[ccp4bb] Different chains in the dimer
Dear All, I have a protein which has the function unit as a dimer. I got two structures of it. One is the native structure, one is the mutant structure. Both structures are dimer in ASU. In the native structure the two chain look the same, but in mutant structure one chain still keep the same structure as the native one, and another chain is different. We expect to see the difference, but we wonder why one chain is different and another one is the same? Is this because of crystallographic packing? Thanks!
[ccp4bb] 2008 Kyoto IUCr Crystallographic Computing School - Sharing our knowledge (fwd)
Forwarded on behalf of Lachlan Cranswick. --dvd -- Forwarded message -- 2008 Kyoto IUCr Crystallographic Computing School - Sharing our knowledge (preliminary announcement) Kyoto Crystallographic Computing School Kansai Seminar House, Kyoto, Japan Monday 18th - Saturday 23rd August 2008 (just prior to the Osaka IUCr 2008 congress) http://www.iucr.org/iucr-top/comm/ccom/kyoto2008/ School Organisers: Prof Anthony Spek (Utrecht), Prof. Min Yao (Sapporo), Dr Ralf Grosse-Kunstleve (Berkeley), Dr Harry Powell (Cambridge), Prof. Atsushi Nakagawa (Osaka), Lachlan Cranswick (Chalk River) --- Introduction: During the first conference on crystallographic computing held at the Pennsylvania State College, USA in 1950, Ray Pepinsky's noted that solving the major computing problems would 'require many minds. Our aim here is to share what we know - to cross-pollinate our minds.' School Aims: To have the crystallographic computing experts of the present, help train and inspire a generation of experts for the future. This will be achieved by the use of an excellent (and full) program of lectures and tutorials. Speakers are listed at: http://www.iucr.org/iucr-top/comm/ccom/kyoto2008/speakers.html --- The Venue The 2008 Crystallographic Computing School will be held at the Kansai Seminar house; at the town of Kyoto. The 2008 Crystallographic Computing School will be held at the Kansai Seminar house in Kyoto. Kyoto is the cultural center of Japan with its long history. The modern city treasures its heritage with its 1600 Buddhist temples, 400 Shinto shrines, and its exquisite gardens. Kyoto is also a center of Japanese tradition. The Kansai Seminar House The Kansai Seminar House was originally derived from movement initiated in Germany by the Christian Churches soon after World War II. The influence reached Japan in the 1960's and the Kansai Seminar House was founded in Shugakuin, Kyoto in 1967. Contributions came from the Christian Academy Movement of Germany, churches affiliated with NCC-USA, and Japan. http://www.iucr.org/iucr-top/comm/ccom/kyoto2008/venue.html --- Costs (and accomodation) We hope, pending completion of sponsorship contributions, that we may be able to keep the entire costs for participants (including accommodation and meals) below the equivalent of 500 Euros. http://www.iucr.org/iucr-top/comm/ccom/kyoto2008/registration.html --- Existing sponsors are listed on the webpage and currently include: IUCr2008 Osaka and International Union of Crystallography (IUCr) http://www.iucr2008.jp/ Cambridge Crystallographic Data Centre: http://www.ccdc.cam.ac.uk/ Hampton Research: http://www.hamptonresearch.com/ Rigaku: http://www.rigaku.com/ --- Promotional Poster: (please feel free to print this out and pin up on your department notice-board) http://www.iucr.org/iucr-top/comm/ccom/kyoto2008/files/kyoto2008.pdf ---
[ccp4bb] extra line of stain in our gel
Hello, When we develop our gel we recently keep getting a horizontal line of stain at 5 mol weight in all lanes of our gel. (This is not a feature of interest except that the protein that we are interested happens to be 5 mol weight). I would appreciate any ideas on how we can get rid of this line. Thanks. Mike
Re: [ccp4bb] extra line of stain in our gel
Hi Mike Sometimes keratin from your skin (fingers, nails, etc) appear in the gels as a band around 48-52 kDa. Take care in the handling of the electrophoresis should be a solution. Other possible reason could be a contamination of the sample loading buffer, also with keratin or with other proteins. Hope it helps Cheers Francisco Hello, When we develop our gel we recently keep getting a horizontal line of stain at 5 mol weight in all lanes of our gel. (This is not a feature of interest except that the protein that we are interested happens to be 5 mol weight). I would appreciate any ideas on how we can get rid of this line. Thanks. Mike - Francisco J. Enguita, Ph.D. Host-pathogen Interactions Group Macromolecular Crystallography Laboratory ITQB EAN, Av. da República 2781-901 Oeiras Portugal Phone : +351-21-4469663 Fax : +351-21-4433644 E-mail : [EMAIL PROTECTED] -
[ccp4bb] NCS-related: Parameter for map/structure quality?
Dear Crystallographers, The recent conversation about NCS got me thinking about something I have been wondering about for a while. Imagine a structure with twenty-fold NCS which diffracts to 2A versus a no-NCS structure of the same resolution--obviously the first model will be better than the other, because there are about twenty times the number of reflections to describe the same underlying structure, all other things (e.g., solvent content) being equal. Is there a indication of this in the statistical measures usually reported in Tables I*, or anywhere else? Regards, Jacob Keller (*there's the old plural of Table I problem) *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
Re: [ccp4bb] an over refined structure
Bottom line: thin shells are not a perfect solution, but if NCS is present, choosing the free set randomly is *never* a better choice, and almost always significantly worse. hmmm ... I wonder if that is true. For low order NCS (two- three- fold, even five-fold) I don't believe that thin shells are better, since they are a systematic omission of data (whcih can affect maps) and in my experience they do not add much. I have only limited experience on this but I somehow tried both and I seem to have settled with random Rfree. With an NCS axis parallel to a crystallographic one (or when translation NCS is there) that might be a whole different ball game though ... not sure. A. Together with multicopy refinement, randomly chosen test sets were almost certainly a major contributor to the spuriously good Rfree values associated with the retracted MsbA and EmrE structures. ehm ... I think 16 models systematically displaced along a direction parallel to helix axes contributed much more to that ... as the authors basically said in the original publication if my recollection is not bad. A.
Re: [ccp4bb] an over refined structure
Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. The same effect applies to a large extent even if the NCS is not crystallographic. Bottom line: thin shells are not a perfect solution, but if NCS is present, choosing the free set randomly is *never* a better choice, and almost always significantly worse. Together with multicopy refinement, randomly chosen test sets were almost certainly a major contributor to the spuriously good Rfree values associated with the retracted MsbA and EmrE structures. Best wishes, Dean Dirk Kostrewa wrote: Dear CCP4ers, I'm not convinced, that thin shells are sufficient: I think, in principle, one should omit thick shells (greater than the diameter of the G-function of the molecule/assembly that is used to describe NCS-interactions in reciprocal space), and use the inner thin layer of these thick shells, because only those should be completely independent of any working set reflections. But this would be too expensive given the low number of observed reflections that one usually has ... However, if you don't apply NCS restraints/constraints, there is no need for any such precautions. Best regards, Dirk. Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: It is important when using NCS that the Rfree reflections be selected is distributed thin resolution shells. That way application of NCS should not mix Rwork and Rfree sets. Normal random selection or Rfree + NCS (especially 4x or higher) will drive Rfree down unfairly. Doug Ohlendorf -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: Tuesday, February 05, 2008 3:38 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/ should not use Rfactors as a criteria for structure correctness. As Ian points out - choosing a different Rfree set of reflections can change Rfree a good deal. certain NCS operators can relate reflections exactly making it hard to get a truly independent Free R set, and there are other reasons to make it a blunt edged tool. The map is the best validator - are there blobs still not fitted? (maybe side chains you have placed wrongly..) Are there many positive or negative peaks in the difference map? How well does the NCS match the 2 molecules? etc etc. Eleanor George M. Sheldrick wrote: Dear Sun, If we take Ian's formula for the ratio of R(free) to R(work) from his paper Acta D56 (2000) 442-450 and make some reasonable approximations, we can reformulate it as: R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) where s is the fractional solvent content, d is the resolution, p is the effective number of parameters refined per atom after allowing for the restraints applied, d^3 means d cubed and sqrt means square root. The difficult number to estimate is p. It would be 4 for an isotropic refinement without any restraints. I guess that p=1.5 might be an appropriate value for a typical protein refinement (giving an R-factor ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R-factor ratio of 0.277/0.215 = 1.29 is well within the allowed range! However it should be added that this formula is almost a self-fulfilling prophesy. If we relax the geometric restraints we increase p, which then leads to a larger 'allowed' R-factor ratio! 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. +49-551-39-2582 *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] *** -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: [EMAIL PROTECTED]
Re: [ccp4bb] an over refined structure
Actually the bottom lines below were my argument in the case that you DO apply strict NCS (although the argument runs into some questionable points if you follow it out). In the case that you DO NOT apply NCS, there is a second decoupling mechanism: Not only the error in Fo may be opposite for the two reflections, but also the change in Fc upon applying a non-symmetrical modification to the structure is likely to be opposite. So there is no way of predicting whether |Fo-Fc| will move in the same direction for the two reflections. I completely agree with Dirk (although I am willing to listen to anyone explain why I am wrong). Ed Edward Berry wrote: Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely to improve the fit at all reflections. For sure R-free will go down when you apply NCS- but this is because you drastically improve your data/parameters ratio. Best, Ed
Re: [ccp4bb] an over refined structure
Here I will disagree. R-free rewards you for putting in atom in density which an atom belongs in. It doesn't necessarily reward you for putting the *right* atom in that density, but it does become difficult to do that under normal circumstances unless you have approximately the right structure. However in the case of multi-copy refinement at low resolution, the refinement is perfectly capable of shoving any old atom in density corresponding to any other old atom if you give it enough leeway. Remember that there's a big difference between R-free for a single copy (45%) and a 16-fold multicopy (38%) in MsbA's P1 form, and almost the same amount (41% vs 33%) with MsbA's P21 form. (These are E.coli and V.cholerae respectively). Both single copy and multicopy refinements were NCS-restrained, as far as I know. So there's evidence, w/o simulation, that the 12-fold or 16-fold multicopy refinements are worth 7-8% in R-free, and I'm doubtful that NCS can generate that sort of gain in either crystal form. I've certainly never seen that in my own experience at low resolution. I've been meaning to put online the Powerpoint from the CCP4 talk with all these numbers in it, but I regret it's sitting on my iBook at home as of writing. Phil Jeffrey Dean Madden wrote: It is true that multicopy refinement was essential for the suppression of Rwork. However, the whole point of the Rfree is that it is supposed to be independent of the number of parameters you're refining. Simply throwing multiple copies of the model into the refinement shouldn't have affected Rfree, IF IT WERE TRULY FREE. It was almost certainly NCS-mediated spillover that allowed the multicopy, parameter-driven reduction in Rwork to pull down the Rfree values as well. The experiment is probably not worth the time it would take to do, but I suspect that if MsbA and EmrE test sets had been chosen in thin shells, then Rfree wouldn't have shown nearly the improvement it did. Dean Phil Jeffrey wrote: While NCS probably played a role in the first crystal form of MsbA (P1, 8 monomers), this is also the one that showed the greatest improvement in R-free once the structure was correctly redetermined (7% or 14% depending on which refinement protocols you compare). The other crystal form of MsbA and the crystal forms of EmrE didn't have particularly high-copy NCS (2 dimers, 4 monomers, dimer, 2 tetramers) and the R-frees were somewhat comparable in all cases (31-36% for the redetermined structures). The *major* source of the R-free suppression in all these cases with the inappropriate use of multi-copy refinement at low resolution. Phil Jeffrey Princeton Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. The same effect applies to a large extent even if the NCS is not crystallographic. Bottom line: thin shells are not a perfect solution, but if NCS is present, choosing the free set randomly is *never* a better choice, and almost always significantly worse. Together with multicopy refinement, randomly chosen test sets were almost certainly a major contributor to the spuriously good Rfree values associated with the retracted MsbA and EmrE structures. Best wishes, Dean Dirk Kostrewa wrote: Dear CCP4ers, I'm not convinced, that thin shells are sufficient: I think, in principle, one should omit thick shells (greater than the diameter of the G-function of the molecule/assembly that is used to describe NCS-interactions in reciprocal space), and use the inner thin layer of these thick shells, because only those should be completely independent of any working set reflections. But this would be too expensive given the low number of observed reflections that one usually has ... However, if you don't apply NCS restraints/constraints, there is no need for any such precautions. Best regards, Dirk. Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: It is important when using NCS that the Rfree reflections be selected is distributed thin resolution shells. That way application of NCS should not mix Rwork and Rfree sets. Normal random selection or Rfree + NCS (especially 4x or higher) will drive Rfree down unfairly. Doug Ohlendorf -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: Tuesday, February 05, 2008 3:38 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/
Re: [ccp4bb] an over refined structure
While NCS probably played a role in the first crystal form of MsbA (P1, 8 monomers), this is also the one that showed the greatest improvement in R-free once the structure was correctly redetermined (7% or 14% depending on which refinement protocols you compare). The other crystal form of MsbA and the crystal forms of EmrE didn't have particularly high-copy NCS (2 dimers, 4 monomers, dimer, 2 tetramers) and the R-frees were somewhat comparable in all cases (31-36% for the redetermined structures). The *major* source of the R-free suppression in all these cases with the inappropriate use of multi-copy refinement at low resolution. Phil Jeffrey Princeton Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. The same effect applies to a large extent even if the NCS is not crystallographic. Bottom line: thin shells are not a perfect solution, but if NCS is present, choosing the free set randomly is *never* a better choice, and almost always significantly worse. Together with multicopy refinement, randomly chosen test sets were almost certainly a major contributor to the spuriously good Rfree values associated with the retracted MsbA and EmrE structures. Best wishes, Dean Dirk Kostrewa wrote: Dear CCP4ers, I'm not convinced, that thin shells are sufficient: I think, in principle, one should omit thick shells (greater than the diameter of the G-function of the molecule/assembly that is used to describe NCS-interactions in reciprocal space), and use the inner thin layer of these thick shells, because only those should be completely independent of any working set reflections. But this would be too expensive given the low number of observed reflections that one usually has ... However, if you don't apply NCS restraints/constraints, there is no need for any such precautions. Best regards, Dirk. Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: It is important when using NCS that the Rfree reflections be selected is distributed thin resolution shells. That way application of NCS should not mix Rwork and Rfree sets. Normal random selection or Rfree + NCS (especially 4x or higher) will drive Rfree down unfairly. Doug Ohlendorf -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: Tuesday, February 05, 2008 3:38 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/ should not use Rfactors as a criteria for structure correctness. As Ian points out - choosing a different Rfree set of reflections can change Rfree a good deal. certain NCS operators can relate reflections exactly making it hard to get a truly independent Free R set, and there are other reasons to make it a blunt edged tool. The map is the best validator - are there blobs still not fitted? (maybe side chains you have placed wrongly..) Are there many positive or negative peaks in the difference map? How well does the NCS match the 2 molecules? etc etc. Eleanor George M. Sheldrick wrote: Dear Sun, If we take Ian's formula for the ratio of R(free) to R(work) from his paper Acta D56 (2000) 442-450 and make some reasonable approximations, we can reformulate it as: R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) where s is the fractional solvent content, d is the resolution, p is the effective number of parameters refined per atom after allowing for the restraints applied, d^3 means d cubed and sqrt means square root. The difficult number to estimate is p. It would be 4 for an isotropic refinement without any restraints. I guess that p=1.5 might be an appropriate value for a typical protein refinement (giving an R-factor ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R-factor ratio of 0.277/0.215 = 1.29 is well within the allowed range! However it should be added that this formula is almost a self-fulfilling prophesy. If we relax the geometric restraints we increase p, which then leads to a larger 'allowed' R-factor ratio! 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. +49-551-39-2582 *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail:
Re: [ccp4bb] an over refined structure
Hi Ed, This is an intriguing argument, but I know (having caught such a case as a reviewer) that even in cases of low NCS symmetry, Rfree can be significantly biased. I think the reason is that the discrepancy between pairs of NCS-related reflections (i.e. Fo-Fo') is generally significantly smaller than |Fo-Fc|. (In general, Rsym (on F) is lower than Rfree.) Thus, moving Fc closer to Fo will also move its NCS partner Fc' closer to Fo' *on average*, if they are coupled. Dean Edward Berry wrote: Actually the bottom lines below were my argument in the case that you DO apply strict NCS (although the argument runs into some questionable points if you follow it out). In the case that you DO NOT apply NCS, there is a second decoupling mechanism: Not only the error in Fo may be opposite for the two reflections, but also the change in Fc upon applying a non-symmetrical modification to the structure is likely to be opposite. So there is no way of predicting whether |Fo-Fc| will move in the same direction for the two reflections. I completely agree with Dirk (although I am willing to listen to anyone explain why I am wrong). Ed Edward Berry wrote: Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely to improve the fit at all reflections. For sure R-free will go down when you apply NCS- but this is because you drastically improve your data/parameters ratio. Best, Ed -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: [EMAIL PROTECTED]
[ccp4bb] One small step for a crystallographer, one giant leap for mankind!
One small step for a crystallographer, one giant leap for mankind! (*) -- For those of you who didn't see it, the following was posted to the PDB mailing list last week: Announcement: Experimental Data Will Be Required for Depositions Starting February 1, 2008 Effective February 1, 2008, structure factor amplitudes/intensities (for crystal structures) and restraints (for NMR structures) will be a mandatory requirement for PDB deposition. http://www.pdb.org/pdb/static.do?p=general_information/news_publications/news/news_2007.html#20071204 Not a decade too soon, either. --dvd (*) See the update at the bottom of this page: http://www.snopes.com/quotes/onesmall.asp for a fine (non-crystallographic) example of an attempt at interpreting ambiguous data to match a predetermined conclusion.
Re: [ccp4bb] an over refined structure
Dear Ed, I don't see how you decouple symmetry mates in the case of a wrong space group. Symmetry mates should agree with each other typically within R_sym or R_merge percent, eg; about 2-5% . Observed and calculated reflections agree within R_Factor of each other, so about 20-30%. The experimental errors are pretty much negligible and overfitting is not a question about error bars; it is about how hard to push a round peg into a square hole? Cheers, Jon Edward Berry wrote: Actually the bottom lines below were my argument in the case that you DO apply strict NCS (although the argument runs into some questionable points if you follow it out). In the case that you DO NOT apply NCS, there is a second decoupling mechanism: Not only the error in Fo may be opposite for the two reflections, but also the change in Fc upon applying a non-symmetrical modification to the structure is likely to be opposite. So there is no way of predicting whether |Fo-Fc| will move in the same direction for the two reflections. I completely agree with Dirk (although I am willing to listen to anyone explain why I am wrong). Ed Edward Berry wrote: Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely to improve the fit at all reflections. For sure R-free will go down when you apply NCS- but this is because you drastically improve your data/parameters ratio. Best, Ed
Re: [ccp4bb] an over refined structure
Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely to improve the fit at all reflections. For sure R-free will go down when you apply NCS- but this is because you drastically improve your data/parameters ratio. Best, Ed
Re: [ccp4bb] Announcement: two crystallographic wikis
Dear Kay, That looks to be an excellent start. I should like to add SHELXL as well as SHELXC/D/E, there are regularly questions about it in CCP4bb. I'm not quite clear about how to generate enough material to make it useful, obviously you don't want to simply copy the SHELX manual, and similar comments would apply to other programs. To make it more complicated, crystallographic programs are moving targets (though some move faster than others). Perhaps the best way would be to go through the CCP4bb archive and try to extract a useful consensus, though even that would be a lot of work. 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. +49-551-39-2582 On Thu, 7 Feb 2008, Kay Diederichs wrote: Dear all, I want to bring two crystallographic wikis to your attention, asking for your contribution: 1) CCP4 user community wiki: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page This wiki was planned by Tassos and me, and Artem and Clemens joined us lately. Unfortunately this has almost no contents so far, but we hope that people who post answers on the CCP4 mailing list will take the time and compose/edit the corresponding articles of the wiki. The intention is that the wiki will capture the breadth of topics on CCP4BB, which will make it a useful resource e.g. for frequently asked questions, offloading some of the question/answer traffic on CCP4BB to a more permanent mode of storage. 2) XDSwiki: http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDS for XDS users. This has quite a bit of information already. The purpose of this wiki is to provide an accurate resource for XDS users, and to provide explanations and hints for the successful use of XDS. It has Tips and Tricks for XDS usage, explanations of its output, and recommendations for the parameters in XDS.INP (if it appears useful to deviate from their defaults). Hoping that these wikis will be useful, and that many people contribute! Kay Diederichs -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz
[ccp4bb] Announcement: two crystallographic wikis
Dear all, I want to bring two crystallographic wikis to your attention, asking for your contribution: 1) CCP4 user community wiki: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page This wiki was planned by Tassos and me, and Artem and Clemens joined us lately. Unfortunately this has almost no contents so far, but we hope that people who post answers on the CCP4 mailing list will take the time and compose/edit the corresponding articles of the wiki. The intention is that the wiki will capture the breadth of topics on CCP4BB, which will make it a useful resource e.g. for frequently asked questions, offloading some of the question/answer traffic on CCP4BB to a more permanent mode of storage. 2) XDSwiki: http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDS for XDS users. This has quite a bit of information already. The purpose of this wiki is to provide an accurate resource for XDS users, and to provide explanations and hints for the successful use of XDS. It has Tips and Tricks for XDS usage, explanations of its output, and recommendations for the parameters in XDS.INP (if it appears useful to deviate from their defaults). Hoping that these wikis will be useful, and that many people contribute! Kay Diederichs -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] an over refined structure
Agreed, and this is even more true if you consider R-merge is calculated on I's and Rfree on F's, Rmerge of 5% should contribute 2.5% to Rfree; and furthermore errors add vectorially so it would be more like ,025/sqrt(2). I guess I have to take all those other errors that have to do with the inability of a simple atomic model to account for the diffraction of a crystal, lump them together and assume they have nothing to do with NCS and are not affected by the simple modification under consideration. I am thinking about the CHANGE in |Fo-Fc| at two sym-related reflections when the refinement program moves a single atom from position 1 to position 2. If we do not apply NCS, this is the only atom that will move, and for Fc we can definitely say there is no reason to expect the two Fc's to move in the same direction, therefore there is no coupling in the case we do not apply NCS. If we apply strict NCS then granted the sym related Fc's are equal before and after the change, so they move in the same direction. As I said, the argument is weaker now. If there are systematic errors contributing to the gap between Rfree and 0.5*Rmerge/sqrt(2), and if these systematic errors follow the NCS, then initial Fo-Fc is likely to be of the same sign at the related reflections and larger than the change in Fc, so |Fo-Fc| would go in the same direction. But to justify this you would have to explin why the systematic errors follow ncs. Crystal morphology related to ncs resulting in similar absorption errors? But how large are absorbtion errors, and is there any reason for morphology to follow NCS? After reading Dean Madden's latest- We might need some assumption here that we are reasonably close to the refined structure. If we start with random atoms then shoving the atoms around in a way that fits the density better might be seen as improving the structure from the point of modeling the density, but not from the point of approximating the real structure. But in this case the change in sign of Fc is completely decoupled between sym-related reflections, and if you enforce symmetry you will be enforcing the wrong symmetry and worsening both the structure and the fit to the density. I think Gerard Kleywegt has an example of enforcing NCS on a an erroneous structure, and it was not very effective at reducing Rfree? And in that case the structure may have had some resemblence to the density at low resolution,the NCS may have been somewhat correct. I guess there are two questions depending whether you are at the beginning at the beginning of a refinement and may have a completely wrong structure, or whether refinement isnearly complete and you want to know whether the further improvement you get on applg NCS is real. Jon Wright wrote: Dear Ed, I don't see how you decouple symmetry mates in the case of a wrong space group. Symmetry mates should agree with each other typically within R_sym or R_merge percent, eg; about 2-5% . Observed and calculated reflections agree within R_Factor of each other, so about 20-30%. The experimental errors are pretty much negligible and overfitting is not a question about error bars; it is about how hard to push a round peg into a square hole? Cheers, Jon Edward Berry wrote: Actually the bottom lines below were my argument in the case that you DO apply strict NCS (although the argument runs into some questionable points if you follow it out). In the case that you DO NOT apply NCS, there is a second decoupling mechanism: Not only the error in Fo may be opposite for the two reflections, but also the change in Fc upon applying a non-symmetrical modification to the structure is likely to be opposite. So there is no way of predicting whether |Fo-Fc| will move in the same direction for the two reflections. I completely agree with Dirk (although I am willing to listen to anyone explain why I am wrong). Ed Edward Berry wrote: Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely
Re: [ccp4bb] an over refined structure
If you think about it, there is an analogy to relaxing geometrical constraints, which also allows the refinement to put atoms into density. The reason it usually doesn't help Rfree is that the density is spurious. At least some of the incorrect structure determinations of the early 90's (that spurred the introduction of Rfree etc.) had high rms deviations, suggesting that this is how the overfitting occurred. Nevertheless, once hit with a bit of simulated annealing, the Rfree values of such models deteriorated significantly. If memory serves, the incorrect structures of the 1990's would have had relaxed geometry precisely because they needed to do that to reduce R, and R used to be the primary indicator of structure quality in the days before R-free was introduced. There's quite a big difference between the latitude afforded by relaxing geometry and the degree of freedom allowed by multicopy refinement. Simply increasing the RMS bond length deviations from 0.012 to 0.035 Angstrom would move atoms on average by only a fraction of a bond length, which is not really enough to jump between different atom locations. In any event, the MsbA statistics can be simply explained from an expectation of what happens if you overfit your (wrong) structure using techniques inappropriate for the resolution: R-work goes down R-free goes down less (R-free - R-work) goes up and this happens in general with use of multicopy refinement at anything less than quite high resolution - I'm thinking in particular of a comment in Chen Chapman (2001) Biophys J vol. 8, 1466-1472. So I see no reason to suggest NCS is having a particularly extreme, perhaps unprecedented, effect. Phil Jeffrey (still working on converting Micro$loth Powerpoint to html)
Re: [ccp4bb] an over refined structure
Dear CCP4ers, I'm not convinced, that thin shells are sufficient: I think, in principle, one should omit thick shells (greater than the diameter of the G-function of the molecule/assembly that is used to describe NCS- interactions in reciprocal space), and use the inner thin layer of these thick shells, because only those should be completely independent of any working set reflections. But this would be too expensive given the low number of observed reflections that one usually has ... However, if you don't apply NCS restraints/constraints, there is no need for any such precautions. Best regards, Dirk. Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: It is important when using NCS that the Rfree reflections be selected is distributed thin resolution shells. That way application of NCS should not mix Rwork and Rfree sets. Normal random selection or Rfree + NCS (especially 4x or higher) will drive Rfree down unfairly. Doug Ohlendorf -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: Tuesday, February 05, 2008 3:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/ should not use Rfactors as a criteria for structure correctness. As Ian points out - choosing a different Rfree set of reflections can change Rfree a good deal. certain NCS operators can relate reflections exactly making it hard to get a truly independent Free R set, and there are other reasons to make it a blunt edged tool. The map is the best validator - are there blobs still not fitted? (maybe side chains you have placed wrongly..) Are there many positive or negative peaks in the difference map? How well does the NCS match the 2 molecules? etc etc. Eleanor George M. Sheldrick wrote: Dear Sun, If we take Ian's formula for the ratio of R(free) to R(work) from his paper Acta D56 (2000) 442-450 and make some reasonable approximations, we can reformulate it as: R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) where s is the fractional solvent content, d is the resolution, p is the effective number of parameters refined per atom after allowing for the restraints applied, d^3 means d cubed and sqrt means square root. The difficult number to estimate is p. It would be 4 for an isotropic refinement without any restraints. I guess that p=1.5 might be an appropriate value for a typical protein refinement (giving an R- factor ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R-factor ratio of 0.277/0.215 = 1.29 is well within the allowed range! However it should be added that this formula is almost a self-fulfilling prophesy. If we relax the geometric restraints we increase p, which then leads to a larger 'allowed' R-factor ratio! 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. +49-551-39-2582 *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: [EMAIL PROTECTED] ***
Re: [ccp4bb] extra line of stain in our gel
When we develop our gel we recently keep getting a horizontal line of stain at 5 mol weight in all lanes of our gel. (This is not a feature of interest except that the protein that we are interested happens to be 5 mol weight). I would appreciate any ideas on how we can get rid of this line. Are these somewhat fuzzy bands? If yes then it's [human] keratins. Usually it is either SDS or bME stocks that get contaminated with keratins. Usually, just changing stocks and working cleanly solves the issue but occasionally bME as bought already contains them. Ochs, D. (1983) Protein contaminants of sodium dodecyl sulfatepolyacrylamide gels. Anal. Biochem. 135, 470–474. Dima
Re: [ccp4bb] an over refined structure
Hi Phil, Here I will disagree. R-free rewards you for putting in atom in density which an atom belongs in. It doesn't necessarily reward you for putting the *right* atom in that density, but it does become difficult to do that under normal circumstances unless you have approximately the right structure. However in the case of multi-copy refinement at low resolution, the refinement is perfectly capable of shoving any old atom in density corresponding to any other old atom if you give it enough leeway. ... So there's evidence, w/o simulation, that the 12-fold or 16-fold multicopy refinements are worth 7-8% in R-free, and I'm doubtful that NCS can generate that sort of gain in either crystal form. I've certainly never seen that in my own experience at low resolution. Remember that there are two things at work here: putting atoms into real density (which does reduce Rfree) and putting atoms into noise (overfitting, which shouldn't help Rfree). At low res, there's a lot of noise. If you think about it, there is an analogy to relaxing geometrical constraints, which also allows the refinement to put atoms into density. The reason it usually doesn't help Rfree is that the density is spurious. At least some of the incorrect structure determinations of the early 90's (that spurred the introduction of Rfree etc.) had high rms deviations, suggesting that this is how the overfitting occurred. Nevertheless, once hit with a bit of simulated annealing, the Rfree values of such models deteriorated significantly. I would argue that 12-fold or 16-fold multicopy refinements simply permitted overfitting of noise. In other words, it is worth 7-8% in R*work*, but not Rfree. In this case, the main reason Rfree also dropped is because the test set was coupled *by NCS* to the overfit working set. Use of a random test set in the presence of NCS could easily prevent the Rfree value from serving as a warning of overfitting. Of course, to be absolutely sure, one would have to repeat the multicopy refinements of the inverted structures with a test set chosen in thin shells, and then see if Rfree dropped as before. I think only the original authors would be in a position to do that properly. Dean -- Dean R. Madden, Ph.D. Department of Biochemistry Dartmouth Medical School 7200 Vail Building Hanover, NH 03755-3844 USA tel: +1 (603) 650-1164 fax: +1 (603) 650-1128 e-mail: [EMAIL PROTECTED]
[ccp4bb] Tag, you're in! Flag, V5, etc
Hello, I'd appreciate it if anyone could provide information (experiences or publications) on the following: 1. Put a tag such as FLAG, V5, etc etc, at the N- and/or C-termini, in order for specific detection, but not interfering with protein folding/structure; 2. Is a linker between the tag and target protein needed? What linkers (length, specific sequences) would you suggest? 3. What are the choices of such tags and why would you recommend them? Thank you for your time and sharing in advance. Yongfu Li
[ccp4bb] Solved: PDB file column-cut-paste issues
Thanks everyone for all your suggestions. What's the issue? Each residue has one CSV value (per residue value) and the PDB line contains many lines of B-values per residue. This would leave us with no easy one-to-one line correlation between the B-value column and the CSV column. From what I understand, that would rule out a simple cut-and- paste in nedit solution. Basically, the student wanted to colour the molecule by chemical shift. It took a couple of PhDs in the lab to figure out the solution. The SOLUTION: Many folks have apparently had the same issue before and therefore, someone (aka Warren Delano, I guess), wrote a script for precisely the same and posted an example script on the PymolWiki. All that was needed was a more extensive Google search :) Your suggestions and tips will be diligently used elsewhere. Cheers, Raji
[ccp4bb] PDB file column-cut-paste issues
Hi People, I post this on behalf of my colleague. My colleague has a file containing chemical-shift values for the 150 aa in his structure. He also has the PDB file for the crystal structure. Now, he would like to replace the B-factor column with the CS values to make some figures. It would be easy to yank out the column from the PDB file and paste the column containing the CS values. However, there is only one value per residue. I don't want to ask him to use Moleman to reset B-factor column per residue with the CS value and do this 150 times!! Also, the number of lines per amino-acid type is different!!! How do we do this in a less than manual way? Thanks. Raji
Re: [ccp4bb] an over refined structure
Dean Madden wrote: Hi Ed, This is an intriguing argument, but I know (having caught such a case as a reviewer) that even in cases of low NCS symmetry, Rfree can be significantly biased. I think the reason is that the discrepancy between pairs of NCS-related reflections (i.e. Fo-Fo') is generally significantly smaller than |Fo-Fc|. (In general, Rsym (on F) is lower than Rfree.) Thus, moving Fc closer to Fo will also move its NCS partner Fc' closer to Fo' *on average*, if they are coupled. OK, I see that now, the systematic errors must be related to NCS in this case because we know if we reduced the data in the higher space group, our Rsyms would be OK. I stand educated. But it is difficult to go from there to real ncs where the large unaccounted errors may not be related to ncs. Furthermore if you don't enforce NCS the structural changes are asymmetric and there is no reason to believe Fc will move in the same direction, even in this artificial case. So Dirk's assertion still stands, I believe. Dean Edward Berry wrote: Actually the bottom lines below were my argument in the case that you DO apply strict NCS (although the argument runs into some questionable points if you follow it out). In the case that you DO NOT apply NCS, there is a second decoupling mechanism: Not only the error in Fo may be opposite for the two reflections, but also the change in Fc upon applying a non-symmetrical modification to the structure is likely to be opposite. So there is no way of predicting whether |Fo-Fc| will move in the same direction for the two reflections. I completely agree with Dirk (although I am willing to listen to anyone explain why I am wrong). Ed Edward Berry wrote: Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. I don't think this is right- remember Rfree is not just based on Fc but Fo-Fc. Working in your lower symmetry space group you will have separate values for the Fo at the two ncs-related reflections. Each observation will have its own random error, and like as not the error will be in the opposite direction for the two reflections. Hence a structural modification that improves Fo-Fc at one reflection is equally likely to improve or worsen the fit at the related reflection. The only way they are coupled is through the basic tenet of R-free: If it makes the structure better, it is likely to improve the fit at all reflections. For sure R-free will go down when you apply NCS- but this is because you drastically improve your data/parameters ratio. Best, Ed
Re: [ccp4bb] Tag, you're in! Flag, V5, etc
If circumstances require a C-terminal tag, the intein system from New England Biolabs has worked very well for us. The pTYB1 plasmid encodes a fusion between your protein of interest, a viral intein (think protein intron) and a chitin binding domain. The fusion adsorbs to a chitin resin and all other proteins are washed away. The intein is engineered to not catalyze protein ligation but rather to release the protein in a thiol-directed manner. To cleave your protein from the fusion, simply add DTT, plug the column, and incubate overnight. The next day, push your unbound protein off the column: native protein with no tag residues left! The CBD-intein remain bound to the resin You don't get a lot of protein (5-10mg is more common than 50mg) and it is diluted to the volume of your column. On the other hand, it is pure enough to go right into trials upon concentration. At least that's been our experience. Andy (No affiliation with NEB) On 2/7/08 4:33 PM, James Whisstock [EMAIL PROTECTED] wrote: Hiya We usually add all N-terminal tags with a TeV cleavable linker. C-terminal tags always seem a pain to remove cleanly, because most highly specific recognition sequences (such as TeV) take advantage of P rather than P' specificity so you are usually left with a five (or so) residue tail. Actually has anyone any neat tricks for C-terminal tag removal? J -- Andrew M. Gulick, Ph.D. --- (716) 898-8619 Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203 --- Senior Research Scientist Hauptman-Woodward Institute Assistant Professor Dept. of Structural Biology, SUNY at Buffalo http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html http://labs.hwi.buffalo.edu/gulick
Re: [ccp4bb] PDB file column-cut-paste issues
Hi Raji, I think phenix.pdbtools can do it (if I correctly understood what you want to do): http://www.phenix-online.org/documentation/pdbtools.htm Example: phenix.pdbtools model.pdb set_b_iso=25.3 selection=chain A and resname ALA and name CA this will set all B=25 for all CA atoms in all ALA residues of chain A. Cheers, Pavel. Raji Edayathumangalam wrote: Hi People, I post this on behalf of my colleague. My colleague has a file containing chemical-shift values for the 150 aa in his structure. He also has the PDB file for the crystal structure. Now, he would like to replace the B-factor column with the CS values to make some figures. It would be easy to yank out the column from the PDB file and paste the column containing the CS values. However, there is only one value per residue. I don't want to ask him to use Moleman to reset B-factor column per residue with the CS value and do this 150 times!! Also, the number of lines per amino-acid type is different!!! How do we do this in a less than manual way? Thanks. Raji
Re: [ccp4bb] an over refined structure
A few comments that you might find useful: 1. yes, even if you don't apply NCS restraints/constraints there will be correlations between reflections in cases of NCS symmetry or pseudo-crystallographic NCS symmetry. 2. Fabiola, Chapman, et al., published a very nice paper on the topic in Acta D. 62, 227-238, 2006. 3. From my experience, the effects for low NCS symmetry are usually small, except cases of pseudo-symmetry which can be easily addressed by defining the test set in the high-symmetry setting. For high NCS symmetry, the effects are more significant, but then the structure is usually much better determined, anyway, due to averaging. 4. At least the first one of the mentioned MsbA and EmrE structures had a very high Rfree in the absence of multi-copy refinement ( ~ 45%)! So, the Rfree indicated that there was a major problem. 5. The Rfree should vary relatively little among test sets (see my Acta D 49, 24-36, 1993 paper) - if there are large variations for different test set choices then the test set may be too small or there may be systematic problems with some of the reflections causing them to dominate the R factors (outliers at low resolution, for example). Axel Brunger On Feb 7, 2008, at 9:57 AM, Dean Madden wrote: Hi Dirk, I disagree with your final sentence. Even if you don't apply NCS restraints/constraints during refinement, there is a serious risk of NCS contaminating your Rfree. Consider the limiting case in which the NCS is produced simply by working in an artificially low symmetry space-group (e.g. P1, when the true symmetry is P2): in this case, putting one symmetry mate in the Rfree set, and one in the Rwork set will guarantee that Rfree tracks Rwork. The same effect applies to a large extent even if the NCS is not crystallographic. Bottom line: thin shells are not a perfect solution, but if NCS is present, choosing the free set randomly is *never* a better choice, and almost always significantly worse. Together with multicopy refinement, randomly chosen test sets were almost certainly a major contributor to the spuriously good Rfree values associated with the retracted MsbA and EmrE structures. Best wishes, Dean Dirk Kostrewa wrote: Dear CCP4ers, I'm not convinced, that thin shells are sufficient: I think, in principle, one should omit thick shells (greater than the diameter of the G-function of the molecule/assembly that is used to describe NCS-interactions in reciprocal space), and use the inner thin layer of these thick shells, because only those should be completely independent of any working set reflections. But this would be too expensive given the low number of observed reflections that one usually has ... However, if you don't apply NCS restraints/constraints, there is no need for any such precautions. Best regards, Dirk. Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf: It is important when using NCS that the Rfree reflections be selected is distributed thin resolution shells. That way application of NCS should not mix Rwork and Rfree sets. Normal random selection or Rfree + NCS (especially 4x or higher) will drive Rfree down unfairly. Doug Ohlendorf -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: Tuesday, February 05, 2008 3:38 AM To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/ should not use Rfactors as a criteria for structure correctness. As Ian points out - choosing a different Rfree set of reflections can change Rfree a good deal. certain NCS operators can relate reflections exactly making it hard to get a truly independent Free R set, and there are other reasons to make it a blunt edged tool. The map is the best validator - are there blobs still not fitted? (maybe side chains you have placed wrongly..) Are there many positive or negative peaks in the difference map? How well does the NCS match the 2 molecules? etc etc. Eleanor George M. Sheldrick wrote: Dear Sun, If we take Ian's formula for the ratio of R(free) to R(work) from his paper Acta D56 (2000) 442-450 and make some reasonable approximations, we can reformulate it as: R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) where s is the fractional solvent content, d is the resolution, p is the effective number of parameters refined per atom after allowing for the restraints applied, d^3 means d cubed and sqrt means square root. The difficult number to estimate is p. It would be 4 for an isotropic refinement without any restraints. I guess that p=1.5 might be an appropriate value for a typical protein refinement (giving an R-factor ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R- factor ratio of 0.277/0.215 = 1.29 is well within
Re: [ccp4bb] Solved: PDB file column-cut-paste issues
Raji Edayathumangalam wrote: Thanks everyone for all your suggestions. What's the issue? Each residue has one CSV value (per residue value) and the PDB line contains many lines of B-values per residue. This would leave us with no easy one-to-one line correlation between the B-value column and the CSV column. From what I understand, that would rule out a simple cut-and- paste in nedit solution. Basically, the student wanted to colour the molecule by chemical shift. The SOLUTION: Many folks have apparently had the same issue before and therefore, someone (aka Warren Delano, I guess), wrote a script for precisely the same and posted an example script on the PymolWiki. Oh dear - too late :-(. You can do it in Coot too! (The solution is on the Coot Wiki now) http://xanana.ucsc.edu/~wgscott/xtal/wiki/index.php/Coot#Example_Scheme_Script_7:_Applying_arbitrary_value_to_.22B.22_factor_column Paul.
Re: [ccp4bb] Solved: PDB file column-cut-paste issues
Ooh..la la! Where were you 12 hrs ago when we were suffering brain damage! Cheers! Raji PS: Thanks! Oh dear - too late :-(. You can do it in Coot too! (The solution is on the Coot Wiki now) http://xanana.ucsc.edu/~wgscott/xtal/wiki/index.php/Coot#Example_Scheme_Script_7:_Applying_arbitrary_value_to_.22B.22_factor_column Paul. -End of Included Message--
Re: [ccp4bb] Tag, you're in! Flag, V5, etc
At least that's been our experience. Andy (No affiliation with NEB) Hi, everyone: Just to give both sides to this story, though, we've had phenomenally bad luck with the NEB Intein system (tagged on either the C- or N- terminus). The protein expressed and purified beautifully, and post-cleavage was pretty much a single band on a silver-stained gel. However, it was intractably misfolded, while purification with other, more 'traditional' tags (His6 or - dare I say it - *untagged* purification!) yielded fully soluble, folded protein. A few other labs around MIT have had similar experiences. So, for any technique there will be circumstantial data affirming or refuting its utility; take any success or failure story with the required ug of NaCl. =) Good luck purifying, Dave
