Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
Dear Ian, what I see are two minima, symmetrically aranged around the special position. One minimum is closer the molecule A, the other closer to the symmetry-related molecule B and waters will randomly be either in minimum A, or in minimum B. Crystal packing prevents the water from moving the protein molecules such that it can make perfect hydrogen bonds with both protein at the same time. Cheers, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ian Tickle Sent: Thursday, December 09, 2010 6:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms The forces acting on an atom at a special position must have the same symmetry on average as the special position, so the atom will be in equilibrium, and there will at first sight be no net force (i.e. zero energy gradient) to push the atom away from the special position. However there are 2 solutions to this: metastable equilibrium where the energy is a maximum, and stable equilibrium where it's a minimum. In the metastable case it will be like a pencil balanced on its point where a small disturbance, such as from random thermal motion, will be sufficient to move it off the special position to an asymmetric position of lower energy thus breaking the symmetry, and of course the initial displacement will be completely random leading to the disorder you observe. In the case of stable equilibrium the atom will simply respond to the disturbance by executing random thermal motion centred on the special position. Which case you see in practice will obviously depend on the precise arrangement of forces acting on the atom. Cheers -- Ian On Thu, Dec 9, 2010 at 4:12 PM, herman.schreu...@sanofi-aventis.com wrote: Even with the famous waters on true Wyckoff positions, I usually observe an elongated or even partly split density, suggesting that the water is disordered, being sometimes closer to one monomer, sometimes closer to the symmetry-related monomer. Since the position of proteins in a crystal is in general not determined by a single water-mediated hydrogen bond, the water will in general not be able to make perfect hydrogen bonds to both symmetry-related monomers at the same time. I think therefore that even waters should generally be considered to be disordered and only in exceptional cases will occupy true Wyckoff positions. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ian Tickle Sent: Thursday, December 09, 2010 3:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms cases out there (and so far I have heard of a disulfide bond on a 2-fold connecting two homodimers). I'm slightly puzzled by this example. If the S-S bond is on the special position, then the rest of the molecule can't have 2-fold symmetry, so would have to be rotationally disordered with occupancy = 0.5 to avoid clashing with its symmetry mate: * X -- C * \ * S | S * \ * C -- X * where the *'s indicate the 2-fold axis (i.e. vertically in the plane of the page). In this case, for the reasons I gave in my previous post there's no reason for the disordered S atoms to be exactly on the 2-fold; it would be pure coincidence if they were. If you mean instead that the 2-fold is _perpendicular_ to the S-S bond (i.e. coming straight out of the page in the diagram), the molecule does indeed have 2-fold symmetry and can be ordered with occupancy = 1, but then the S atoms are not on special positions, so this would not be an example of protein atoms _on_ a special position. One could imagine an example, say where the same side-chain on each monomer is cross-linked (e.g. LYS with glutaraldehyde), forming the homodimer: X -- C -- N = C -- C -- C -- C -- C = N -- C -- X Here the central C atom could be on a 2-fold (i.e. axis perpendicular to the page) special position without rotational disorder. I've no idea whether such a structure actually exists! Cheers -- Ian
[ccp4bb] Structure containing nickel ions from Ni-NTA column
Dear, I was wondering if anybody has experienced before the leakage of Ni- ions (from a Ni-NTA column) and additionally binding to specific sites in the protein structure..? Many thanks Regards Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 --
[ccp4bb] Space group vs. gap between Rwork and Rfree?
Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] Structure containing nickel ions from Ni-NTA column
Hi Kristof, depending on what you termed specific sites the answer is definitely yes. I experienced such a case in the following reference related to the AcrB structure but I remember that this situation arose also in the case of the LTC4 structure. -There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization. Veesler D, Blangy S, Cambillau C, Sciara G. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Oct 1;64(Pt 10): 880-5. -Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase. Martinez Molina D, Wetterholm A, Kohl A, McCarthy AA, Niegowski D, Ohlson E, Hammarberg T, Eshaghi S, Haeggström JZ, Nordlund P. Nature. 2007 Aug 2;448(7153):613-6. All the best David Le 10 déc. 10 à 12:17, Kristof Van Hecke a écrit : Dear, I was wondering if anybody has experienced before the leakage of Ni- ions (from a Ni-NTA column) and additionally binding to specific sites in the protein structure..? Many thanks Regards Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Can you exclude space groups with screw axes (I4132, I213)? It's worth trying molecular replacement in all possible space groups to check, or try the York Zanuda server (http://www.ysbl.york.ac.uk/YSBLPrograms/index.jsp) Phil On 10 Dec 2010, at 11:17, Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] A script is needed to renumber image
Quickie 1-liners are also possible in Perl, and it's a lot more
flexible than awk to boot:
perl -e 'for(*) {rename $_,$1.($2-720).$3 if /(.+?)(\d+)(.img)$/}'
This (as well as I suspect some of the shell scripts posted) would
fail if you had asked to rename to the range 001..360 since the
leading zeroes will not appear. This can be fixed by a bit of
defensive programming: replace '($2-720)' with
'sprintf(%03d,$2-720)'.
-- Ian
On Fri, Dec 10, 2010 at 1:51 AM, wu donghui wdh0...@gmail.com wrote:
Dear all,
I need a script to renumber my image. My initial image number ranges from
*_1081.img to *_1440.img. There are 360 images in total. I want to renumber
these images with the ranges from *_361.img to *_720.img, that means every
initial image-720, but I don't know how to do it. Below is my script draft.
#! /bin/csh -f
@ i = 1081
while ( $i = 1081 )
while ($i = 1440 )
echo mv CD267A_3_pk_1_$i.img CD267A_3_pk_1_$i-720.img
@ i++
end
exit
Can anyone help me for this script? Thank you very much.
Best regards,
Donghui
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Hi Petr, Usually IDXREF suggests more than one Bravais lattice that is consistent with your diffraction images; hence it is (sometimes) worthwhile trying to INTEGRATE in all possible Bravais lattices and this allows you to eliminate a number of possibilities (poor profiles during integration, instability of refinement, poor Rmerge values at the level of CORRECT). This leaves you with a smaller number of choices for the Bravais lattive. And I personally (at the end) integrate in the correct Bravais lattice instead of integrating in P1 and then doing the space group assignment at the level of CORRECT. I get improved Rmerge values then. I've had a case where one of the students here was wrongly advising my student about an orthorhombic space group, and my advice to carry out integration in all Bravais lattices suggested by IDXREF, integration led in orthorhombic gave a molecular replacement solution that did not refine properly and a molecular replacement solution that refined in a hexagonal space group. The Rmerge values (at the level of CORRECT) vere very similar both for P222 and for P6 integration. HTH, Fred. Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Some refinement of my question: I am working on mutant variant (point mutation, not on the interface) of one protein already deposited in space group I432. Screw axes are excluded, NCS operator is equivalent to missing symmetry operation. To Fred, I always process data in P1 first. After that and all analysis, I completely process the data again in suggested space groups in different directories to avoid any mixture of the files. I do not know, maybe the problem is only in refinement, but I can not make the gap between Rfree and Rwork somehow reasonably stable. Many thanks to all of you for your suggestions and interest. Petr 2010/12/10 Vellieux Frederic frederic.velli...@ibs.fr: Hi Petr, Usually IDXREF suggests more than one Bravais lattice that is consistent with your diffraction images; hence it is (sometimes) worthwhile trying to INTEGRATE in all possible Bravais lattices and this allows you to eliminate a number of possibilities (poor profiles during integration, instability of refinement, poor Rmerge values at the level of CORRECT). This leaves you with a smaller number of choices for the Bravais lattive. And I personally (at the end) integrate in the correct Bravais lattice instead of integrating in P1 and then doing the space group assignment at the level of CORRECT. I get improved Rmerge values then. I've had a case where one of the students here was wrongly advising my student about an orthorhombic space group, and my advice to carry out integration in all Bravais lattices suggested by IDXREF, integration led in orthorhombic gave a molecular replacement solution that did not refine properly and a molecular replacement solution that refined in a hexagonal space group. The Rmerge values (at the level of CORRECT) vere very similar both for P222 and for P6 integration. HTH, Fred. Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
Does one regard the metal atom in a metalloprotein as being part of the protein? If so, a shared metal could occupy a special position in a dimer for example. In Acta Cryst. (2008). D64, 257-263 Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination I. Dokmanic, M. Sikic and S. Tomic ( http://scripts.iucr.org/cgi-bin/paper?S090744490706595X ) it says there are 25 cases of metal atoms in special positions. Also Acta Cryst. (2002). D58, 29-38 The 2.6 Å resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic `special position' D. Cobessi, L.-S. Huang, M. Ban, N. G. Pon, F. Daldal and E. A. Berry ( http://scripts.iucr.org/cgi-bin/paper?S0907444901017267 ) though the 'special position' is justifiably in quotation marks in this example as disorder is present. Colin
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Dear Petr, An Rfree of 33% does happen, especially at low resolution. However my experience is that with such high Rfrees there usually is some problem. The problem might be disorder in the protein or the crystal. In this case there is not much you can do. However, before giving up, I would at least check the following (if you have not already done it): 1) run phaser with sgalternative all. Although the celldimensions are the same, the spacegroup maybe different and even with the same space group and cell dimensions, the packing may be different. I am currently working on a project were different crystals, grown from the same protein and with the same cell dimensions and space group, have a different packing. In one case, the molecules are 90° rotated with the respect to the reference, in other cases the molecules are tilted by 20-30°. The latter structure (in the wrong orientation) could be refined to an Rfree of ~35%! 2) check for twinning. Your 4-fold symmetry might be broken to become 2-fold, and it might be in a different direction in different parts of the crystal. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Petr Kolenko Sent: Friday, December 10, 2010 1:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Space group vs. gap between Rwork and Rfree? Some refinement of my question: I am working on mutant variant (point mutation, not on the interface) of one protein already deposited in space group I432. Screw axes are excluded, NCS operator is equivalent to missing symmetry operation. To Fred, I always process data in P1 first. After that and all analysis, I completely process the data again in suggested space groups in different directories to avoid any mixture of the files. I do not know, maybe the problem is only in refinement, but I can not make the gap between Rfree and Rwork somehow reasonably stable. Many thanks to all of you for your suggestions and interest. Petr 2010/12/10 Vellieux Frederic frederic.velli...@ibs.fr: Hi Petr, Usually IDXREF suggests more than one Bravais lattice that is consistent with your diffraction images; hence it is (sometimes) worthwhile trying to INTEGRATE in all possible Bravais lattices and this allows you to eliminate a number of possibilities (poor profiles during integration, instability of refinement, poor Rmerge values at the level of CORRECT). This leaves you with a smaller number of choices for the Bravais lattive. And I personally (at the end) integrate in the correct Bravais lattice instead of integrating in P1 and then doing the space group assignment at the level of CORRECT. I get improved Rmerge values then. I've had a case where one of the students here was wrongly advising my student about an orthorhombic space group, and my advice to carry out integration in all Bravais lattices suggested by IDXREF, integration led in orthorhombic gave a molecular replacement solution that did not refine properly and a molecular replacement solution that refined in a hexagonal space group. The Rmerge values (at the level of CORRECT) vere very similar both for P222 and for P6 integration. HTH, Fred. Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] brute force MR
Arnon, We have developed an MR search mechanism which may be helpful in this scenario. It is web accessible and available to any public or academic researchers: https://portal.nebiogrid.org/secure/apps/wsmr/ It can use up to the full set of SCOP domains (100k) to attempt a Phaser MR placement of each domain and then ranks the results, allowing you to identify a single well placed domain. The web-based system does not allow you to fix that domain to continue the search for subsequent domains but we can do this from the command line interface. If you have a particular set of domains you'd like to search against (RCSB or SCOP PDB codes), then we can limit the search to that set. If you decide to use this, please contact us once your first domain search has been completed (these take 2-3 years of serial computing time and will finish in 1-3 days, depending on how many other computations are ahead of it in our queue, the complexity/resolution of your data set, space group, and unit cell size). Regards, Ian Stokes-Rees
Re: [ccp4bb] brute force MR
For anyone who is interested, I meant to include a reference to the PNAS paper that has just come out (web-only early release) describing the wide search MR strategy we've developed: Stokes-Rees, Sliz Protein structure determination by exhaustive search of Protein Data Bank derived databases Proc. Nat'l Academy of Sciences doi:10.1073/pnas.1012095107 http://www.pnas.org/content/early/2010/11/17/1012095107 Ian
Re: [ccp4bb] how to reduce Rfactor R free
Jack (?), On Thu, 2010-12-09 at 21:35 -0800, Jack Russel wrote: So is it now the time to stop further refining the solution . R-values are not the only criteria for this. You should be looking for a) lack of unexplained density b) good geometry c) acceptable R-values This http://xray.bmc.uu.se/gerard/supmat/rfree2000/plotter.html can help you to get some idea of the range of R-values for the structures deposited in the PDB. if not then how can i lower the R factor and Rfree? First, you should probably be looking to lower only Rfree. Otherwise, the answer to your question depends on what you are dealing with. You may have done some of this, but consider - TLS - more conservative geometry restraints - NCS restraints if you got more than one copy in the asu - there is this very cute Coot feature Refine/improve Ramachandran plot - etc, etc, etc Keep in mind that, to paraphrase Daniel Gewirth, there are many ways in which R-values can be manipulated, both deliberately and not. Yet another reason not to go nuts about R-values and to take them, like poison, with a grain of salt. The second question is it possible to have such a large difference between R factor and R free? Well, apparently it is, you have just proven it :). Seriously though, the 10% that you have is on the high side. Some things mentioned above for the reduction of Rfree may help to close the gap, but once again, stuff happens. I'd make sure to have 10% set aside for the test set, at this resolution 5% may be not enough. Could you privately send me the mtz-file from refmac output? I am curious about R-value statistics in this case. Naturally, it will be treated confidentially. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] A script is needed to renumber image
Assuming that your files are named something_.img what follows must
be one line):
echo | awk '{for(i=361;i721;i++) printf mv something_%d.img something_
%d.img\n,i+720,i;}' | bash -sf
I'd try first without piping it to bash just to make sure that it works
right.
On Fri, 2010-12-10 at 09:51 +0800, wu donghui wrote:
Dear all,
I need a script to renumber my image. My initial image number ranges
from *_1081.img to *_1440.img. There are 360 images in total. I want
to renumber these images with the ranges from *_361.img to *_720.img,
that means every initial image-720, but I don't know how to do it.
Below is my script draft.
#! /bin/csh -f
@ i = 1081
while ( $i = 1081 )
while ($i = 1440 )
echo mv CD267A_3_pk_1_$i.img CD267A_3_pk_1_$i-720.img
@ i++
end
exit
Can anyone help me for this script? Thank you very much.
Best regards,
Donghui
--
I'd jump in myself, if I weren't so good at whistling.
