Re: [ccp4bb] disordered helix
Hard to say without seeing the maps and experimenting. My first check would be to set the NTD occupancies to 0.0 - refine the CTD alone, then look at the maps in COOT. Or maybe let an automatic modelling building program such as Buccaneer try to rebuild the NTD section, with starting phases from the CTD. Eleanor On 13 May 2013 09:04, atul kumar atulsingh21...@gmail.com wrote: Dear all, I have solved the structure of my protein by molecular replacement at 2.9A, with Rfactor and Rfree 18 and 22 respectively. Overall everything seems fine, its a two domain protein NTD and CTD, the NTD have high average B factor compared to CTD. A helix of NTD seems to be disordered, I tried different geometric weights but the refined structure does not seem to follow proper geometry for this helix. The B-factor of this helix is very high compared to overall B factor for NTD and omit map shows only some partial density in this region( off course not conclusive). All the homologous structure have helix in this region although with high B-factor. Should I submit the current pdb or need more refinement? thanks and regards Atul Kumar
Re: [ccp4bb] Fwd: Re: [ccp4bb] reference for true multiplicity?
The traditional benefit was in reducing absorbtion errors. This obviously depends on crystal path, and seeing it is hard to model well, one way to mitigate the errors was to average equivalents collected at different settings. The error was still there, but assuming random distribution about the true intensity, averaging helps.. On 15 May 2013 10:20, Colin Nave colin.n...@diamond.ac.uk wrote: Oh – I seemed to have diverted Frank’s thread. Fortunately most languages themselves are highly redundant, with following characters and words being quite predictable. The entropy and redundancy of English language was analysed by Shannon (with the help of his wife) and he obtained figures of about 1 bit per character rather than log base 2 (27), a redundancy of around 75%. I guess this redundancy helps us put things in context. However, in order to avoid future misunderstandings, I would like to suggest that further communications with CCP4BB be done in Latin which I believe has less ambiguity. I hope people will adopt this helpful suggestion. OK – perhaps not a good idea. More relevant to Frank’s question, I was referring to cases where, for a particular reflection, the path of x-rays through the crystal was altered to average out systematic errors. What type of systematic errors would be mitigated by this? There is one potential addition to the list (absorption errors, detector calibration) I produced but it applies to synchrotron sources rather than the type of x-ray source used in the Acta D paper. I will let others have a think before suggesting it. Colin
Re: [ccp4bb] Calcium ions in enzymes
I would think a Google search would make some suggestions for you. There are lots of cases of proteins which require Calcium to function, but it is a bit chicken-and-egg-y - can the protein only function after it folds correctly, and is the Ca essential for that folding? On 31 May 2013 11:54, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: My work with colicin M class bacteriocins shows that they require Ca2+ (or Mg or Mn) for catalysis: 1 Grinter, R., Roszak, A. W., Cogdell, R. J., Milner, J. J. and Walker, D. (2012) The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins. J. Biol. Chem. 287, 38876-3 Best Regards, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wei Liu [ we...@me.com] Sent: 31 May 2013 11:25 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Calcium ions in enzymes Dear all, As we all know, many proteins contain calcium ions. Does anyone know if there are reported cases where calcium ions play a catalytic role rather than a structural role in enzymes? Best Wei Liu
Re: [ccp4bb] Hi
I don't really understand what your space group is? space group mC??? Eleanor On 10 Jun 2013, at 19:57, Wei Shi wrote: Hi all, I was trying to solve the structure of a protein in several different datasets using xds and phenix. I could solve the structure from one dataset in space group P4. For another dataset, I could solve the structure using the monomer of the structure I got from the first dataset as search model and solve the structure in space group mC. For the third dataset, in IDXREF.LP, the space group of the highest symmetry is hp: 101.2, 101.3, 58.8, 90, 90, 120. According to Mathiews coefficient, 1 monomer is expected in the asymmetric unit. But I couldn't get the molecular replacement solution using the same method as for the second dataset. I also tried several other search models (eg. deletion of the potential flexible region in the search model) and tried to find the solution in all possible pointgroup. I also tried to process the data in oC (C222), the space group of the second highest symmetry in IDXREF.LP, but, still I could not get right molecular replacement solution. I don't whether this means that there is a big conformational change for the structure in the third dataset or the space group I use is not right. Let me know if any of you would have any comments or suggestions for me. Thank you so much! Best, Wei
Re: [ccp4bb] Fwd: [ccp4bb] pdbset
If you are resetting B factors you must recalculate the AniosUs later But if you are getting negative B values something in your procedure is less than optimal! Are you refining aniso Us with too limited data? You need high resolution data to attempt this. Are you trying to refine B factors and occupancies at the same time? You need superb data to do this, and as a rule it isnt recommended. (Maybe for metal atoms, or some special ligand, but not a good general practice..) Or have you set occupancies too low, and the B factor is trying to compensate for that? Eleanor On 11 June 2013 10:42, Swastik Phulera swastik.phul...@gmail.com wrote: Dear Tim,Fred Negative B factors and occupancies higher than one came in from shelxl, Also getting rid of Ansiou at first would lead me to an additional step of placing them in their right place later on (is there a short cut that I may take). On Tue, Jun 11, 2013 at 2:09 PM, Tim Gruene t...@shelx.uni-ac.gwdg.dewrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Swastik Phulera, unless you need the ANISOU cards, you can first remove them with e.g. grep -v ^ANISOU youfile.pdb yournewfile.pdb before running pdbset. (I hope you don't work on a Windows machine, then you would probably first find a way to install 'grep', a command common on unixoid operating systems). By the way: how did you get negative B-values into your PDB-file? Best, Tim On 06/11/2013 10:22 AM, Swastik Phulera wrote: -- Forwarded message -- From: Swastik Phulera swastik.phul...@gmail.com Date: Tue, Jun 11, 2013 at 1:51 PM Subject: Re: [ccp4bb] pdbset To: Tim Gruene t...@shelx.uni-ac.gwdg.de Dear Tim, Miguel Thanks for your suggestions, the program does work now, but it seems that it cant handle AnsioU s . It gives an error: PDBSET: *** AnisoU present: cannot reset B *** Is there any other program which would set minimum bfactors for me. Also I am looking for a program that would set the maximum occupancy to a desired value (It seems that pdbset can only play with the minimum values).. On Tue, Jun 11, 2013 at 12:11 PM, Tim Gruene t...@shelx.uni-ac.gwdg.dewrote: Dear Swastik Phulera, after the word 'output.pdb' you must first hit the Enter-key which takes you into the program pdbset. Then you type B_reset Minimum 0 END and the program runs. If you wish to do it without interaction, e.g. in a script, you can use the shell construct '': pdbset XYZIN input.pdb XYZOUT output.pdb eof B_reset MINIMUM 0 eof Best, Tim On 06/11/2013 08:15 AM, Swastik Phulera wrote: Dear All, I am trying to use pdbset from the terminal and am constantly getting an error: [XYZ@NCCS3 110613]$ pdbset XYZIN input.pdb XYZOUT output.pdb B_reset MINIMUM 0 CCP4 library signal ccp4_general:Use: logical name file name (Error) raised in ccp4fyp pdbset: Use: logical name file name pdbset: Use: logical name file name Times: User: 0.0s System:0.0s Elapsed: 0:00 Does any one have any idea what's wrong here? Swastik Phulera - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRtuJQUxlJ7aRr7hoRAhcWAKDGPXBS3iJhzzbaYBK4GnJGjijyngCgqYZI B26VKBDFO5FQNJJQd6plsc8= =mDut -END PGP SIGNATURE- -- -- स्वस्तिक फुलेरा वरिष्ठ अनुसंधान कर्ता संरचनात्मक जीवविज्ञान प्रयोगशाला डी. एन. ए. फिंगरप्रिंटिंग एवं निदान केंद्र हैदराबाद, ५००, ००१ और राष्ट्रीय कोशिका विज्ञान केंद्र पुणे विश्वविद्यालय परिसर, गणेशखिंड पुना, ४११,००७
Re: [ccp4bb] str solving problem
As others say - the Rfactors look pretty good for MR, mine usually start over 50% even with a better model and one hopes they then decrease.. But you say you took the Balbes model into phaser? and I think Balbes automatically runs cycles of refinement so any comment on R factors may not mean much. Have you found both molecules in the asymmetric unit? You only give LLG for one? Eleanor On 19 June 2013 17:44, Eugene Valkov eugene.val...@gmail.com wrote: Yes, I would agree with Francis that diffraction shows contribution from several lattices, which could lead to misindexing. However, it should be feasible to get a model that refines from this sort of data. Pramod - could you please post your data processing statistics from your scaling program? Better if you have several for different spacegroups. Also, I have no idea how HKL200 does this, but could you please provide an indexing solution table from Mosflm that shows penalties associated with each type of space group? Was there a sharp penalty drop at some point or was it more gradual? When you index spots in Mosflm, do your predictions agree with the spots? Or is there a substantial portion that are missed? I would consider altering thresholds in Mosflm for indexing (see the manual). Eugene On 19 June 2013 17:34, Francis E. Reyes francis.re...@colorado.eduwrote: On Jun 17, 2013, at 12:36 PM, Pramod Kumar pramod...@gmail.com wrote: I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO no ice ring is appeared, diffraction pattern looks ok, misindexing in any direction is not conclusive to me (plz see the imj attachment) The diffraction does not look ok... there's hints of multiple lattices... which is not a problem if the two lattice orientations do not perfectly overlap (i.e. their spots are separable). Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices (and even worse if you have twinning/pseudosymmetry issues). Try a program that uses all spots for indexing, across all images (XDS for example) and you might get the true space group. Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder -- Dr Eugene Valkov MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 407840
Re: [ccp4bb] AW: Twinning problem - almost solved.
At your resolution that seems to me a reasonable gap between R and Rfree? Eleanor On 21 Jun 2013, at 12:28, herman.schreu...@sanofi.com wrote: Dear Bulletin Board, After some headbanging (Refmac5 had helpfully created gap records for all insertions and deletions present in the structure), I got refmac5 running with the TWIN option. Refmac5 also found the k,h,-l domain and rejected the other possible domains because they were too small. The Rfactor's are now extremely good: ~14% and the Rfree's are for me acceptable: ~24%. Since I found the difference between R and Rfree somewhat large, I have been playing with the weighting. By using a weight of 0.01, I can bring the Rfactor up to 18%, but the Rfree stays about the same or even gets a little worse. My question: is there a way to bring R and Rfree closer together, or is it related to the twinned data and is it something we have to live with? Best regards, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Miller, Mitchell D. Gesendet: Donnerstag, 20. Juni 2013 17:43 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Twinning problem You are welcome. Let me also for the benefit of others who may search the archives in the future, let me correct two errors below - (typo and a miss-recollection). Specially, I was thinking that phenix.refine was now able to refine multiple twin laws, but according to Nat Echols on the phenix mailing list http://phenix-online.org/pipermail/phenixbb/2013-March/019538.html phenix.refine only handles 1 twin law at this time. (My typo was that and our second structure was 3nuz with twin fractions 0.38, 0.32, 0.16 and 0.14 -- not 2nuz). A useful search for deposited structures mentioning tetartohedral http://www.ebi.ac.uk/pdbe-srv/view/search?search_type=all_texttext=TETARTOHEDRALLY+OR+TETARTOHEDRAL Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.com Sent: Thursday, June 20, 2013 8:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] AW: Twinning problem Dear Mitch (and Philip and Phil), It is clear that I should give refmac a go with the non-detwinned F's and just the TWIN command. Thank you for your suggestions, Herman -Ursprüngliche Nachricht- Von: Miller, Mitchell D. [mailto:mmil...@slac.stanford.edu] Gesendet: Donnerstag, 20. Juni 2013 16:18 An: Schreuder, Herman RD/DE Betreff: RE: Twinning problem Hi Herman, Have you considered the possibility of your crystals being tetartohedral twinned. That is more than one of the twin laws may apply to your crystals. E.g. in P32 it is possible to have tetartohedral twinning which would have 4 twin domains - (h,k,l), (k,h,-l), (-h,-k,l) and (-k,-h,-l). Perfect tetartohedral twinning of P3 would merge in P622 and each twin domain would have a faction of 0.25. We have had 2 cases like this (the first 2PRX was before there was support for this type of twinning except for in shelxl and we ended up with refined twin fractions of 0.38, 0.28, 0.19, 0.15 for the deposited crystal and a 2nd crystal that we did not deposit had twin fractions of 0.25, 0.27, 0.17, 0.31). The 2nd case we had was after support for twining (including tetartohedral twinning) was added to refmac (and I think phenix.refine can also handle this). For 2NUZ, it was P32 with refined twin fractions of 0.25, 0.27, 0.17, 0.31. Pietro Roversi wrote a review of tetartohedral twinning for the CCP4 proceedings issues of acta D http://dx.doi.org/10.1107/S0907444912006737 I would try refinement with refmac using the original (non-detwinned F's) with just the TWIN command to see if it ends up keeping twin fractions for all 3 operators (4 domains) -- especially with crystals 1 and 3 which appear to have the largest estimates of the other twin fractions. Regards, Mitch == Mitchell Miller, Ph.D. Joint Center for Structural Genomics Stanford Synchrotron Radiation Lightsource 2575 Sand Hill Rd -- SLAC MS 99 Menlo Park, CA 94025 Phone: 1-650-926-5036 FAX: 1-650-926-3292 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.com Sent: Thursday, June 20, 2013 6:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Twinning problem Dear Bulletin Board, Prodded by pdb annotators, which are very hesitant to accept coordinate files when their Rfactor does not correspond with our Rfactor, I had a look again into some old data sets, which I suspect are twinned. Below are the results of some twinning tests with the Detwin program (top value: all reflections, lower value: reflections Nsig*obs (whatever that may mean). The space group is P32, the resolution is 2.3 - 2.6 Å and data are reasonable
Re: [ccp4bb] R too low?
You have obviously solved this problem, but one thing that can change apparent Rfactors is the number of reflections accepted.. If one gives you 5% more very weak reflections say, then those will inevitably have high Rfactors and this can increase the apparent Rfactor without changing the map appearance much.. Eleanor On 27 June 2013 21:26, Roberts, Sue A - (suer) s...@email.arizona.eduwrote: Hello Everone, Thanks for all the help. The key to finding the problem was following up on Tim Gruene's suggestion to compare the data sets directly. It appears that an error occurred during conversion from I to F - until I find the log file for the conversion, I can't guess what was done. Longer version: When I compared the good and bad data sets, R was about 0.15, instead of the 0.07 I was expecting. Yesterday, I reintegrated the images using the same program that generated the bad data (CrystalClear - sorry to be opaque but I didn't want to inspire a lot of discussion about various integration programs when I was pretty sure the program wasn't at fault.), and ended up with a data set that agreed with the good data (XDS). (Yeah, I should've done this before sending a message to ccp4bb). The R for scaling the new CC dataset and the XDS dataset was 0.07 and refinement behaved as expected and agreed with that of XDS. I have been unable to find the log file for the conversion from integrated I to mtz F (it's on some computer somewhere, I'm sure), but I did find the original ScalAveraged.ref file for the bad data and reimported that using the import scaled data task in ccp4i. That data set is also good. So, I conclude that something was done wrong during import to ccp4. Tim suggested that perhaps the data was converted twice to amplitudes, perhaps that's it. Anyway, now I know where the problem arose. Several people suggested checking statistics using phenix polygon and other analysis tools in phenix. I agree that those are nice tools (and we had done that), however, they only tell you how your statistics are different from the median and often don't give any hints as to how any problems might have arisen. Again, thanks for all the help. Sue On Jun 26, 2013, at 8:54 AM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sue, if you made your rmsd (bonds) 20-30 times smaller I would agree they were not too loose. 0.14A is pretty high. So two suggestions: a) check the molprobity report of your PDB if its geometry is sane b) check the CC plot of one data set against the other one to check if the problem is due to two different data or due to the PDB file (xprep can do this plot conveniently). Did you check if you converted the data twice to amplitudes, or maybe not at all? Best, Tim On 06/26/2013 05:44 PM, Roberts, Sue A - (suer) wrote: Hello Everyone I have two data sets, from the same crystal form (space group P32) of the same protein, collected at 100 K at SSRL, about 2.2 A resolution, that refining to R = 0.14, Rf = 0.26 (refmac/TLS). This is a molecular replacement solution, from a model with about 40% homology (after MR density was apparent for some missing or misbuilt residues, so I don't think the structure is stuck in the wrong place. The Fo-Fc map is essentially featureless. The 2Fo-Fc map doesn't look as good as it should - for instance, there are very few water molecules to be found. The data reduction statistics look OK, the resolution cutoff is pretty conservative. There is one molecule in the asymmetric unit, so no NCS. There is no twinning either. It seemed to me that the R is too low, not Rf too high. More normally, R ends up about .18 - .20 for a data set at this resolution. I reprocessed the images with a different data processing program and redid the MR. The data reduction statistics look similar, the resolution is the same, but now the structure refines to R = 0.20, Rf = 0.24 (same free R set of reflections chosen, still refmac/TLS.) The maps look more normal. Further rebuilding took us to R = 0.18, Rf = 0.22 So, the question I have (and that I've been asked by the student and PI) is: What was the problem with the original data set? What should I be looking for in the data reduction log files, for instance, or in the refinement log? The large R - free R spread is characteristic of overfitting, but the geometry is not too loose (rmsd bonds = 0.14), there are plenty of reflections (both working and free). Can anyone point me toward a reason R would be low? Thanks Sue Dr. Sue A. Roberts Dept. of Chemistry and Biochemistry University of Arizona 1041 E. Lowell St., Tucson, AZ 85721 Phone: 520 621 8171 or 520 621 4168 s...@email.arizona.edu http://www.cbc.arizona.edu/xray or http://www.cbc.arizona.edu/facilities/x-ray_diffraction - -- - -- Dr Tim Gruene Institut fuer anorganische
Re: [ccp4bb] pseudotranslation issues
With that translational vector the data set is approximately I centred. If you integrate it in I centred lattice then you must consider space groups I 4 or I41. In point P4 you might need to consider SGs P4, P41, P42, P43 Eleanor On 1 July 2013 11:19, rajakumara eerappa reera...@gmail.com wrote: Dear Fulvio Saccoccia As you suspected it might be I4 instead of P4. Since fractional coordinates are (0.5, 0.5, 0.479) and, 0.479 is almost 0.5. Try to integrate and scale in I4 and see how chi-square behaves. Raj On Fri, Jun 7, 2013 at 11:17 AM, Fulvio Saccoccia fulvio.saccoc...@uniroma1.it wrote: Dear ccp4bb users, I have a data set collected at 2.2A resolution that I can integrate in P4. Aimless, Phaser and Xtriage recognized a pseudotranslation (34.6% of origin peak) at fractional coordinates 0.500 0.500 0.479 (ORTH 46.7 46.7 37.4). However labelit.index run with sublattice_allow=true search did not detect the pseudotranslation vector. So far, any attempts to phase by molecular replacement was unsuccesfull and I suspect that it could be due to incorrect assignement of space group: Now, some questions arise: 1) does the pseudo translation exist or do not? 2) if translational NCS is present, could it mimic a P41/P42/P43 space group? 3) in any case, any advices in order to properly deal with tNCS will be of great help? I thank you in advance Best wishes -- Fulvio Saccoccia, PhD Sapienza University of Rome Dept. of Biochemical Sciences A. Rossi Fanelli Piazzale Aldo Moro, 5 00185 Rome (Italy)
Re: [ccp4bb] Self rotation function and translation peak
Well - translation peaks are listed in the data processing log file - look for the word translation. If there is an off-origin peak 25% of the origin you probably have two 9or more) molecules in similar orientations but displaced from each other. Self rotation peaks are listed in various programs. Do you have an example - it is easier to comment on a particular result rather than try to describe general ones. Eleanor On 5 Jul 2013, at 23:27, Faisal Tarique wrote: Dear All In case we have more than one molecule per asymmetric unit, how to look for the results of the self-rotation function calculation and translation peak in the native patterson. -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Refinement of partly occupied water molecules
You are desribing the reason many people limit their refinement to lower resolution! I think it is probably universally true that there are multiple conformations for sidechain/water networks at the surface, which we just dont model properly. If you are going to tackle the fine details you need to judge how the nearby side chains are fitted as well as the water. And of course then you can probably also see stuff present in the crystallisation media. The rewards are a lower R factor, and a better understanding of mobility I guess. Eleanor On 12 July 2013 09:08, Stefan Krimmer krim...@staff.uni-marburg.de wrote: Dear all, I have a question concerning the refinement of partly occupied water molecules: in some of my macromolecular crystal structures with resolutions between 1.1 - 1.4 Å, several round positive Fo-Fc electron density blobs are detectable which show after assignment of a water molecule to these blobs and subsequent refinement with Phenix.refine a good-looking 2Fo-Fc electron density. However, there also occurs a small negative Fo-Fc electron density detectable inside the 2Fo-Fc density blob. The negative Fo-Fc electron density disappears if the occupancy of the water molecule is automatically refined by Phenix.refine (occupancy manually set to a value below 100% followed by refinement) or manually set to 50% and fixed for this value (Fix occupancy option in phenix.refine). Therefore, I think these positions are partly occupied by water molecules, but I am not sure how I should handle it/how it is generally handled. Which one of the two options described above is the better one? I would be thankful for any advice and/or literature about this topic. Thank you for your help! Stefan
Re: [ccp4bb] NCS information from different crystal parameters?
Hard to comment without more information, but check how many molecules you expect per asymmetric unit. If more than one look at the self rotation function - it might help. And you can with difficulty do a cross rotation between two data sets, which sometimes suggests how crystal 1 is related to crystal 2. But if you have the Se sites for both crystals, it is probably easier to try to match them and get transformation matrices to use for cross-averaging that way. Is that your case? Eleanor. On 12 July 2013 03:33, Yuan SHANG shangyuan5...@gmail.com wrote: Dear all, Currently, I was stuck in a coiled-coil crystal. I have two Se-derivative crystals in similar crystallization conditions. And the cell parameters of these two are list below : Crystal A: P21, a=69,b=43,c=135, beta=100.7 Crystal B: P21, a=29,b=230,c=42, beta=92.2 Assuming the crystal packings in A and B are overall similar, there seems to be another P21 axis in crystal A, which may help to determine the NCS parameters during phasing as there are two moleculars in the ASU. Could anyone help to tell me how to derive the NCS parameters? Best regards, Yuan SHANG
Re: [ccp4bb] Methods to reduce the model bias and increase the SAD phase
I check ha sites by 1) looking at the shelxe plots ( these are displayed in the CCP4 GUI task - prob also in phenix? ) If there is good contrast for one hand rather than the other the sites are likely to be correct - if not - hmmm; there might be a reason but they could be wrong.. Then I check the sites by giving the strongest ones to phaser_ep and seeing if it suggests more which agree with SHELX ones.. Bias is not usually a problem if the initial build is correct but if the Rfactors are not coming down that may not be the case. Can you see sidechains - espec MET near your Se sites? Eleanor On 12 July 2013 04:12, Yuan SHANG shangyuan5...@gmail.com wrote: Dear All, I got a Se-crystal diffracted to 3.3A. The protein is predicted to be a coiled coil. After I got the heavy atom positions from a shelxd based programs from AutoRickShaw, I run phaser and phenix.autobuild to get a initial phase shown in the attachment. I fit a standard helix into the density maps. After that, I did a two step iteration to improve my phases similar to the methods mentioned in Roversi's paper With phases: how two wrongs can sometimes make a right. Step 1: Run DM with current model, heavy atom positions, the initial density maps to get a new DM map. Step 2: In the new DM map, some new density appears. Fit new helixes in this new map. The iteration ends when I found no obvious helix densities. However, when I run refinement, I still got very bad R/Rfree values. It seemed I got a lot of model bias during the process above. Now, I have some questions that puzzled me a lot. 1. Is there any programs that could help to compare and check the heavy atom positions in shelxd solutions? Especially in cases when the solutions are not obvious. SitCOM seemed very good, is there a 32-bit distribution of this programs? Or any others have similar functions? 2. How to reduce the model bias and phase errors in my situations? any suggestions? 3. Should I collect a MAD data instead of SAD data? How much would that could help? Best regards Many thanks! Yuan
Re: [ccp4bb] TLS refinement refmac
Oh dear - you should always start refinement against the original processed data - but others have told you that already.. The TLS files will not be appropriate for the rescaled FPs - it is surprising that the Rfactor actually goes down though! Or are you doing several cycles of refinement in thesecond pass too - in that case one would hope the Rfactor would continue to fall. Eleanor On 17 July 2013 11:00, Guenter Fritz guenter.fr...@uni-konstanz.de wrote: Am 17.07.2013 11:59, schrieb Guenter Fritz: Hi Stefan and Gottfried, thanks a lot for the answers. This is the point. Wouldn't it make more sense to add an extra column that contains the changed Fs? Best, Guenter Hi, it is strongly advised to use the original mtz e.g. scala.mtz as the refmac input mtz in all refmac runs, as this contains the original Fs - Refmac applies some aniso corrections to the Fs and puts them into the output.mtz. so the output Fs are not the same as in the input F - therefore one should use the scala.mtz cheers Stefan -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Guenter Fritz Gesendet: Mittwoch, 17. Juli 2013 10:39 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] TLS refinement refmac Dear all, one gets different R values, if you re-read in the mtz written out by refmac after TLS refinement. I think this issue had been a while ago in ccp4bb, but I can't find the right track. Here are the details. 1st run: If we do TLS + restr. refinement in refmac we get: InitialFinal R factor0.3010 0.2170 R free0.3175 0.2695 2nd run: Now, if we use the same input pdb and the same input tls paramter file, but use the mtz written out by the first run, we get: InitialFinal R factor0.2274 0.1903 R free0.2482 0.2540 Apparently in the mtz file written out by the 1st refmac must contain some information that is re-read in the 2nd run. But one just defines FP, SIGFP and Rfree flags. Do FPs change in the output mtz after TLS refinement?? Any help to clarify this is appreciated. Thanks, Guenter
Re: [ccp4bb] TLS refinement refmac
Well - most refinement procedures output the Fobs and the Fcalc on the same (more-or-less) absolute scale.. After data processing the observations are on a more or less arbitrary scale, so that seems sensible. It gets more problematic when you start correcting for anisotropy.. Eleanor On 17 July 2013 15:59, Phoebe A. Rice pr...@uchicago.edu wrote: EEK, it seems to me that anything called simply FP should be unadulterated FP. If the software modifies a column in some way, it should give it a new label, shouldn't it? ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Eleanor Dodson [eleanor.dod...@york.ac.uk] *Sent:* Wednesday, July 17, 2013 6:05 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] TLS refinement refmac Oh dear - you should always start refinement against the original processed data - but others have told you that already.. The TLS files will not be appropriate for the rescaled FPs - it is surprising that the Rfactor actually goes down though! Or are you doing several cycles of refinement in thesecond pass too - in that case one would hope the Rfactor would continue to fall. Eleanor On 17 July 2013 11:00, Guenter Fritz guenter.fr...@uni-konstanz.dewrote: Am 17.07.2013 11:59, schrieb Guenter Fritz: Hi Stefan and Gottfried, thanks a lot for the answers. This is the point. Wouldn't it make more sense to add an extra column that contains the changed Fs? Best, Guenter Hi, it is strongly advised to use the original mtz e.g. scala.mtz as the refmac input mtz in all refmac runs, as this contains the original Fs - Refmac applies some aniso corrections to the Fs and puts them into the output.mtz. so the output Fs are not the same as in the input F - therefore one should use the scala.mtz cheers Stefan -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Guenter Fritz Gesendet: Mittwoch, 17. Juli 2013 10:39 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] TLS refinement refmac Dear all, one gets different R values, if you re-read in the mtz written out by refmac after TLS refinement. I think this issue had been a while ago in ccp4bb, but I can't find the right track. Here are the details. 1st run: If we do TLS + restr. refinement in refmac we get: InitialFinal R factor0.3010 0.2170 R free0.3175 0.2695 2nd run: Now, if we use the same input pdb and the same input tls paramter file, but use the mtz written out by the first run, we get: InitialFinal R factor0.2274 0.1903 R free0.2482 0.2540 Apparently in the mtz file written out by the 1st refmac must contain some information that is re-read in the 2nd run. But one just defines FP, SIGFP and Rfree flags. Do FPs change in the output mtz after TLS refinement?? Any help to clarify this is appreciated. Thanks, Guenter
Re: [ccp4bb] Twin or underestimation of symmetry
This is a bit puzzling. Sticking to point groups: P3 P6 are sub groups of P6/mmm so data which merges in P6/mmm will always satisfy P3 and P6. And twinning in P3 or P6 will make the data seem to have higher symmetry. Four way twinning is unusual, but possible of course. But if you really have twinning, you should see it indicated in the pointless/aimless plots, or via Xtriage.. If those tests do not show it and you dont have a non-crystallographic translation twinning is unlikely. Your choice of SGs seem puzzling too. If the data is P31 (or P32) then you should see intensities for l=3n, and absences for l=3n+-1 If the data is P61 (or P65) then you should only see intensities for l=6n. If the data is P6322 you should only see intensities for l=2n. What are the absences along 00l? Eleanor On 29 July 2013 18:37, Jeffrey D Brodin jbro...@ucsd.edu wrote: Hi everyone, I have a dataset that's been giving me some trouble and wanted to get your ideas on the best way to proceed. The data extend to ~2.6 Å and scale integrate/scale well in P622. According to Pointless, the symmetry is either P622 or P6322, however, neither Molrep norm Phaser finds molecular replacement solutions in these space groups. If I run Phaser and let it check all other space groups it finds a solution in P6122, but I can only refine it to an Rfree value of ~35%. The data also scales well in P3 and P6 and I get molecular replacement solutions in space groups of either P31 or P61. However, the Refinement again stalls at an Rfree value of ~35%. I tried adding twin refinement in Refmac and it seems to be helping; The R/Rfree values dropped to 20.9/27.3 and the map appears much better compared to the previous refinements. The refined twin fractions are 0.26, 0.24, 0.24 and 0.25. Am I doing something wrong with the refinement or is this a legitimate way to treat the data. Thank you in advance for your help. * *
Re: [ccp4bb] R factor geeting stuck
that doesnt seem too bad an Rfactor to me! What do you expect? Eleanor On 27 August 2013 09:59, Afshan Begum afshan...@yahoo.com wrote: Hi ccp4 experts, I have collected diffraction images to 0.96 Angstrom resolution to the edge of the detector. One data set give me the full completeness and below i have pasted my statistic values got from XDS. I have cut off data at 1.12 A which seems to be quite nice regarding all necessary parameter to consider. But the problem is the Rfree is 0.176 and Rwork is 0.151 but the maps look very good. Even If I cut off the data to 1.15 Angstrom the R factors not improved. The space group of my complex is P21212. I used anisotropic Bfactors, add Hydrogen on and also used TLS but unfortunately i can not reduce able to reduce R factor in a good way. so, could you people kindly give me some suggestion how can i improve my data quality. RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 1.12 104562 21143 21268 99.4% 37.0% 37.8% 103982 4.0441.4%91.8* 00.767 15016 1.04 86588 21777 23100 94.3% 98.4% 95.8%84962 1.44 113.0%74.1*-10.700 10431 I would be thankful for your valuable comments AFSHAN
Re: [ccp4bb] Only refine Bs in Refmac?
What has happened to the CCP4 update procedure? Shouldnt this mean latest versions are on that web site? e On 4 September 2013 22:57, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Hi refine bref bonly should be what you are looking for. You may need to use the latest available version (5.8) from our LMB site: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ With best regards Garib On 4 Sep 2013, at 19:59, hari jayaram wrote: Hi, How does one only refine Bs in refmac without changing the model coordinates . Is this accomplished using a zero cycle refinement with b-refinement set. I have never had to do this till now and didnt know how to set it up. Thanks Hari Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Low resolution, twinning, pseudo-translation
First - you probably have the best solution you can hope for - Rs of 27% 33% are not great, but unlikely if you had a wrong answer. A query though: t is surprising to have a pseudo translation if you expect one molecule in the asymmetry unit .. an you elborate a bit on that? Is there a good reason to try to get more information from this 3A twinned data, if the same structure is already solved. If there is a good reason then it is worth persisting, but it will be difficult Eleanor On 5 September 2013 13:09, Ashu Kumar kumar1980a...@gmail.com wrote: Dear all, I am trying to solve the structure of a protein at ~3 A. Though the structure is already solved in a different space group. I thought it would be a straight forward MR problem. But there are a couple of problems: The possible point group is P3 (2 molecules / asu) and pointless and phenix.xtriage predicted it to be P321 ( 1 molecule /asu). But it shows 37% twinning with P3 whereas twinning is 0.055 % in P321. Several attempts to get a sensible MR solution, in either of the space groups, failed. R and Rfree were always in the range of 35-40 %. In both the cases there is a pseudotranslation of ~17%. Even different runs of the same inputs gave different solutions on multiple trials. After many trials, molrep gave one solution in P3 with 2 molecules/asu which when refined in Phaser with twinning corrections gave R and Rfree of 0.27 and 0.33 respectively. The map looks good and there is nothing really to do in terms of model-building. While my attempts to get better dataset are ON, I was wondering how to ensure that the solution/symmetry which I got are correct (or find out that they are wrong.) and also what next with this data sets in either cases to reach to a correct model? Any suggestion would be highly appreciated. Thanks Ashu
Re: [ccp4bb] About twin refinement in Refmac
It seems you cannot read either your data or a scratch mtz file - the log file should tell you its name. There is obviously something funny - see the discussion of free r reflections.. You dont say your pointgroup - presumably P/mmm or P3m or P6/mmm - they have that twin law. But with perfect twinning (both fractions 0.5) are you sure you dont have a higher symmetry spacegroup? Eleanor On 10 September 2013 12:08, miyatake miyat...@riken.jp wrote: Dear all, I run a twin refinement using Refmac, but it was in failure saying the message below; Why did it happen? H. Miyatake - free R flag 0 Number of free reflections 12993 Number of all reflections 14430 Warning == Number of free refletions is more than half all reflections Warning == Switching to free R flag = 1 Filtering out small twin domains, step 1 Twin operators with Rmerge 0.440 will be removed Symmetry operator K, H, -L : R_merge =0.068: twin or higher symmetry -- Filtering out small twin domains, step 2 Twin domains with fraction7.000E-02 are removed Twin operators with estimated twin fractions Twin operator: H, K, L: Fraction = 0.500; Equivalent operators: K, -H, L; -H, -K, L; -K, H, L Twin operator: K, H, -L: Fraction = 0.500; Equivalent operators: H, -K, -L; -K, -H, -L; -H, K, -L -- Error in LRREWD: mindx 1 not open for read! Error in LRREFL: mindx 1 not open for read! Error in LRREFM: mindx 1 not open for read! Error in LRREFL: mindx 1 not open for read! Error in LRREFM: mindx 1 not open for read! Error in LRREFL: mindx 1 not open for read! Error in LRREFM: mindx 1 not open for read!
Re: [ccp4bb] Why nobody comments about the Nobel committee decision?
This is amazing - I am so glad the Nobel committee has recognised this ground breaking, rather unglamorous work requiring great intelligence, very hard work, and a lot of disappointments! Congratulations to them all, and to the whole field. Eleanor Dodson On 10 October 2013 09:26, Alexandre OURJOUMTSEV sa...@igbmc.fr wrote: Hello to everybody, Alex, it was a great idea to initiate the conversation sending congratulations to our colleagues ! Bob, it was another great idea, when congratulating the Winners, to remind us of the framework. As one of my colleagues pointed out, we shall also give a lot of credits to Shneior Lifson who was in the very origins of these works, ideas and programs (see the paper by M.Levitt The birth of computational structural biology, Nature Structural Molecuar Biology, 8, 392-393 (2001); http://www.nature.com/nsmb/journal/v8/n5/full/nsb0501_392.html ). Older crystallographers may remember a fundamental paper by Levitt Lifson (1969). With best wishes, Sacha Urzhumtsev -Message d'origine- De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Sweet, Robert Envoyé : mercredi 9 octobre 2013 23:52 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] השב: [ccp4bb] Why nobody comments about the Nobel committee decision? It deserves comment!! I've been too busy talking with my friends about it to think of CCP4. This morning on NPR I heard Karplus's name and started to whoop and holler, and by the time they got to Arieh I realized they had a Hat Trick!! It's a spectacular thing that this field should get recognition! An interesting feature to me is that, at least when I was following the field, these three use physics to do their work, modeling with carefully estimated spring constants, etc., and eventually QM results. Those who use phenomenology -- hydrophobic volumes, who likes to lie next to whom, etc. -- are extremely effective (you know who they are), and they deserve credit. But they (we, some years ago) stand on the shoulders of the achievements of these three. It's good to remember the late, great, Tony Jack, cut down before reaching his prime. Bob From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Nat Echols [nathaniel.ech...@gmail.com] Sent: Wednesday, October 09, 2013 5:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] השב: [ccp4bb] Why nobody comments about the Nobel committee decision? Levitt also contributed to DEN refinement (Schroder et al. 2007, 2010). -Nat On Wed, Oct 9, 2013 at 2:29 PM, Boaz Shaanan bshaa...@bgu.ac.ilmailto:bshaa...@bgu.ac.il wrote: Good point. Now since you mentioned contributions of the recent Nobel laureates to crystallography Mike Levitt also had a significant contribution through the by now forgotten Jack-Levitt refinement which to the best of my knowledge was the first time that x-ray term was added to the energy minimization algorithm. I think I'm right about this. This was later adapted by Axel Brunger in Xplor and other progrmas followed. Cheers, Boaz הודעה מקורית מאת Alexander Aleshin aales...@sanfordburnham.orgmailto:aales...@sanfordburnham.org תאריך: 10/10/2013 0:07 (GMT+02:00) אל CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK נושא [ccp4bb] Why nobody comments about the Nobel committee decision? Sorry for a provocative question, but I am surprised why nobody comments/congratulations laureates with regard to recently awarded Nobel prizes? However, one of laureates in chemistry contributed to a popular method in computational crystallography. CHARMM - XPLOR - CNS - PHENIX-… Alex Aleshin
Re: [ccp4bb] changes in small sections of secondary structure
Being of an untrusting disposition, I would ask your collaborator for the reflection data as well as the PDBs - it is always a good idea to look at the maps when there is some unexpected structural feature! - The DB provides an electron density server, if the structures have been deposited, otherwise it is straightforward to generate a map yourself and look at it in coot. Eleanor Dodson On 21 October 2013 13:53, Antony Oliver antony.oli...@sussex.ac.uk wrote: Dear Mahesh, Are all the structures at similar resolution? Definition of secondary structure is, and can be, affected by the level of geometric restraints/constraints used in the refinement process. Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On Oct 21, 2013, at 11:55 AM, Mahesh Lingaraju wrote: Hello experts Thanks for your insights. For one of the structures, it turned out to be a rendering issue by pymol like matt pointed out. For the other, the residues are clearly in a less than ideal position. Even if I see deviation from the RMSD plots, i cannot be sure that the structure were refined ideally at those positions ( those are not my structures, i just have the pdb files from my collaborator). Thanks again, Mahesh P.S from what all of you are saying it sounds like those changes are not real, if I find that they could be Ill let everyone know.
Re: [ccp4bb] AW: [ccp4bb] Molecular Replacement using low sequence identity templates
I dont know about LLG scores - they seem extremely variable depending on the degree of sequence similarity you assign. However when you get an R/Rfree of 40%/47% that is a pretty good sign that at least something is correct. It isnt clear whether that is after you have placed 2 copies of the second component? Anyway - maybe try again with the refined component 2 and look for component 1 again? Eleanor On 18 October 2013 15:51, herman.schreu...@sanofi.com wrote: Dear Jan, There are a few things a would do in this case. The first is to check the processing to make sure the space group is really C2 and, although unlikely, not some other space group. The second thing would be to try to place the first component. From your email it is not clear to me whether or not you were able to place the first component after the second component had been placed. In your case, I would give both components to phaser and ask phaser to first place component 2 and then component 1. It might be that the correct solution gets rejected because of clashes, so I would also try to trim the first component, or to increase the number of allowed clashes in Phaser. Although you expect two copies of your heterodimer, you may have a crystal with a high solvent content and only one dimer in the asymmetric unit. In this case the crystal packing should make sense i.e. continuous crystal contacts in all three dimensions. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan Félix Gesendet: Freitag, 18. Oktober 2013 13:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates Dear all, I have a question regarding Molecular Replacement using low sequence identity templates. I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex (space group C 1 2 1, no twinning detected using xtriage). For the first component homologs are available, but for the other the best found template only has 20 % sequence similarity. Strangely, I cannot place the first component directly, but the second component can be placed (after trimming the template with chainsaw) using phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 NCS copies of the heterodimer are expected based on the unit cell parameters, only 1 copy of the second component gets placed. If I try to place the first component based on the .sol file of the first MR solution, the TFZ score for the second placement is only about 3.5, but if I then try to place this second MR solution (2 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000. However, none of the MR solutions I obtained seems to refine in PHENIX. Using autobuild does not lower the R/Rfree values which seem to get stuck at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the model.. Also, in every MR solution only half of the asymmetric unit seems to be filled, but phaser fails to place more units..As I am seriously starting to doubt the actual content of the crystals, I tried Nearest Cell to search for similar space group, but without any hits. So here is my question. Is it possible to get TFZ/LLG values this high in C 1 2 1 with a completely incorrect model by chance, or can I assume that this MR solution points out that what I think is in the crystal is actually there? And secondly, as I am a bit stuck here, are there any new strategies I can try to tackle this problem? Off course, experimental phasing is an option, but the crystals grew slowly over e few months and I only had 1 drop with 1 crystal, so reproducing the crystals might be though.. Thanks for any tips and best regards, Jan
Re: [ccp4bb] [ccp4bb] Molecular Replacement using low sequence identity templates
I guess my experience is out of date - please ignore comments on LLG! Eleanor On 21 October 2013 15:40, Randy Read rj...@cam.ac.uk wrote: Hi Eleanor, Yes, the initial LLG scores in Phaser are highly dependent on the assigned sequence identity, which is translated into an initial estimate of the effective RMSD of the model. However, the latest versions of Phaser refine the RMSD estimate at the end of the job and, assuming that two runs find the same solution and the refinement manages to converge (which it usually does these days), the LLG at the end should be pretty reproducible regardless of the assigned sequence identity. Best wishes, Randy On 21 Oct 2013, at 14:42, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I dont know about LLG scores - they seem extremely variable depending on the degree of sequence similarity you assign. However when you get an R/Rfree of 40%/47% that is a pretty good sign that at least something is correct. It isnt clear whether that is after you have placed 2 copies of the second component? Anyway - maybe try again with the refined component 2 and look for component 1 again? Eleanor On 18 October 2013 15:51, herman.schreu...@sanofi.com wrote: Dear Jan, There are a few things a would do in this case. The first is to check the processing to make sure the space group is really C2 and, although unlikely, not some other space group. The second thing would be to try to place the first component. From your email it is not clear to me whether or not you were able to place the first component after the second component had been placed. In your case, I would give both components to phaser and ask phaser to first place component 2 and then component 1. It might be that the correct solution gets rejected because of clashes, so I would also try to trim the first component, or to increase the number of allowed clashes in Phaser. Although you expect two copies of your heterodimer, you may have a crystal with a high solvent content and only one dimer in the asymmetric unit. In this case the crystal packing should make sense i.e. continuous crystal contacts in all three dimensions. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan Félix Gesendet: Freitag, 18. Oktober 2013 13:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates Dear all, I have a question regarding Molecular Replacement using low sequence identity templates. I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex (space group C 1 2 1, no twinning detected using xtriage). For the first component homologs are available, but for the other the best found template only has 20 % sequence similarity. Strangely, I cannot place the first component directly, but the second component can be placed (after trimming the template with chainsaw) using phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 NCS copies of the heterodimer are expected based on the unit cell parameters, only 1 copy of the second component gets placed. If I try to place the first component based on the .sol file of the first MR solution, the TFZ score for the second placement is only about 3.5, but if I then try to place this second MR solution (2 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000. However, none of the MR solutions I obtained seems to refine in PHENIX. Using autobuild does not lower the R/Rfree values which seem to get stuck at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the model.. Also, in every MR solution only half of the asymmetric unit seems to be filled, but phaser fails to place more units..As I am seriously starting to doubt the actual content of the crystals, I tried Nearest Cell to search for similar space group, but without any hits. So here is my question. Is it possible to get TFZ/LLG values this high in C 1 2 1 with a completely incorrect model by chance, or can I assume that this MR solution points out that what I think is in the crystal is actually there? And secondly, as I am a bit stuck here, are there any new strategies I can try to tackle this problem? Off course, experimental phasing is an option, but the crystals grew slowly over e few months and I only had 1 drop with 1 crystal, so reproducing the crystals might be though.. Thanks for any tips and best regards, Jan -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K
Re: [ccp4bb] Unusual electron density - any guesses??
Well I would start by flipping the carbonyl oxygen then see if the side chain can use the density - what are the B values for the neighbouring stuff? Eleanor On 25 October 2013 19:00, Patel, Joe joe.pa...@astrazeneca.com wrote: Is that a glycine in the sequence next to the Glu/Gln? Have you tried building a 50% occ of the backbone in that region in two conformations, and then a water molecule further up into the feature. The density over the carbonyl looks weak and you have some negative density there that might indicate mixed conformation. Just an idea, hard to tell from still images if my idea would work. Joe P -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jose Artur Brito Sent: Friday, October 25, 2013 1:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Unusual electron density - any guesses?? Dear All, I'm refining an X-ray structure to 1.6A resolution in BUSTER-TNT v2.10. The model is pretty much finished but I see a strange electron density that I can't imagine what it is. Please take a look at four snapshots in http://www.itqb.unl.pt/~jbrito/ITQB/ . Any pointers/guesses are most welcome. In short, I see an oblong piece of density coming straight out of the main-chain!! It doesn't refine as a chain of waters and any small piece of PEG doesn't refine properly either (actually, not sure if this would make any sense but since the crystallization condition is PEG3350 and gave it a try!!). The crystallization condition is PEG3350, Bis.Tris buffer, (NH4)2SO4 and NaI. The protein was purified from recombinant expression in E. coli with trivial reagents: Tris and Bis.Tris buffers, NaCl, glycerol, ... Wishing you all an excellent weekend, best regards, Jose -- * José Artur Brito, PhD* * * * Post-Doctoral Fellow * * Membrane Protein Crystallography Lab * * Instituto de Tecnologia Química e Biológica * * Oeiras - Portugal* * * * Tel.: +351.21.446.97.61 * * Fax: +351.21.443.36.44 * * * * E-mail: jbr...@itqb.unl.pt * * URL: http://mx.itqb.unl.pt *
Re: [ccp4bb] Is it possible for a ligand cif file from the CCP4 library to contain errors ?
There are errors in every branch of human endeavour I am sure! Please if you find something wrong. send your suggested correction to Garib Paul, and once there is a fix notify us all via the BB. CCP4 has always depended heavily on the users noticing the bugs Thank you for your observation. Eleanor On 29 October 2013 10:21, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jodie, except for the matter if there is 'right' and 'wrong' in science or rather 'good' and 'bad' models, Garib Murshudov maintains the cif-library and if you send him a compilation of possible fixes he would surely appreciate your input. Best, Tim On 10/29/2013 10:15 AM, Jodie Johnston wrote: Hi I wanted to check is it possible that there could be errors in the library cif files associated with CCP4 and coot ? I have noticed a couple of potential errors in a ligand cif file from the CCP4 library. Perhaps it is my error - I shall need to triple check it again but, if it appears not to be, is it possible to talk to someone about correcting the cif file ? Many Thanks Jodie - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.15 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFSb4wRUxlJ7aRr7hoRAu21AJ9u78CgI0EV2shsvQS7ghl1FbddxwCgjxfJ EfmUgIVb9xKGdzmxSaRsBbw= =7+vC -END PGP SIGNATURE-
Re: [ccp4bb] Comparison of Water Positions across PDBs
Well - years ago I wrote a program called watertidy to do just this - but it asigned waters as OH0 OH1 OH2 with a according to what atom it was H bonded too, and those names are not now permitted.. My way now is to read in a completed homologous structure - use SSM to fit it over the new one - extract the HOHs from structure 1 - add them to structure 2 - do some refinement and use COOT to decide which ones are valid - the density fit picture shows wonderful red bars for wrong'uns - then add more again using coot.. Eleanor On 30 October 2013 06:49, Joel Sussman joel.suss...@weizmann.ac.il wrote: For detailed examination of this topic, see: Koellner, G., Kryger, G., Millard, C. B., Silman, I., Sussman, J. L. Steiner, T. (2000). Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica. Journal of Molecular Biology 296, 713-735. http://www.ncbi.nlm.nih.gov/pubmed/10669619 best regards, Joel On 30 Oct 2013, at 01:35, Ed Pozharski epozh...@umaryland.edu wrote: http://www.ccp4.ac.uk/html/watertidy.html On 10/29/2013 04:43 PM, Elise B wrote: Hello, I am working on a project with several (separate) structures of the same protein. I would like to be able to compare the solvent molecules between the structures, and it would be best if the waters that exist in roughly the same position in each PDB share the same residue number. Basically, I want to compare solvent molecule coordinates and assign similar locations the same name in each structure. What would be the best strategy for re-numbering the water molecules such that those with similar coordinates in all the structures receive the same residue number? I'd appreciate any suggestions. Elise Blankenship -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] A photograph of the Arndt-Wonacott rotation camera?
one in their book, i am sure. eleanor On 30 Oct 2013, at 16:05, Gerard Bricogne wrote: Dear all, Apologies for such a retro and non-biological question, but would anyone have a photograph of an Arndt-Wonacott rotation camera that he/she would be willing to share? I collected data on the first two prototypes in the early seventies, then on one of the first commercial models, but I cannot find any images of this ground-breaking piece of equipement on the Web. I found images for the Enraf-Nonius precession camera and the CAD-4 diffractometer, but not for the A-W rotation camera. This would be for use as visual material in presentations, and I would gratefully acknowledge the source of it. Thank you in advance! With fingers crossed ... . Gerard. -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
Re: [ccp4bb] SA-omit map
What does a straight difference map look like? i.e. omit one nucleotide at a time, do a few cycles of refinement and then inspect the weighted difference map - SA may be too violent for your structure. Eleanor On 4 Nov 2013, at 06:36, dengzq1987 wrote: Dear all, Recently, I received the comments from referees, they asked for the SA-omit map of the ssDNA of our protein-DNA complex. They said that simulated annealing omit map better than a biased 2Fo-Fc. The ssDNA consists of seven thymidine nucleotide. Our data diffracted to 2.65A，but the data quality is not good and twin. We tried to produce SA-omit map using phenix. The map is really bad. Does anyone have suggestion to refine the map? Thank you! Bests, zq Deng 2013-11-04 dengzq1987
Re: [ccp4bb] shelx anamalous data
It is easy enough if you still ant to do it. I would feed both into f2mtz separately to make a mysadIplus.mtz and a mysadImin.mtz then use CAD to combine and change the default labels to something like I(+) SIGI(+) and I(-) SIGI(-) But as George says Why do you want it? That will change the way you proceed. Eleanor On 14 Nov 2013, at 21:08, George Sheldrick wrote: I'm not sure why you want to do that. If you wish to look at a map or poly-Ala trace from SHELXE, just read the .pdb and then .phs files into Coot directly. If you want to use them to make pictures with PYMOL, use Tim Gruene's SHELX2map. For further information please go to the SHELX homepage (Google knows where it is). George On 11/14/2013 10:09 PM, Yarrow Madrona wrote: I'm sorry, I have not used shelx before and didn't realize in my last post that the anamolous data is kept separate. I am planning on converting both the mysad.phs and mysad.pha to mtz files and then merge them. However, I am not sure of the column lables in mysad.pha. Does anyone know how to get this info? -Yarrow -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] I want to dock/align an EM envelope (MRC) into a DM averaging and/or solvent envelope (MSK).
There are ways - the EM map is your model equiv to a PDB file and you need to generate a set of structure factors from your map - the EM density is put into a large box and transformed by a program such as sfall. . Is that possible for you? There are problems of scaling etc etc.. On 15 November 2013 00:58, Francis Reyes francis.re...@colorado.edu wrote: Is there a single tool or suite of tools that addresses this? Or a CCP4 workflow if need be. Thanks! F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] SELF-ROTATION FUNCTION FROM MOLREP
First Q - how good is your data - is there no possibility of twinning or any other distraction? Second Q - To compare those results properly we need to know how the P2 and the P222 cell align - are the cell dimensions more or less the same? But the 2 plots you attach (and the list above) show both very strong 222 symmetry so the most likely assumption is that the pointgroup is P222. The next peak down the list is only 0.13 for one and 0.17 for the other pointgroup, which only borders on significance.. But this doesnt really prove anything - for example, if there is a flexible linker the 2 domains of each molecule may be in different relative orientations . Do you have any MR search model for the 2 domains? I would search with them and see what they predict - on the whole self rotation functions are most comprehensible AFTER the structure is solved! Eleanor On 18 November 2013 09:58, Monica Mittal monica.mitta...@gmail.com wrote: Dear CCp4 users, May anyone help me in interpreting the self-rotation function from molrep. Data can be indexed,scaled equally well in P21 and P222. This protein has two domain linked by a long flexible linker and get cleaved during crystallization. After crystallizing it in many condition, i believed that i found new condition where it may be full length. May anyone please suggest me by looking at the self-rotation function, how many molecule exist in ASU. Matthews coefficient suggest that there would be 2 copy of full length protein or four of truncated protein in P21 space group. For your interpretation, please find attached images of rotation function around K=180 Molrep self rotation peaks are P21 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -0.00 180.000.79 | | 335.78 -0.00 180.000.17 | | 490.00 -170.05 180.000.17 | | 558.71 180.00 180.000.14 | | 6 108.34 180.00 180.000.14 | | 7 117.39 180.00 180.000.13 | | 890.00 90.00 108.540.13 | | 990.00 -90.00 108.540.13 | | 10 144.50 -0.00 180.000.13 | | 1137.57 25.59 179.980.12 | | 12 148.100.00 180.000.12 | | 13 121.736.36 179.620.11 | | 1463.31 -42.27 180.000.11 | | 1590.00 90.00 162.290.11 | | 1690.00 -90.00 162.290.11 | | 1771.12 136.88 180.000.10 | | 1899.17 -180.00 180.000.10 | | 19 144.80 -161.87 180.000.10 | | 2090.00 -138.24 180.000.10 | | 2182.27 -76.45 180.000.10 | | 2290.00 90.00 115.940.10 | | 2390.00 -90.00 115.940.10 | | 24 159.16 27.07 180.000.10 | | 2595.08 -35.92 179.840.10 | | 26 113.86 -139.44 179.540.10 | | 2790.23 -0.00 89.320.10 | | 2820.18 -156.63 179.980.10 | | 29 116.90 -40.40 179.500.10 | | 3063.12 139.60 179.500.10 P222 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -21.41 180.000.13 | | 3 125.320.00 180.000.13 | | 4 151.18 -90.00 180.000.12 | | 590.00 -180.00 90.000.12 | | 6 141.32 26.82 180.000.12 | | 790.00 -42.59 180.000.11 | | 8 127.22 -25.21 180.000.11 | | 937.04 -14.48 180.000.11 | | 1059.17 -129.14 180.000.10 | | 1156.66 -174.09 180.000.09 | | 1240.85 144.82 180.000.09 | | 1354.16 -167.88 180.000.09 | | 14 109.29 -61.09 180.000.09 | | 15 123.12 -84.90 180.000.09 | | 1664.66 -93.79 180.000.09 | | 17 113.09 108.59 179.890.09 | | 1893.84 37.37 179.940.08 | | 1990.00 -33.41 180.000.08 | | 20 131.95 90.00 90.750.08 | | 2153.78 -144.59 180.000.08 | | 2249.15 -43.11 180.000.08 | | 2362.98 -56.60 180.000.08 | | 2461.97 138.06 179.910.08 | | 2596.77 59.47 179.900.08 | | 26 118.74 40.91 179.850.07 | | 27 100.14 57.97 179.860.07 | | 2859.74 -139.98 180.000.07 | | 2958.97 -141.14 180.000.07 | | 30 103.38 59.92 179.810.07 Many Thanks in advance for your kind help. THANK YOU
Re: [ccp4bb] translational pseudo symmetry
I guess you have checked that P43212 is a better match than P41212? (And that you are running REFMAC against an mtz file with the same symmetry as the input PDB - you may need to change the SG in the mtz header by hand. mtzutils hklin P41212.mtz hklout P43212.mtz symm P43212 end Or vice versa.. Sorry - THIS IS CRAZY but there you are.. Re the pseudo translation -Randy summs up the situation very clearly. I would build my model by hand actually but I am sure PHASER does itwell too! Something I dont understand but maybe it is to do with your patterson sampling. Peak 3 is a consequence of Pk 1 and Pk2 - Pk 5 is the consequence of Pk 1 and Pk4 but the peak heights dont exactly fit.. Eleanor On 18 November 2013 10:19, Randy Read rj...@cam.ac.uk wrote: Dear Dan, First, you don't want to reprocess in the smaller cell. What xtriage is saying is that, if *and only if* the translation detected in the Patterson map were an exact crystallographic translation, then you would get the smaller cell. However, in order for that to be a plausible hypothesis, the Patterson peaks would have to be near to 100% of the origin peak. You actually seem to have a very interesting case, where the Patterson peaks are related by multiples of approximately the same translation. If you take a translation of 1/2,1/2,1/6 and multiply it by 1, 2 and 3, you get something close to the three biggest peaks in your Patterson (taking account of lattice translations), and these are related by the Patterson inversion centre to what you get if you multiply by 4 and 5. So the six molecules should be related to each other by something close to a repeated translation of 1/2,1/2,1/6. (You should check this in the solution that you already have.) If this were exact, you would have a smaller cell, but it's not exact, and one way in which it is not exact is that the translations along z are not exactly multiples of 1/6. This is reminiscent of a structure that we recently collaborated with Mariusz Jaskolski and Zbyszek Dauter to solve (paper accepted for publication in Acta D). In that case, there are seven translations of approximately 0,0,1/7. The difficulty with cases like this is figuring out how to break the exact symmetry. Any solution that has approximately the right translations will basically fit the data, but you need to find the right combination of deviations from the exact symmetry to get an optimal answer. If you get the wrong deviations from exact symmetry, the refinement will stall, and this may be the problem that you're facing. You can deal with problems like this in Phaser by using the TNCS NMOL 6 command (to say that there are 6 copies related by repeated applications of the same translation). You should tell Phaser to use the 1/2,1/2,0.174 vector (TNCS TRA VECTOR 0.5 0.5 0.174), and hopefully this will break the symmetry in a way that subsequent rigid-body refinement can deal with. I'm happy to give you more advice on this, off-line, because this kind of case isn't something that we've figured out how to deal with automatically yet. The optimal approach probably involves getting a deeper understanding of commensurate modulation, which is another way of thinking about pseudo-translations. Best wishes, Randy Read On 18 Nov 2013, at 09:19, #CHEN DAN# chen0...@e.ntu.edu.sg wrote: Dear experts, I am working on one dataset (2.5A) which was processed using space group P43212 ( 107.9, 107.9, 313.7; 90, 90, 90). After running MR with 6 molecules in ASU and one round of refmac, the R factors are high (38%/45%). I ran phenix.xtriage and found that translational pseudo symmetry is likely present. It suggested that the space group is I4122 with the unit cell about 1/3 smaller (I paste the patterson analyses below). I tried to reprocess the data to get the suggested space group and unit cell using HKL2000. But the index always gives a long c axis about 313A. Could you provide any suggestions on how to proceed? Patterson analyses -- Largest Patterson peak with length larger than 15 Angstrom Frac. coord.:0.5000.5000.174 Distance to origin : 93.757 Height (origin=100) : 55.763 p_value(height) :3.018e-05 The reported p_value has the following meaning: The probability that a peak of the specified height or larger is found in a Patterson function of a macro molecule that does not have any translational pseudo symmetry is equal to 3.018e-05. p_values smaller than 0.05 might indicate weak translational pseudo symmetry, or the self vector of a large anomalous scatterer such as Hg, whereas values smaller than 1e-3 are a very strong indication for the presence of translational pseudo symmetry. The full list of Patterson peaks is: x y zheight p-value(height) ( 0.500, 0.500, 0.174 ) : 55.763 (3.018e-05) ( 0.500, 0.500,
Re: [ccp4bb] Weird MR result
Hmm - why should a translational peak not be along the 40 axis? Anyway othercell shows this Conversion of cell 40, 32, 101, 90, 101, 90 can give Laue groups C m m m 40.0 198.3 32.0 90.0 90.0 89.60.42 [h,h+2l,-k] Possible spacegroups: C 2 2 21 C 2 2 2 or C 1 2/m 1 40.0 198.3 32.0 90.0 90.0 89.60.42 [h,h+2l,-k] or C 1 2/m 1 198.3 40.0 32.0 90.0 90.0 90.40.42 [-h-2l,h,-k] or P 1 2/m 1 40.0 32.0 101.3 90.0 101.8 90.00.84 [-h,-k,h+l] That is so close to your input cell that twinning is a very likely option. What does thedata processing suggest - look at pointless or Xtriage or something Eleanor On 15 November 2013 18:18, Niu Tou niutou2...@gmail.com wrote: It may be helpful to add some information during index. HKL2000 could find four reasonable solutions: 40, 32, 101, 90, 101, 90 for P1 and P2 200, 40, 32, 90, 90, 90 for C2 and C222 It looks very strange to me since these two unit cells look differently, but during refinement the predicated spots are identical, and they produced similar quality data--at least from those output parameters. All solutions (including P21, C2221) have the 95% off origin peak and several minor ones. The 95% peak is at (0.5 0 0) on the 40 line, so if cut it into half, that dimension will be too small (20 only). HKL2000 also did not give any solution with one dimension as 20. Maybe I did not get a right index yet, I wonder any expert can tell something from these information? On Fri, Nov 15, 2013 at 6:41 AM, Melanie Vollmar melanie.voll...@sgc.ox.ac.uk wrote: Dear Niu, I had an interesting pseudo-translation case recently where my off-origin peak was located near the centre of the unit cell (fractions a=0.5, b=0.46, c=0.5) of a P222 symmetry. Processing and phasing in P222 looked reasonable and the model could be built. I had background density which I thought of as water. I got suspicious when I identified density for a helix which was near my build main chain but could not be joined and built or be accounted for by looking at symmetry mates. Moreover I got stuck in refinement with R/Rfree 25/30%. I could identify which part of the protein caused me the trouble on crystal packing and the appearance of the off-origin peak. In my case it was the C terminus. So I used a new construct with swapped purification tag (N to C terminus). This altered the peptide sequence for the C terminus and allowed the protein to pack nicely into I222. This turned my off-origin peak into a true symmetry operator. I also had reasonable processing and phasing results for P2 and C2. So besides the strength of your off-origin peak it may be off some use to look at the location. HTH Melanie From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Niu Tou [niutou2...@gmail.com] Sent: 14 November 2013 23:58 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Weird MR result Dear Phil, I used PHASER to do the task. I have double checked and both files have the same prefix, so they are from the same output. I have also checked the headers again, they have the same spacegroup. Actually I was trying to search for two different molecules but only one was found. The spacegeoup is P2 and I am quite sure it is not P21 from system absence. One possibility is that the space group was wrong, since there is a 95% off origin peak. There are several choices from data processing, P1, P2, C2 C222, all have this large off origin peak. I wonder if this 95% peak can tell some information? It will not surprise me if this result is incorrect, however how could these regular density be? Best, Niu On Thu, Nov 14, 2013 at 5:47 PM, Phil Jeffrey pjeff...@princeton.edu wrote: Hello Niu, 1. We need extra information. What program did you use ? What's the similarity (e.g. % identity) of your model. What's your space group ? Did you try ALL the space groups in your point group in ALL the permutations (e.g. in primitive orthorhombic there are 8 possibilities). 1a. My best guess on limited info is that you've got a partial solution in the wrong space group with only part of the molecules at their correct position. 2. I recently had a very unusual case where I could solve a structure in EITHER P41212 or P43212 with similar statistics, but that I would see interpenetrating electron density for a second, partial occupancy molecule no matter which of these space groups I tried (and it showed this when I expanded the data to P1). Might conceivably be a 2:1 enantiomorphic twin, in retrospect, but we obtained a more friendly crystal form. I hope you don't have something like that, but it's possible. Phil Jeffrey Princeton On 11/14/13 5:22 PM, Niu Tou wrote: Dear All, I have a strange MR case which do not know how to interpret, I wonder if any one had similar experiences. The output model does not fit into the map at all, as shown in picture
Re: [ccp4bb] translational pseudo symmetry
You would get a different MR solution in P41212 than in P43212 so you shouldnt test the SAME pdb in both SGS? Not sure I am understanding this though. Eleanor On 19 November 2013 05:02, #CHEN DAN# chen0...@e.ntu.edu.sg wrote: Hi Eleanor, I checked P43212 and P41212 by changing the header of mtz file and running refmac for the same PDB input. P43212 is a better match than P41212. Sincerely, Dan From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Eleanor Dodson eleanor.dod...@york.ac.uk Sent: Monday, November 18, 2013 8:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] translational pseudo symmetry I guess you have checked that P43212 is a better match than P41212? (And that you are running REFMAC against an mtz file with the same symmetry as the input PDB - you may need to change the SG in the mtz header by hand. mtzutils hklin P41212.mtz hklout P43212.mtz symm P43212 end Or vice versa.. Sorry - THIS IS CRAZY but there you are.. Re the pseudo translation -Randy summs up the situation very clearly. I would build my model by hand actually but I am sure PHASER does itwell too! Something I dont understand but maybe it is to do with your patterson sampling. Peak 3 is a consequence of Pk 1 and Pk2 - Pk 5 is the consequence of Pk 1 and Pk4 but the peak heights dont exactly fit.. Eleanor On 18 November 2013 10:19, Randy Read rj...@cam.ac.uk wrote: Dear Dan, First, you don't want to reprocess in the smaller cell. What xtriage is saying is that, if *and only if* the translation detected in the Patterson map were an exact crystallographic translation, then you would get the smaller cell. However, in order for that to be a plausible hypothesis, the Patterson peaks would have to be near to 100% of the origin peak. You actually seem to have a very interesting case, where the Patterson peaks are related by multiples of approximately the same translation. If you take a translation of 1/2,1/2,1/6 and multiply it by 1, 2 and 3, you get something close to the three biggest peaks in your Patterson (taking account of lattice translations), and these are related by the Patterson inversion centre to what you get if you multiply by 4 and 5. So the six molecules should be related to each other by something close to a repeated translation of 1/2,1/2,1/6. (You should check this in the solution that you already have.) If this were exact, you would have a smaller cell, but it's not exact, and one way in which it is not exact is that the translations along z are not exactly multiples of 1/6. This is reminiscent of a structure that we recently collaborated with Mariusz Jaskolski and Zbyszek Dauter to solve (paper accepted for publication in Acta D). In that case, there are seven translations of approximately 0,0,1/7. The difficulty with cases like this is figuring out how to break the exact symmetry. Any solution that has approximately the right translations will basically fit the data, but you need to find the right combination of deviations from the exact symmetry to get an optimal answer. If you get the wrong deviations from exact symmetry, the refinement will stall, and this may be the problem that you're facing. You can deal with problems like this in Phaser by using the TNCS NMOL 6 command (to say that there are 6 copies related by repeated applications of the same translation). You should tell Phaser to use the 1/2,1/2,0.174 vector (TNCS TRA VECTOR 0.5 0.5 0.174), and hopefully this will break the symmetry in a way that subsequent rigid-body refinement can deal with. I'm happy to give you more advice on this, off-line, because this kind of case isn't something that we've figured out how to deal with automatically yet. The optimal approach probably involves getting a deeper understanding of commensurate modulation, which is another way of thinking about pseudo-translations. Best wishes, Randy Read On 18 Nov 2013, at 09:19, #CHEN DAN# chen0...@e.ntu.edu.sg wrote: Dear experts, I am working on one dataset (2.5A) which was processed using space group P43212 ( 107.9, 107.9, 313.7; 90, 90, 90). After running MR with 6 molecules in ASU and one round of refmac, the R factors are high (38%/45%). I ran phenix.xtriage and found that translational pseudo symmetry is likely present. It suggested that the space group is I4122 with the unit cell about 1/3 smaller (I paste the patterson analyses below). I tried to reprocess the data to get the suggested space group and unit cell using HKL2000. But the index always gives a long c axis about 313A. Could you provide any suggestions on how to proceed? Patterson analyses -- Largest Patterson peak with length larger than 15 Angstrom Frac. coord.:0.5000.5000.174 Distance to origin : 93.757 Height (origin=100) : 55.763 p_value(height) :3.018e-05 The reported
Re: [ccp4bb] Orientation of molecules
If you suspect your MR solution is crystallographically correct but it does not represent the biological entity - eg - you will generate a dimer if you move molecule B by a symmetry operator such -x,y-1/2,1-z then the easy way is to submit the coordinates to PISA - either in the CCP4 GUI or send them to PDBe to search for quaternary structure, and let PISA sort out the best orientations of possible crystal symmetry Eleanor But Phils suggestion should do the same thing.. On 21 November 2013 18:24, Phil Jeffrey pjeff...@princeton.edu wrote: * Open molecular replacement solution in Coot * Display crystal packing (DrawCell Symmetry), perhaps as Calphas only * Find the symmetry-related instance of copyB that is in the correct position relative to copyA according to your preferences * Use FileSave Symmetry Coordinates to write the structure transposed by that operator (note: select the menu option, then click on the copyB instance) * Since Coot will write the entire structure transposed by that symop, assemble the desired solution from copyA from the mol.rep. solution and copyB from the transposed solution. I'm a Luddite so I use emacs and/or grep for this. Phil Jeffrey Princeton 11/21/13 1:11 PM, Appu kumar wrote: Dear All, I think i have not explained my problem precisely. This may be weird one but let me elaborate more. I have have a protein moleculeA, having N-term, and C-term end. Structurally, it is dimer with anti-parallel arrangement i.e N-terminal of one copyA of molecule form dimer in such a way that it copyB would be arranged in antiparallel fashioned (N-term of copyA is besides C-term of CopyB). So when i am searching for two copy of molecule in phaser it is giving me two copy of molecule in parallel arrangement. So my question is, how to tell phaser that after fixing the orientation of first copy, to change the orientation of 2nd copy with respect to first one so that their n-teminal and c-terminal lies beside each other. I am looking for your valuable suggestion. Thank you
Re: [ccp4bb] Refinement of data with pseudo translation symmetry
Well - your R values will probably appear higher than normal - there will be zones where all reflections are weak.. but the maximum likelihood targets are meant to deal with this reasonably well. It seems to work and the maps usually look OK! Eleanor On 25 November 2013 22:31, Niu Tou niutou2...@gmail.com wrote: Dear All, Does any body know if the existence of pseudo translation symmetry will affect refinement ? If it does, is there any keyword or method to avoid it? Thanks! Best, Niu
Re: [ccp4bb] AW: [ccp4bb] Phaser output problem
Things to check - number of molecules to search for. You can use Matthewscoeff to get a suggestion - MOLREP does it autromatically . Space group - has Phaser chosen as best SG an alternative to that in your header? Eleanor On 16 December 2013 11:03, herman.schreu...@sanofi.com wrote: Looks like you need to search for more molecules. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Amanda Blythe *Gesendet:* Montag, 16. Dezember 2013 11:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Phaser output problem I am trying to solve a structure by molecular replacement using Phaser. The pdb fits the density really well but the output shows strange crystal packing. I've attached an image, does anyone know what is causing this? Amanda PhD student Laboratory 4.26, Bayliss Building School of Chemistry and Biochemistry M313 The University of Western Australia 35 Stirling Highway Crawley WA 6009 Australia *T* (+61 8) 6488 3163 *E* lewis...@student.uwa.edu.au image001.jpg
Re: [ccp4bb] Heavy atom sites
I would find the sites from the PHIC - you need to use CAD to add Fcalc PHIC and FOM to the original data with Fnative Fderiv DANOderiv etc I usually then use SCALEIT to scale native and derivative to Fcalc - then you know you are roughly on an absolute scale Then feed those sites into Phaser_EP and it will refine occupancy etc and give you phasing power Eleanor On 16 December 2013 09:21, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Hello David, I would use the SAD target function of refmac5 for the anomalous occupancy. As of isomorphous occupancy and phasing power, I don't know. Best, Tim On 12/15/2013 10:29 PM, David Schuller wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Heavy atom sites
Yes - mlphare did, but so does Phaser_ep E On 16 December 2013 18:46, Bosch, Juergen jubo...@jhsph.edu wrote: Didn't mlphare use to print those values in the log file ? Jürgen On Dec 15, 2013, at 4:29 PM, David Schuller dj...@cornell.edu wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] R-factors from SfCHECK versus R-factors from PHENIX
SFCHECK is a very quick and dirty report generator - On 15 January 2014 21:42, Pavel Afonine pafon...@gmail.com wrote: Hi Ursula, you will find answers here: http://www.phenix-online.org/papers/he5476_reprint.pdf Pavel On Wed, Jan 15, 2014 at 1:38 PM, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote: I am submitting a structure to the PDB database. The SfCHECK summary report provided by the PDB validation shows an R-factor for model vs structure factors of 0.32, while the R-factor from the refinement program PHENIX is 0.21. I am not familiar with SfCHECK, but I am puzzled how these programs can calculate such different R-factors. I would be thankful for some explanation. Ursula -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220 (510) 642 8766
Re: [ccp4bb] Two P1 xtals with same xtal contacts give 2 different asymmetric units
You dont say whether the unit cells are the same? In cases like this I sbmit both sets of coordinates to PISA - to get the preferred biological unit and check whether each structure has similar contacts. PISA is distributed with CCP4 but the version at the EBI pdb gives prettier results! Eleanor On 23 January 2014 15:19, Yong Wang wang_yon...@lilly.com wrote: Gabriel, Could this be just different but equivalent way of defining the asu? Ignoring one of the two tetramers and just focusing on the one tetramer that looks different in your case, the following picture assumes objects ABCD form a tetramer and repeat themselves in P1. You can have one trimer (ABC) plus a D from a symmetry related object as enclosed in the blue box. Then the other equivalent assembly is two dimers (BD and AC) as enclosed in the red box. This assumes that the “void” you referred to actually contains electron density for one monomer, not real void as having empty density. The equivalent assembly of asu can happen to any crystal form but if you try to keep the equivalent molecules together, it may appear more easily in P1 due to the arbitrary origin shift in P1. Cheers, *Yong Wang, Ph.D. Research Advisor, Discovery Chemistry Research* Eli Lilly Company Phone: 317-655-9145 Lilly Corporate Center DC 0403 Fax: 317-651-6333 Indianapolis, IN 46285 wang_y...@lilly.com CONFIDENTIALITY NOTICE: This e-mail message from Eli Lilly and Company (including all attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Gabriel Moreno *Sent:* