighbor Frank suggests can be highly beneficial. Cheers, N. Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Friday, November 18, 2011 2:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystal orientation during data collection I believe you achieve completeness more quickly (fewer crystals) if you just take random orientations. At least, that's what I learnt from Dave Stuart. phx On 18/11/2011 04:20, Frank Murphy wrote: Yanwu, I surmise from your question that you are inquiring how to go about collecting from many crystals optimally. Merging data ex post facto is a totally different kettle of fish. In my opinion, the most robust way to go about this is to use a kappa goniometer as Jim suggested (I am most familiar with the MK3). Since you intend to collect from many crystals, align the first and all subsequent crystals to the same easily attainable (or seemingly so) orientation, and then collect the sweep suggested by your data collection strategy program of choice. To achieve this at NE-CAT, we have a GUI-based system that used STAC for orientation determination and BEST for strategy generation. As Jim suggested, more options than STAC exist. If anyone is unable to get to a kappa goniometer, they can employ Mosflm or XDS (Xplan) to generate strategies for data collection from a crystal taking into account previously collected data. This is not nearly as robust a solution, but is a workable substitute (and also automated at NE-CAT). I know there are other ways to achieve similar results, but I have suggested the methods I am most familiar with... Yours, Frank Murphy Begin forwarded message: From: yanwu huo Date: November 17, 2011 4:00:06 PM CST To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal orientation during data collection Reply-To: yanwu huo Hi, I worked on a crystal sensitive to radiation damage, So I need to merge many crystal to obtain complete dataset, Does anyone know such program that can tell crystal orientation after first frame exposure. Thank you in advance. -- Thank you very much and all the best, Yanwu Huo Postdoctoral Associate Department of Molecular Biology and Genetics Cornell University Ithaca, NY, 14853 Email:yh...@cornell.edu
Re: [ccp4bb] negative density in difference map [SEC=UNCLASSIFIED]
I wish I could answer this! One possibility is that the side-chain B values are too tightly restrained - Ian Tickle recommends releasing these somewhat.. Here are the default refmac values. THERMAL FACTORS Weight= 1.00 Main chain bond (1-2 neighbour) 1.5A**2 Main chain angle (1-3 neighbour) 2.0A**2 Side chain bond 3.0A**2 Side chain angle 4.5A**2 and you could change them say to: Under GUI - check geometric parameters and alter the Bfactor values data line becomes: temp 1.0 1.5 2.0 4.0 6.0 The trouble is that resyraints for ARG or MET say should be looser than those for SER or VAL. But I often finish up with some inexplicable red blobs - sometimes floating in space.. Eleanor On 11/23/2011 08:39 AM, DUFF, Anthony wrote: Delete (set occupancies to zero) the side chain back to CA. Do a few rounds of refinement and calculate Fo-Fc and examine. It is possible that the side chain is disordered, or ordered in multiple conformations. Compare the density for alternate confonformers against the density for CA. Alternatively, your side chain B-factor restaints might be too tight. Anthony From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina Edgooms [careinaedgo...@yahoo.com] Sent: Wednesday, 23 November 2011 6:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] negative density in difference map Good morning CCP4 members I have a question about a 2F0-Fc difference map that I calculated with Refmac. In some instances it gives me negative (red) density around part of a side chain and no positive density in sight. Furthermore the entire residue fits well into the blue density of the complete map, including the part with negative density. I am struggling to interpret this. Does the fact that it fits the blue density mean that the side chain is in the correct place or does the red blob on part of the side chain (eg on the sulphur in a Met residue) mean that something funky is happening with this side chain? Thanks for any assistance Careina
Re: [ccp4bb] negative density in difference map [SEC=UNCLASSIFIED]
Hi Careina Since my name came up I felt compelled to comment, though I don't have a definitive answer. Over-restraining of B factors is certainly a possibility and could well explain otherwise inexplicable difference density. IMO B factors are generally over-restrained. They are a bit like the R factors: tight restraints keep the B factors low and people (referees in particular!) seem to feel happier when they see low values, even though that is not necessarily optimal (this mind-set is of course known as "wishful thinking" - see Wikipedia article). Personally I never restrain 1-3 B factors, and only use the 1-2 restraints, and even then I relax those from the default values. The 1-3 B restraints are clearly not independent of the 1-2 ones, and restraints should ideally be independent of each other, otherwise you are applying them more than once, IMO the overall B factor restraint weight should be optimised in the same way that the X-ray weight should be, by minimising Rfree or -LLfree. X-plor, CNS (and I would guess phenix.refine) have had this option for many years (optimize_rweight.inp in CNS), and it seems an excellent thing to do, Disorder is also an obvious possibility as Anthony said, and I would follow his sensible advice. Cheers -- Ian Cheers -- Ian On 23 November 2011 10:58, Eleanor Dodson wrote: > I wish I could answer this! > One possibility is that the side-chain B values are too tightly restrained - > Ian Tickle recommends releasing these somewhat.. > > Here are the default refmac values. > THERMAL FACTORS > Weight= 1.00 > Main chain bond (1-2 neighbour) 1.5A**2 > Main chain angle (1-3 neighbour) 2.0A**2 > Side chain bond 3.0A**2 > Side chain angle 4.5A**2 > > and you could change them say to: > > > Under GUI - check geometric parameters and alter the Bfactor values > > data line becomes: temp 1.0 1.5 2.0 4.0 6.0 > > The trouble is that resyraints for ARG or MET say should be looser than > those for SER or VAL. > > But I often finish up with some inexplicable red blobs - sometimes floating > in space.. > Eleanor > > > On 11/23/2011 08:39 AM, DUFF, Anthony wrote: >> >> >> Delete (set occupancies to zero) the side chain back to CA. Do a few >> rounds of refinement and calculate Fo-Fc and examine. >> >> >> > > >> It is possible that the side chain is disordered, or ordered in multiple >> conformations. Compare the density for alternate confonformers against the >> density for CA. >> >> >> >> Alternatively, your side chain B-factor restaints might be too tight. >> >> >> >> Anthony >> >> >> >> >> >> >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina >> Edgooms [careinaedgo...@yahoo.com] >> Sent: Wednesday, 23 November 2011 6:54 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] negative density in difference map >> >> Good morning CCP4 members >> >> I have a question about a 2F0-Fc difference map that I calculated with >> Refmac. In some instances it gives me negative (red) density around part of >> a side chain and no positive density in sight. Furthermore the entire >> residue fits well into the blue density of the complete map, including the >> part with negative density. >> I am struggling to interpret this. Does the fact that it fits the blue >> density mean that the side chain is in the correct place or does the red >> blob on part of the side chain (eg on the sulphur in a Met residue) mean >> that something funky is happening with this side chain? >> >> Thanks for any assistance >> Careina >> >
[ccp4bb] [CCP4] identify a rotation centre: domain rotation
Dear members, I would like to have your ideas if there is any way to identify a rotation centre of domain in two different states using CCP4 or other program. The situation is: the domain of the protein will rotate between two different states (depending on substrate binding) around 8 degree, and it is (nearly) clearly that the domain is rotated around a rotation centrel. So the question is how to identify this "rotation centre" in this 3D model? The ideal is to identify a region of residues in the domain which are most closed to the rotation centre. The tool I am using right now is the "superpose" tool in CCP4 package. The output which I think mightbe uesful is: CENTROID OF "WORKING" MOLECULE : 157.812 152.396 -70.778 CENTROID OF "WORKING" MOLECULE :(fractional) 157.812 152.396 -70.778 CENTROID OF "REFERENCE" MOLECULE: 157.251 151.877 -70.874 CENTROID OF "REFERENCE" MOLECULE:(fractional) 157.251 151.877 -70.874 Distance between CENTROIDS :0.770 Direction cosines of vector between CENTROIDS: 0.729 0.674 0.124 I would say the “CENTROID" it mentioned above, such as (157.251 151.877 -70.874), is possibly near to the "rotation centre". I would like to have your opinion though. Thank you. King regards, Wenhe
Re: [ccp4bb] [CCP4] identify a rotation centre: domain rotation
I guess the rotation centre is approximately the mid point between the two centroids.. But these look surprisingly similar? I would hsave expected after an 8 degree rotation there would be some greater difference.. Eleanor On 11/23/2011 02:45 PM, WENHE ZHONG wrote: Dear members, I would like to have your ideas if there is any way to identify a rotation centre of domain in two different states using CCP4 or other program. The situation is: the domain of the protein will rotate between two different states (depending on substrate binding) around 8 degree, and it is (nearly) clearly that the domain is rotated around a rotation centrel. So the question is how to identify this "rotation centre" in this 3D model? The ideal is to identify a region of residues in the domain which are most closed to the rotation centre. The tool I am using right now is the "superpose" tool in CCP4 package. The output which I think mightbe uesful is: CENTROID OF "WORKING" MOLECULE : 157.812 152.396 -70.778 CENTROID OF "WORKING" MOLECULE :(fractional) 157.812 152.396 -70.778 CENTROID OF "REFERENCE" MOLECULE: 157.251 151.877 -70.874 CENTROID OF "REFERENCE" MOLECULE:(fractional) 157.251 151.877 -70.874 Distance between CENTROIDS :0.770 Direction cosines of vector between CENTROIDS: 0.729 0.674 0.124 I would say the “CENTROID" it mentioned above, such as (157.251 151.877 -70.874), is possibly near to the "rotation centre". I would like to have your opinion though. Thank you. King regards, Wenhe
Re: [ccp4bb] negative density in difference map
On Tue, Nov 22, 2011 at 11:54 PM, Careina Edgooms wrote: > I have a question about a 2F0-Fc difference map that I calculated with > Refmac. In some instances it gives me negative (red) density around part of > a side chain and no positive density in sight. Furthermore the entire > residue fits well into the blue density of the complete map, including the > part with negative density. > I am struggling to interpret this. Does the fact that it fits the blue > density mean that the side chain is in the correct place or does the red > blob on part of the side chain (eg on the sulphur in a Met residue) mean > that something funky is happening with this side chain? It could mean that the sidechain is correctly placed, but poorly ordered, and the B-factor restraints are preventing the B-factors for those atoms from going as high as they probably should. I don't think this is terribly uncommon. I've seen it before, and each time I tried truncating the residue(s) to alanine, I got positive difference density back after refinement. If you see the same thing, my inclination would be to leave it alone and not worry about it as long as everything else looks sensible. The negative density around Met S could be radiation damage - also not uncommon, since these and carboxyl groups tend to get hit the hardest (unless you have heavier atoms). -Nat
Re: [ccp4bb] negative density in difference map
On 23 November 2011 07:54, Careina Edgooms wrote: > I have a question about a 2F0-Fc difference map that I calculated with Refmac. On 23 November 2011 15:40, Nat Echols wrote: > The negative density around Met S could be radiation damage. But you wouldn't expect to see -ve density in the 2Fo-Fc map from radiation damage right? The Fo-Fc map for sure. Cheers -- Ian
Re: [ccp4bb] negative density in difference map
On Wed, Nov 23, 2011 at 7:57 AM, Ian Tickle wrote: > On 23 November 2011 07:54, Careina Edgooms wrote: >> I have a question about a 2F0-Fc difference map that I calculated with >> Refmac. > > On 23 November 2011 15:40, Nat Echols wrote: >> The negative density around Met S could be radiation damage. > > But you wouldn't expect to see -ve density in the 2Fo-Fc map from > radiation damage right? The Fo-Fc map for sure. I assumed that's what the original poster meant. (and I apologize for the redundant comments, GMail groups messages by subject, so every time someone changes the subject line, it becomes a new thread, which I usually miss. That said, I thought after reading Garib's Refmac paper published earlier this year that it was now using distance-based B-factor restraints, instead of bond connectivity - is this correct or did I misunderstand?) -Nat
Re: [ccp4bb] negative density in difference map
Sounds like rad dam to me. See Burmeister, W. (2000)."Structural changes in a cryo-cooled protein crystal owing to radiation damage", Acta Cryst. D 56, 328-341. The first sign of a Met loosing its S-CH3 group will be a negative difference peak on the S. -James Holton MAD Scientist On 11/22/2011 11:54 PM, Careina Edgooms wrote: Good morning CCP4 members I have a question about a 2F0-Fc difference map that I calculated with Refmac. In some instances it gives me negative (red) density around part of a side chain and no positive density in sight. Furthermore the entire residue fits well into the blue density of the complete map, including the part with negative density. I am struggling to interpret this. Does the fact that it fits the blue density mean that the side chain is in the correct place or does the red blob on part of the side chain (eg on the sulphur in a Met residue) mean that something funky is happening with this side chain? Thanks for any assistance Careina
[ccp4bb] Postdoctoral position, institut Pasteur, Paris, France
Postdoctoral position, institut Pasteur, Paris, France Deadline for application: 15th of January 2012. A 3 year postdoctoral position funded by the ERC (European Research Council) is available in the G5 Unit « structural biology of bacterial secretion » (http://www.pasteur.fr/research/sbbs) in the Structural Biology and Chemistry department at Institut Pasteur (Paris, France). Our research aim is to decipher the molecular mechanisms that support DNA transfer events between bacteria during natural transformation and conjugation. The person to be recruited will work more specifically on the purification and structural characterization using X-ray crystallography of individual components and protein complexes isolated from the DNA uptake system found in Streptococcus pneumoniae. Interested candidates should have obtained a Ph.D. in molecular and structural biology and should have relevant research publications in this field. Experience in membrane protein biochemistry and X-ray crystallography is highly desirable but not mandatory. The position will be available in 2012. The starting date will be decided in agreement with the selected candidate, preferably during the first semester of 2012. Contact: Applications in the form of a CV, publication list, a summary of research activities (2 pages max) and contact details of two referees, should be sent by Email to Dr Rémi Fronzes (remi.fron...@pasteur.fr). Selected candidates will be individually interviewed in January and February. The final decision will be made before the end of february 2012.
Re: [ccp4bb] negative density in difference map
There may be several reasons. 1) Artefacts (some of them) a) effect of mask: if there are large holes inside molecule and there should be no electron density (for example hydrophobic holes) then in older versions masks would put a constant density there and as a result difference map would have negative densities. This has been dealt with in the newer version b) Effect of TLS refinement. If you are using TLS then you can try without TLS and compare densities. If it is effect of TLS then without TLS you should not see negative density. TLS model is a simple model assumes that all atoms in the group oscillates as a rigid group. Loops and other flexible parts may not obey this assumption 2) Genuine density Alternative conformations (Met almost always are in more than one conformations). In these cases you may be able to see alternative conformation if you look at very low level of electron density. And you may be able to see if it is case by looking at the B values. If neigbouring atoms have large differences in B values then it may be because of alt conformation Regards Garib On 23 Nov 2011, at 07:54, Careina Edgooms wrote: > Good morning CCP4 members > > I have a question about a 2F0-Fc difference map that I calculated with > Refmac. In some instances it gives me negative (red) density around part of a > side chain and no positive density in sight. Furthermore the entire residue > fits well into the blue density of the complete map, including the part with > negative density. > I am struggling to interpret this. Does the fact that it fits the blue > density mean that the side chain is in the correct place or does the red blob > on part of the side chain (eg on the sulphur in a Met residue) mean that > something funky is happening with this side chain? > > Thanks for any assistance > Careina Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] negative density in difference map
I assumed that since this topic came up fairly recently, in fact 3 weeks ago (see thread starting from http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg23628.html), it wasn't just a re-run of the same question. Perhaps the original poster could clarify whether we are talking about unexplained -ve density in the 2Fo-Fc map or in the Fo-Fc map? Cheers -- Ian On 23 November 2011 16:03, Nat Echols wrote: > On Wed, Nov 23, 2011 at 7:57 AM, Ian Tickle wrote: >> On 23 November 2011 07:54, Careina Edgooms wrote: >>> I have a question about a 2F0-Fc difference map that I calculated with >>> Refmac. >> >> On 23 November 2011 15:40, Nat Echols wrote: >>> The negative density around Met S could be radiation damage. >> >> But you wouldn't expect to see -ve density in the 2Fo-Fc map from >> radiation damage right? The Fo-Fc map for sure. > > I assumed that's what the original poster meant. > > (and I apologize for the redundant comments, GMail groups messages by > subject, so every time someone changes the subject line, it becomes a > new thread, which I usually miss. That said, I thought after reading > Garib's Refmac paper published earlier this year that it was now using > distance-based B-factor restraints, instead of bond connectivity - is > this correct or did I misunderstand?) > > -Nat >
Re: [ccp4bb] dark progression of radiation damage
I think I need to clarify couple of things in my recent post about "exploding" crystals during re-mounting by a robot. First, it was a bit over-dramatization - what I meant by "explosion" was actually bubbling often observed when heavily exposed crystal is warmed up. Second, this bubbling was observed only after a HEAVILY OVEREXPOSED crystal was re-mounted and not during a regular screening procedure. Third and more import, as couple of people pointed out to me, the observed bubbling was probably a result of something being wrong in the robot operation, rather than intrinsic feature of that or any other robot. This indeed seems to be the case since the observations I was referring to were made a couple of years ago and have not been seen since. During this time the robot operation underwent several upgrades and tweaks, perhaps fixing whatever problem it might have had resulting in occasional bubbling. Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov On 11/20/2011 5:22 PM, Sanishvili, Ruslan wrote: > Hi James, > I don't think the comment you referenced meant to imply "dark progression of radiation damage. If I remember from the recent thread, it was to say that if you can only collect few (3?) shots from one crystal before it's "too dead" and you use 1st of these shots to devise the strategy, then you are wasting your crystals and will never get you data. Of course, you don't have to use so much flux for the image which is meant only for defining the orientation but it was omitted from that comment. > > Now back to the rest of your message. I can add another warning observation: > If a cryo-cooled crystal was exposed long enough (i.e. for data collection) then stored (by a robot) and then mounted again, some times one sees that it had "exploded". Such an explosion, presumably a hydrogen gas escape, can be seen almost always if a crystal is wormed up after long data collection. The fact that robot-stored crystals sometimes display same behavior, indicates that a crystal in the arms of the robot can worm up somewhat. Therefore, comparing diffraction before and after storage is not always valid. > Also beware of comparing diffraction quality from different parts of the crystal as large crystals are almost never homogeneous. > Cheers, > Nukri > > > > > -Original Message- > From: CCP4 bulletin board on behalf of James Holton > Sent: Sun 11/20/2011 2:31 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] dark progression of radiation damage > > > Mark's comment below reminded me of a quandary that is starting to > develop in the rad dam field. The idea of the "free radical cascade" > continuing to damage protein crystals even after the beam has been > turned off seems to have originated on page 253 of Blundell and Johnson > (1976), and I think most of us have had the unpleasant experience of > loosing diffraction after a "delay" in data collection. However, can > one be sure that the incident beam alignment was the same if the "delay > in data collection" was due to a storage ring dump, or a filament > change? Can one be sure that a crystal stored under cryo never ever got > warmed up (like during mounts and dismounts, or perhaps a colleague > making an undocumented late-night rummage through the storage dewar)? > Can one be sure that a crystal at room temperature wasn't just drying > up? Can one be sure that the damage didn't all occur during the first > shot (and the image we saw is just the sum over the decay)? > > I ask because many systematic studies have now been made to try and > quantify the "dark progression" phenomenon, only to find it doesn't seem > to really exist, either under cryo (Garman& McSweeney, 2007; Sliz et > al., 2003; Leiros et al., 2006; Owen et al., 2006), or at room > temperature (Southworth-Davies et al. Structure 2007; Warkentin et al. > Acta D 2011), except at temperatures that are almost never used for data > collection (Warkentin et al. Acta D 2011). Now, there are observations > of radiochemical reactions progressing for several minutes "in the dark" > (Weik et al., 2002, Southworth-Davies& Gaman Acta D 2007 McGeehen et > al., 2009 ), but I don't personally know of anyone (other than Warkentin > et al. 2011) who has demonstrated that _diffraction_ continues to decay > in the dark. > > > So, my question is: does anyone out there have an example system where > one can reproducibly demonstrate "dark progression" of diffraction spot > fading? That is, you can mount the crystal, store it in its "mount" for > at least a few days (to prove that its not just drying up), take at > least two low-dose shots to get an idea of the expected rate of decay, > then wait for "a while" and start shooting again. Do you see > significantly worse diffraction? > > -James Holton > MAD Scientist > > > On 11/18/2011 1:50 AM, Mark J van Raaij wrote: >>I.e. if you collect
Re: [ccp4bb] dark progression of radiation damage
Any cacodylate buffer will cause gas to be produced. One only needs a minute exposure on a modern home lab source to see this happening. I suggest that everyone avoid cacodylate in their crystallization drops that end up being exposed to X-rays. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, Ruslan [rsanishv...@anl.gov] Sent: Wednesday, November 23, 2011 11:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] dark progression of radiation damage I think I need to clarify couple of things in my recent post about "exploding" crystals during re-mounting by a robot. First, it was a bit
Re: [ccp4bb] dark progression of radiation damage
Also, cacodylate contains arsenic which is heavy, and thus has a much larger X-ray absorption cross section than do buffers constituted of lighter atoms. There is therefore a bigger dose (Joules/kg of crystal) absorbed with cacodylate in the buffer than there would be without it (and no extra diffraction strength), so that is another very good reason to avoid it, or to buffer exchange it out before the diffraction experiment. Elspeth -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jim Pflugrath Sent: 23 November 2011 18:11 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] dark progression of radiation damage Any cacodylate buffer will cause gas to be produced. One only needs a minute exposure on a modern home lab source to see this happening. I suggest that everyone avoid cacodylate in their crystallization drops that end up being exposed to X-rays. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, Ruslan [rsanishv...@anl.gov] Sent: Wednesday, November 23, 2011 11:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] dark progression of radiation damage I think I need to clarify couple of things in my recent post about "exploding" crystals during re-mounting by a robot. First, it was a bit
Re: [ccp4bb] dark progression of radiation damage
I understand that absorbed dose increases with presence of heavy atoms, but I don't understand why that should play a role in damaging the crystal, as heavy atoms such as in cacodylate should probably usually not be near enough to protein atoms to cause problems. At 100K, isn't it true that secondary radiation damage plays little role if any? So the only problem I can think of is the case when the cacodylate molecule happens to be within "striking distance" of a protein atom when it turns into a radical (not sure what that distance would be). This should be relatively rare in, say, 55mM cacodylate, when there is only ~1 cacodylate for every 1000 waters, no? Has there been an empirical study comparing similar crystals of the same protein +/- solvent heavy atoms? I guess derivatives are the obvious example--but real derivatives always have ordered, occupied sites. Jacob On Wed, Nov 23, 2011 at 12:28 PM, Elspeth Garman wrote: > Also, cacodylate contains arsenic which is heavy, and thus has a much larger > X-ray absorption cross section than do buffers constituted of lighter atoms. > There is therefore a bigger dose (Joules/kg of crystal) absorbed with > cacodylate in the buffer than there would be without it (and no extra > diffraction strength), so that is another very good reason to avoid it, or to > buffer exchange it out before the diffraction experiment. > > Elspeth > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jim > Pflugrath > Sent: 23 November 2011 18:11 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] dark progression of radiation damage > > Any cacodylate buffer will cause gas to be produced. One only needs a minute > exposure on a modern home lab source to see this happening. I suggest that > everyone avoid cacodylate in their crystallization drops that end up being > exposed to X-rays. > > Jim > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, > Ruslan [rsanishv...@anl.gov] > Sent: Wednesday, November 23, 2011 11:49 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] dark progression of radiation damage > > I think I need to clarify couple of things in my recent post about > "exploding" crystals during re-mounting by a robot. First, it was a bit > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] dark progression of radiation damage
Since "striking distance" is about 3 microns for the primary photoelectron and the largest unit cell in the PDB is ~0.1 microns long, I think that means all bets are off when trying to "connect" energy absorbed by a heavy atom to damage somewhere else in the unit cell. -James Holton MAD Scientist On 11/23/2011 10:49 AM, Jacob Keller wrote: I understand that absorbed dose increases with presence of heavy atoms, but I don't understand why that should play a role in damaging the crystal, as heavy atoms such as in cacodylate should probably usually not be near enough to protein atoms to cause problems. At 100K, isn't it true that secondary radiation damage plays little role if any? So the only problem I can think of is the case when the cacodylate molecule happens to be within "striking distance" of a protein atom when it turns into a radical (not sure what that distance would be). This should be relatively rare in, say, 55mM cacodylate, when there is only ~1 cacodylate for every 1000 waters, no? Has there been an empirical study comparing similar crystals of the same protein +/- solvent heavy atoms? I guess derivatives are the obvious example--but real derivatives always have ordered, occupied sites. Jacob On Wed, Nov 23, 2011 at 12:28 PM, Elspeth Garman wrote: Also, cacodylate contains arsenic which is heavy, and thus has a much larger X-ray absorption cross section than do buffers constituted of lighter atoms. There is therefore a bigger dose (Joules/kg of crystal) absorbed with cacodylate in the buffer than there would be without it (and no extra diffraction strength), so that is another very good reason to avoid it, or to buffer exchange it out before the diffraction experiment. Elspeth -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jim Pflugrath Sent: 23 November 2011 18:11 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] dark progression of radiation damage Any cacodylate buffer will cause gas to be produced. One only needs a minute exposure on a modern home lab source to see this happening. I suggest that everyone avoid cacodylate in their crystallization drops that end up being exposed to X-rays. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, Ruslan [rsanishv...@anl.gov] Sent: Wednesday, November 23, 2011 11:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] dark progression of radiation damage I think I need to clarify couple of things in my recent post about "exploding" crystals during re-mounting by a robot. First, it was a bit
Re: [ccp4bb] [CCP4] identify a rotation centre: domain rotation
Would this work? Take the rot-trans operator from superpose or lsqman and express the rotation matrix as polar coordinates of rotation axis (and angle about it). Get the rotation axis as direction cosines, which will be a vector along the rotation axis of the matrix. Now take the component of the translation vector along that rotation axis, and subtract from the translation vector, to get the component of the translation vector which is orthogonal to rotation axis. Divide that by two and, as a vector from the origin, it defines a point on the rotation axis. Add any multiple of the rotation axis vector to this and it gives another point on the rotation axis. Test these points by operating on them with the orig rot-trans operator, they should just move along the rotation axis by the screw component of the rotation WENHE ZHONG wrote: Dear members, I would like to have your ideas if there is any way to identify a rotation centre of domain in two different states using CCP4 or other program. The situation is: the domain of the protein will rotate between two different states (depending on substrate binding) around 8 degree, and it is (nearly) clearly that the domain is rotated around a rotation centrel. So the question is how to identify this "rotation centre" in this 3D model? The ideal is to identify a region of residues in the domain which are most closed to the rotation centre. The tool I am using right now is the "superpose" tool in CCP4 package. The output which I think mightbe uesful is: CENTROID OF "WORKING" MOLECULE : 157.812 152.396 -70.778 CENTROID OF "WORKING" MOLECULE :(fractional) 157.812 152.396 -70.778 CENTROID OF "REFERENCE" MOLECULE: 157.251 151.877 -70.874 CENTROID OF "REFERENCE" MOLECULE:(fractional) 157.251 151.877 -70.874 Distance between CENTROIDS :0.770 Direction cosines of vector between CENTROIDS: 0.729 0.674 0.124 I would say the “CENTROID" it mentioned above, such as (157.251 151.877 -70.874), is possibly near to the "rotation centre". I would like to have your opinion though. Thank you. King regards, Wenhe
Re: [ccp4bb] dark progression of radiation damage
Regarding "striking distances", there might be some shorter range effects with low energy Auger electrons but for all practical purposes I agree with James. The main reason for this message is to ensure the original question raised by James is not forgotten as it is definitely worth resolving. Blundell and Johnson were presumably confining their remarks to room temperature. The idea of free radicals wandering around a crystal, disturbing atoms and increasing B factors over periods of hours or days does seem unlikely. However, I don't think that is necessarily what Blundell and Johnson were implying by " The chain reaction initiated by fee radical formation". I was about to put down other possibilities when I realised that these are covered in the paper by Warkentin et. al., for example in the section beginning " At temperatures above 200 K, damage arises from processes occurring on many length scales, from diffusion and reaction of radicals to solvent-coupled conformational motions to lattice-scale structural relaxations. These processes must involve an extremely broad range of timescales." The questions are 1. Whether some of the timescales are sufficiently long (at say 290K) to give observable dark progression effects with current data collection techniques (I guess James's original question) 2. Which of the processes (listed in Warkentin et. al. or other processes) is responsible for these timescales. 3. What experimental techniques do we have for studying these processes. 4. Can all this be modelled by in silico simulations (next week's job!) Colin -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of James Holton Sent: 23 November 2011 19:08 To: ccp4bb Subject: Re: [ccp4bb] dark progression of radiation damage Since "striking distance" is about 3 microns for the primary photoelectron and the largest unit cell in the PDB is ~0.1 microns long, I think that means all bets are off when trying to "connect" energy absorbed by a heavy atom to damage somewhere else in the unit cell. -James Holton MAD Scientist On 11/23/2011 10:49 AM, Jacob Keller wrote: > I understand that absorbed dose increases with presence of heavy > atoms, but I don't understand why that should play a role in damaging > the crystal, as heavy atoms such as in cacodylate should probably > usually not be near enough to protein atoms to cause problems. At > 100K, isn't it true that secondary radiation damage plays little role > if any? So the only problem I can think of is the case when the > cacodylate molecule happens to be within "striking distance" of a > protein atom when it turns into a radical (not sure what that distance > would be). This should be relatively rare in, say, 55mM cacodylate, > when there is only ~1 cacodylate for every 1000 waters, no? > > Has there been an empirical study comparing similar crystals of the > same protein +/- solvent heavy atoms? I guess derivatives are the > obvious example--but real derivatives always have ordered, occupied > sites. > > Jacob > > > > On Wed, Nov 23, 2011 at 12:28 PM, Elspeth Garman > wrote: >> Also, cacodylate contains arsenic which is heavy, and thus has a much larger >> X-ray absorption cross section than do buffers constituted of lighter atoms. >> There is therefore a bigger dose (Joules/kg of crystal) absorbed with >> cacodylate in the buffer than there would be without it (and no extra >> diffraction strength), so that is another very good reason to avoid it, or >> to buffer exchange it out before the diffraction experiment. >> >> Elspeth >> >> -Original Message- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> Jim Pflugrath >> Sent: 23 November 2011 18:11 >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] dark progression of radiation damage >> >> Any cacodylate buffer will cause gas to be produced. One only needs a >> minute exposure on a modern home lab source to see this happening. I >> suggest that everyone avoid cacodylate in their crystallization drops that >> end up being exposed to X-rays. >> >> Jim >> >> >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of >> Sanishvili, Ruslan [rsanishv...@anl.gov] >> Sent: Wednesday, November 23, 2011 11:49 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] dark progression of radiation damage >> >> I think I need to clarify couple of things in my recent post about >> "exploding" crystals during re-mounting by a robot. First, it was a bit >> > >
[ccp4bb] Post doctoral position in chromatin structural biology at the University of Toronto
A Post-doctoral position in chromatinstructuralbiology is available at the Structural Genomics Consortium, University of Toronto. The Chromatin Biology and Epigenetics Group at the Structural Genomics Consortium (SGC), University of Toronto, aims to characterize chromatin proteins by X-ray crystallography in combination with other biochemical and biophysical techniques. Please check our lab website for details: http://www.sgc.utoronto.ca/min. This position is available in March 2012 with a highly competitive salary commensurate with experience. Applicants should hold a Ph.D. in molecular biology, biochemistry or structural biology with 0-3 years postdoctoral research experience; prior training in X-ray crystallography is desirable, but not necessary. Interested candidates are invited to send their CV with 3 references to:jr@utoronto.ca. Location: Toronto, ON, Canada Institution: University of Toronto Start Date: March 2012 Contact: Dr. Jinrong Min Email: jr@utoronto.ca
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