Julian, King of Lemurs
Re: [ccp4bb] A script is needed to renumber image
Ben, does this assume that the current folder contains only the files to be renamed? Also, how does one add padding zeros? While Donghui didn't need zero padding, my one liner can be easily corrected to do this in the same way Ian's is, by replacing %d with %0nd, where n is the total number of digits one desires. Ed. On Thu, 2010-12-09 at 21:42 -0500, Ben Eisenbraun wrote: #! /bin/csh -f That's your problem right there! You don't need a script at all. In bash/ksh/zsh right on the command line: c=361 for f in * ; do mv $f CD267A_3_pk_1_$c.img ; c=$(($c+1)) ; done Bill Scott probably has some zsh slickness to do the same thing without the loop, but too much magic gives me the willies. -b -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu | -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] how to reduce Rfactor R free
The other question is, you say it's 3.3Å, by which criteria ? Xtriage might report a 4Å dataset if you take I/sigI 3 and 85% completeness It might also make a difference which data reduction program you used. [/Advertisement on]XDS/XSCALE or d*Trek[/Advertisement off] might squeeze a bit more our of your dataset. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.me.com/bosch_lab/ On Dec 10, 2010, at 12:35 AM, Jack Russel wrote: Hi all, I am new in the field of crystallography. I have just collected a data at 3.3 Å and the solved the structure using MR . the space group comes out to be P2 3. There are 3 molecules in Asymetric unit with the LLG of 300 and tfz 18. But after repeated rounds of refinement with REFMAC5 and model building with coot the R factor had been struck at 30% and R free at 40%. So is it now the time to stop further refining the solution . if not then how can i lower the R factor and Rfree? The second question is it possible to have such a large difference between R factor and R free? Thanks in advance
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
Colin Nave wrote: .. Also Acta Cryst. (2002). D58, 29-38 The 2.6 Å resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic `special position' D. Cobessi, L.-S. Huang, M. Ban, N. G. Pon, F. Daldal and E. A. Berry ( http://scripts.iucr.org/cgi-bin/paper?S0907444901017267 ) though the 'special position' is justifiably in quotation marks in this example as disorder is present. Colin Yes, this was an example of Ian's rotationally disordered with occupancy 0.5 possibility. Heme has a pseudo-two-fold axis passing through the plane of the ring, which is violated only by the position of the methyl and vinyl substituents on either side: rotating through the pseudo-2-fold superimposes methyl on vinyl and vice versa on either side. This axis was sitting on a crystallographic 2-fold. We did trial refinements in a lower symmetry space group so the axis became NCS, and we refined with the heme in one orientation or the other. In either case, difference maps showed positive density for another atom beyond the methyl, and negative density on CB of the vinyl. So we concluded the heme was oriented both ways, and in the average satisfied the symmetry of the higher space group. Putting it with half occupancy in the asymmetric unit allowed crystallographic symmetry to generate the rotated half. Looking at it from the heme's point of view, this makes sense. Its environment is perfectly symmetrical, so there is no way for it to choose one orientation over the other and both are adopted equally. (The two-fold is intrinsic to the biological unit, not generated by crystal packing). I think the heme iron was right on the crystallographic axis. When refining in the lower space-group with two .5 occupancy non-interacting hemes, the propionates (which actually satisfy the covalent symmetry) in the two hemes took on different conformations, not sure if this was significant or just taking liberties where freedom is given. eab
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Hi, At this resolution, such a difference in Rs could be an indicator that the structure is over--fitted. What weight did you choose? Lijun On Dec 10, 2010, at 3:17 AM, Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz Lijun Liu 555 Mission Bay Blvd South CVRB Room 482, MBox 3122 University of California, San Francisco San Francisco, CA 94158 Phone: (415)514-2836
Re: [ccp4bb] A script is needed to renumber image
Hi Ed,
does this assume that the current folder contains only the files to be
renamed?
Indeed it does. I was aiming to show that for simple tasks like this,
a modern shell makes things easy enough that scripts become overkill.
Also, how does one add padding zeros?
You can just add in a printf to go, e.g., from a padded 001 to 360:
c=1 for f in * ; do echo mv $f CD267A_3_pk_1_`printf %03d $c`.img ;
c=$(($c+1)) ; done
Or a more specific set of images:
c=1 for f in CD267A_3_pk_1_*.img ; do echo mv $f CD267A_3_pk_1_`printf
%03d $c`.img ; c=$(($c+1)) ; done
Or an even more specific set of images:
c=1 for f in CD267A_3_pk_1_{1081..1440}.img ; do echo mv $f
CD267A_3_pk_1_`printf %03d $c`.img ; c=$(($c+1)) ; done
I guess once it gets complex enough then you might as well go down James'
path and create a generalized script that can be used in many cases, but
it's definitely more work to write the generalized script than to toss off
one liners.
-ben
--
| Ben Eisenbraun | Software Sysadmin |
| SBGrid Consortium | http://sbgrid.org |
| Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
Hi, Some of you were right, I did not try to modify weights. Previous weight was 0.03 (assigned automatically), actual is 0.01 and Rwork vs Rfree is about 0.25 and 0.33, so 8% of difference - hopefully acceptable. But now I have very low RMSDs. And then you read in this forum quite often, that we should refine against the data and not against the restraints. :) Thanks anyway, Petr 2010/12/10 Lijun Liu lijun@ucsf.edu: Hi, At this resolution, such a difference in Rs could be an indicator that the structure is over--fitted. What weight did you choose? Lijun On Dec 10, 2010, at 3:17 AM, Petr Kolenko wrote: Dear colleagues, I appreciate any help, or any suggestion with my difficult data. Many thanks at least for consideration. I work with datasets at 3.6AA of resolution. Integrated with XDS, scaled with SCALA. After integration and scaling in P1, POINTLESS suggested space group I432: Space group confidence: 0.95 Laue group confidence: 1.000 Total probability: 0.97 After any longer refinement (more than 20 cycles), there was always a big gap between Rwork and Rfree (0.22 and 0.33), although the structure looks quite good at this resolution. I tried also space group I23, here are my statistics: Data processing (I23 vs I432): Rmerge - 0.124 vs 0.134 high resolution Rmerge - 0.649 vs 0.732 low resolution Rmerge - 0.048 vs 0.037 The gap between Rwork and Rfree was stabilized in I23 using tight NCS restraints: Rwork vs Rfree: I23 withouth NCS: 0.228 vs 0.334 I23 with NCS: 0.249 vs 0.296 I43 : 0.228 vs 0.326 My question is, what would you recommend me to close the gap? Or is this Rfree pointing out a lower real symmetry (I23) than suggested by POINTLESS or PHENIX.XTRIAGE (I432)? I have tried different selection of free reflections in I432 dataset already, but with almost the same results. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz Lijun Liu 555 Mission Bay Blvd South CVRB Room 482, MBox 3122 University of California, San Francisco San Francisco, CA 94158 Phone: (415)514-2836 -- Petr Kolenko kole...@imc.cas.cz http://kolda.webz.cz
Re: [ccp4bb] Space group vs. gap between Rwork and Rfree?
On Fri, 2010-12-10 at 19:32 +0100, Petr Kolenko wrote: But now I have very low RMSDs. And then you read in this forum quite often, that we should refine against the data and not against the restraints. :) Well, at 3.6A you must have low rmsds. And at 3.6A you don't really have any data to refine against beyond backbone conformation and maybe some sidechain orientation. You have perhaps 4-5 reflections per residue, right? So you barely have enough to identify backbone torsions with any degree of confidence. We should, of course, refine against the data, but not against the noise in it. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
Good point Colin! 2-Zn insulin is of course a classic example of this, where the two independent Zn2+ ions both sit on the crystallographic 3-fold in R3. It doesn't matter whether you count the metal ion as part of the protein or not: if I understand Gloria's original question correctly, all that matters is that the atom/ion is present in the crystal structure. In fact here are some extracts from the PDB entry (4INS): REMARK 375 ZNZN B 31 LIES ON A SPECIAL POSITION. REMARK 375 ZNZN D 31 LIES ON A SPECIAL POSITION. REMARK 375 HOH B 251 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 44 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 134 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 215 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 269 LIES ON A SPECIAL POSITION. HETATM 835 ZNZN B 31 -0.002 -0.004 7.891 0.33 10.40 ZN HETATM 836 ZNZN D 31 0.000 0.000 -8.039 0.33 11.00 ZN HETATM 885 O HOH B 251 -0.023 -0.033 11.206 0.33 21.05 O etc Hmmm - but shouldn't the occupancy of the Zn be 1.00 if it's on the special position (assuming it's not disordered), though the first Zn above and the water do appear to be disordered since they're not actually on the special position. Fractional occupancy always implies some kind of disorder: occupancy = 1/3 of an atom on a special position would imply occupancy disorder, i.e. it's randomly present in only 1/3 of the unit cells. -- Ian On Fri, Dec 10, 2010 at 1:11 PM, Colin Nave colin.n...@diamond.ac.uk wrote: Does one regard the metal atom in a metalloprotein as being part of the protein? If so, a shared metal could occupy a special position in a dimer for example. In Acta Cryst. (2008). D64, 257-263 Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination I. Dokmanic, M. Sikic and S. Tomic ( http://scripts.iucr.org/cgi-bin/paper?S090744490706595X ) it says there are 25 cases of metal atoms in special positions. Also Acta Cryst. (2002). D58, 29-38 The 2.6 Å resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic `special position' D. Cobessi, L.-S. Huang, M. Ban, N. G. Pon, F. Daldal and E. A. Berry ( http://scripts.iucr.org/cgi-bin/paper?S0907444901017267 ) though the 'special position' is justifiably in quotation marks in this example as disorder is present. Colin
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
The proper occupancy for an atom on a special position depends on how one defines the meaning of the number in that column. In the past, refinement programs, at least I know mine did, simply expanded all atoms in the coordinate file by the symmetry operators to determine the contents of the unit cell. With that operation the occupancy of the atoms on special positions had to be reduced. It is certainly true that there are 1/3 the number of atoms in the unit cell represented by ZN D 31 than, for example, the CA of residue 50. Most modern refinement programs try to handle this automatically, since users proved unreliable at detecting this condition and modifying their coordinate files. They use the interpretation that the site is fully occupied but there are only 1/3 the number of these sites than sites at general positions. Personally I find it disturbing to have the occupancy of B 31 set to 0.33 and that of D 31 set to 1.00 simply because of an insignificant shift in the position of the atom. Dale Tronrud On 12/10/10 13:53, Ian Tickle wrote: Good point Colin! 2-Zn insulin is of course a classic example of this, where the two independent Zn2+ ions both sit on the crystallographic 3-fold in R3. It doesn't matter whether you count the metal ion as part of the protein or not: if I understand Gloria's original question correctly, all that matters is that the atom/ion is present in the crystal structure. In fact here are some extracts from the PDB entry (4INS): REMARK 375 ZNZN B 31 LIES ON A SPECIAL POSITION. REMARK 375 ZNZN D 31 LIES ON A SPECIAL POSITION. REMARK 375 HOH B 251 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 44 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 134 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 215 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 269 LIES ON A SPECIAL POSITION. HETATM 835 ZNZN B 31 -0.002 -0.004 7.891 0.33 10.40 ZN HETATM 836 ZNZN D 31 0.000 0.000 -8.039 0.33 11.00 ZN HETATM 885 O HOH B 251 -0.023 -0.033 11.206 0.33 21.05 O etc Hmmm - but shouldn't the occupancy of the Zn be 1.00 if it's on the special position (assuming it's not disordered), though the first Zn above and the water do appear to be disordered since they're not actually on the special position. Fractional occupancy always implies some kind of disorder: occupancy = 1/3 of an atom on a special position would imply occupancy disorder, i.e. it's randomly present in only 1/3 of the unit cells. -- Ian On Fri, Dec 10, 2010 at 1:11 PM, Colin Nave colin.n...@diamond.ac.uk wrote: Does one regard the metal atom in a metalloprotein as being part of the protein? If so, a shared metal could occupy a special position in a dimer for example. In Acta Cryst. (2008). D64, 257-263 Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination I. Dokmanic, M. Sikic and S. Tomic ( http://scripts.iucr.org/cgi-bin/paper?S090744490706595X ) it says there are 25 cases of metal atoms in special positions. Also Acta Cryst. (2002). D58, 29-38 The 2.6 Å resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic `special position' D. Cobessi, L.-S. Huang, M. Ban, N. G. Pon, F. Daldal and E. A. Berry ( http://scripts.iucr.org/cgi-bin/paper?S0907444901017267 ) though the 'special position' is justifiably in quotation marks in this example as disorder is present. Colin
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
No that surely can't be right. Application of a symmetry operator to a point on a special position which is unchanged by the operator doesn't generate a symmetry copy of the point, because there is no symmetry copy of such a point! For general Wyckoff positions it does, for sure. If you look at the entry for R3 (hexagonal or rhombohedral setting, it doesn't matter which) on the Wyckoff position website I indicated earlier, you'll see a column labelled 'multiplicity' which is 3 for the general position and 1 for the special position. This means that the symmetry operator generates 3 symmetry copies of a general position (including the original), but only 1 copy (i.e. only the original) of the special positions on the 3-fold. That's precisely why such points are called special positions - i.e. you have to treat them specially! If what you describe is what the refinement program or whatever is doing then it would imply there's a programming error, i.e. the program is not treating special positions as special, it's treating them instead as general positions. As I said whenever you see fractional occupancy reported it should imply some kind of disorder, i.e. the atom exists on that site in only the indicated fraction of the unit cells in the lattice. -- Ian On Fri, Dec 10, 2010 at 10:02 PM, Sue Roberts s...@email.arizona.edu wrote: Hi Ian No disorder is involved. The occupancy of an (fully occupied) atom on an n-fold rotation axis is 1/n If a two-fold, 1/2 If a three-fold, 1/3 When you sum over all the atoms in the unit cell, application of the symmetry operations to atoms lying on the rotation axis generates atoms with unchanged coordinates. Hence to generate a fully occupied atom on a n-fold symmetry axis, the original occupancy has to be 1/n. Sue On Dec 10, 2010, at 2:53 PM, Ian Tickle wrote: Good point Colin! 2-Zn insulin is of course a classic example of this, where the two independent Zn2+ ions both sit on the crystallographic 3-fold in R3. It doesn't matter whether you count the metal ion as part of the protein or not: if I understand Gloria's original question correctly, all that matters is that the atom/ion is present in the crystal structure. In fact here are some extracts from the PDB entry (4INS): REMARK 375 ZN ZN B 31 LIES ON A SPECIAL POSITION. REMARK 375 ZN ZN D 31 LIES ON A SPECIAL POSITION. REMARK 375 HOH B 251 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 44 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 134 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 215 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 269 LIES ON A SPECIAL POSITION. HETATM 835 ZN ZN B 31 -0.002 -0.004 7.891 0.33 10.40 ZN HETATM 836 ZN ZN D 31 0.000 0.000 -8.039 0.33 11.00 ZN HETATM 885 O HOH B 251 -0.023 -0.033 11.206 0.33 21.05 O etc Hmmm - but shouldn't the occupancy of the Zn be 1.00 if it's on the special position (assuming it's not disordered), though the first Zn above and the water do appear to be disordered since they're not actually on the special position. Fractional occupancy always implies some kind of disorder: occupancy = 1/3 of an atom on a special position would imply occupancy disorder, i.e. it's randomly present in only 1/3 of the unit cells. -- Ian On Fri, Dec 10, 2010 at 1:11 PM, Colin Nave colin.n...@diamond.ac.uk wrote: Does one regard the metal atom in a metalloprotein as being part of the protein? If so, a shared metal could occupy a special position in a dimer for example. In Acta Cryst. (2008). D64, 257-263 Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination I. Dokmanic, M. Sikic and S. Tomic ( http://scripts.iucr.org/cgi-bin/paper?S090744490706595X ) it says there are 25 cases of metal atoms in special positions. Also Acta Cryst. (2002). D58, 29-38 The 2.6 Å resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic `special position' D. Cobessi, L.-S. Huang, M. Ban, N. G. Pon, F. Daldal and E. A. Berry ( http://scripts.iucr.org/cgi-bin/paper?S0907444901017267 ) though the 'special position' is justifiably in quotation marks in this example as disorder is present. Colin Dr. Sue A. Roberts Dept. of Chemistry and Biochemistry University of Arizona 1041 E. Lowell St., Tucson, AZ 85721 Phone: 520 621 8171 s...@email.arizona.edu http://www.biochem.arizona.edu/xray
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
On Fri, 2010-12-10 at 22:40 +, Ian Tickle wrote: Application of a symmetry operator to a point on a special position which is unchanged by the operator doesn't generate a symmetry copy of the point, because there is no symmetry copy of such a point! Why not? Symmetry-related copy may be considered just that - copy produced by a symmetry operator. What advantage do you gain by restricting it to only those that result in physically different point? There is one excellent point that you made in some past exchanges - that deposited structures are mathematical models and they do not always have to make strict physical sense. This seems to be just such case - one does not have three zincs in that spot with each being gone two thirds of the time, but making the model physically meaningful would require perhaps dropping to P1 and implementing NCS to enforce crystal symmetry. So compromising on the physical meaning of the atom record seems like a small price to pay. This feels odd - I seem to be arguing your side :) Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
An excerpt from the Shelx manual (which I think is a good reference for proper handling of atoms on special positions in refinement of small or large molecules): 4.3 Special position constraints Constraints for the coordinates and anisotropic displacement parameters for atoms on special positions are generated automatically by the program for ALL special positions in ALL space groups, in conventional settings or otherwise. For upwards compatibility with SHELX-76, free variables may still be used for this, but it is better to leave it to the program. If the occupancy is not input, the program will fix it at the appropriate value for a special position. If the user applies (correct or incorrect) special position constraints using free variables etc., the program assumes this has been done with intent and reports but does not apply the correct constraints; accidental application of wrong special position constraints is one of the easiest ways to cause a refinement to 'blow up' ! Also, please check the book of Shmueli and Weiss, Introduction to crystallographic statistics p. 5, last paragraph and the next page where a rigorous treatment of of contribution to the structure factor of atoms in general and special positions in the unit cell is given (eq. 1.2.10). Thus, occupancy of an atom on a 3-fold would be 1/3, 1/2 on a 2-fold, 1/4 on a 4-fold and so forth. Cheers, Boaz - Original Message - From: Ian Tickle ianj...@gmail.com Date: Saturday, December 11, 2010 0:41 Subject: Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms To: CCP4BB@JISCMAIL.AC.UK No that surely can't be right. Application of a symmetry operator to a point on a special position which is unchanged by the operator doesn't generate a symmetry copy of the point, because there is no symmetry copy of such a point! For general Wyckoff positions it does, for sure. If you look at the entry for R3 (hexagonal or rhombohedralsetting, it doesn't matter which) on the Wyckoff position website I indicated earlier, you'll see a column labelled 'multiplicity' which is 3 for the general position and 1 for the special position. This means that the symmetry operator generates 3 symmetry copies of a general position (including the original), but only 1 copy (i.e. only the original) of the special positions on the 3-fold. That's precisely why such points are called special positions - i.e. you have to treat them specially! If what you describe is what the refinement program or whatever is doing then it would imply there's a programming error, i.e. the program is not treating special positions as special, it's treating them instead as general positions. As I said whenever you see fractional occupancy reported it should imply some kind of disorder, i.e. the atom exists on that site in only the indicated fraction of the unit cells in the lattice. -- Ian On Fri, Dec 10, 2010 at 10:02 PM, Sue Roberts s...@email.arizona.edu wrote: Hi Ian No disorder is involved. The occupancy of an (fully occupied) atom on an n-fold rotation axis is 1/n If a two-fold, 1/2 If a three-fold, 1/3 When you sum over all the atoms in the unit cell, application of the symmetry operations to atoms lying on the rotation axis generates atoms with unchanged coordinates. Hence to generate a fully occupied atom on a n-fold symmetry axis, the original occupancy has to be 1/n. Sue On Dec 10, 2010, at 2:53 PM, Ian Tickle wrote: Good point Colin! 2-Zn insulin is of course a classic example of this, where the two independent Zn2+ ions both sit on the crystallographic 3-fold in R3. It doesn't matter whether you count the metal ion as part of the protein or not: if I understand Gloria's original question correctly, all that matters is that the atom/ion is present in the crystal structure. In fact here are some extracts from the PDB entry (4INS): REMARK 375 ZN ZN B 31 LIES ON A SPECIAL POSITION. REMARK 375 ZN ZN D 31 LIES ON A SPECIAL POSITION. REMARK 375 HOH B 251 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 44 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 134 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 215 LIES ON A SPECIAL POSITION. REMARK 375 HOH D 269 LIES ON A SPECIAL POSITION. HETATM 835 ZN ZN B 31 -0.002 -0.004 7.891 0.33 10.40 ZN HETATM 836 ZN ZN D 31 0.000 0.000 -8.039 0.33 11.00 ZN HETATM 885 O HOH B 251 -0.023 -0.033 11.206 0.33 21.05 O etc Hmmm - but shouldn't the occupancy of the Zn be 1.00 if it's on the special position (assuming it's not disordered), though the first Zn above and the water do appear to be disordered since they're not actually on the special position. Fractional occupancy always implies some kind of disorder: occupancy = 1/3 of an atom on a special
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
SHELXL also expects that the occupancy of a fully occupied atom on a threefold axis should be set at 1/3, and will generate this automatically if necessary. It will also generate automatically the necessary constraints for the x, y and z parameters (and for the Uij if the atom is anisotropic). It is essential that this is done correctly if a full-matrix refinement is being performed (e.g. to get esd estimates), otherwise the refinement can explode. The user may change or switch off the tolerance for detecting whether an atom is on a special position (with the SPEC instruction). Setting the occupancy to a fraction avoided a complicated IF construction inside a loop and 35 years ago computers were so slow! I can't change it now because I have to preserve upwards compatibility. Unfortunately the CIF committee decided to use the other definition (i.e. the Zn on the threefold axis has an occupancy of 1.0) and this has caused considerable confusion in the small molecule world ever since; atoms are frequently encountered on special positions in inorganic and mineral structures. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 10 Dec 2010, Ed Pozharski wrote: On Fri, 2010-12-10 at 21:53 +, Ian Tickle wrote: Hmmm - but shouldn't the occupancy of the Zn be 1.00 if it's on the special position Shouldn't 1/3 be better for programming purposes? If you set occupancy to 1.0, then you should specify that symmetry operators do not apply for these atoms, making Fc calculation a bit more cumbersome. If definition of the asu content is you get full content of the unit cell after applying symmetry operators, then occupancy *must* be 1/3, right? The first zinc and the water are on special position, but because they are not excluded from positional refinement (perhaps they should be), they will drift a bit. CNS has distance cutoff for treating atoms as special positions, if it jumps over the limit during, say, simulated annealing, it will cause problems. Perhaps PROLSQ did something similar. It is a good question if it's better to fix these in place or let them wobble a bit to account for some potential disorder. While I see the formal argument that it should be nailed to three-fold axes, it is also true that this is a mathematical compromise to simplify modeling that does not reflect physical reality (i.e. you don't have three partially occupied zinc ions, it's just one). In any event, given that this is a 1.5A structure, (-0.002 0.004) is statistically speaking the same as (0 0). Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms
No disorder is involved. The occupancy of an (fully occupied) atom on an n-fold rotation axis is 1/n If a two-fold, 1/2 If a three-fold, 1/3 When you sum over all the atoms in the unit cell, application of the symmetry operations to atoms lying on the rotation axis generates atoms with unchanged coordinates. Hence to generate a fully occupied atom on a n-fold symmetry axis, the original occupancy has to be 1/n. I guess I have a few small points to add: 1) If a site on special position is not fully occupied, then the occupancy is q/n, where q is actual occupancy (for fully occupied site q=1). Since ATOM record in PDB file does not (directly) tell you whether the atom is on special position or not then it is not straightforward to know whether the occupancy of atom in question is not 1 due to disorder or due to symmetry. 2) Let's consider a hypothetical case... The maximal multiplicity of a spacial position in proteins is 24 (is that right? please correct if not), and the precision of the occupancy field in PDB file is only two digits, like: 1.00. Imagine you have a fair amount of very heavy atoms all seating at special positions with the multiplicity 24, and your structure is relatively small. Then what you will be forced to put in the PDB file is: 1/24 ~ 0.04 and not 0.042. I'm not sure if this rounding error is significant or not (and what is significant), but just to keep in mind another source of mismatch between reported and recomputed R-factors. 3) If I add an atom onto special position using Coot, will it set the occupancy as 1/n automatically or do I have to to edit the PDB file myself (and remember to do so)? (another room for an error) So, having said this, I tend to think that using occupancy 1 (and not 1/n) is a good thing. Pavel.
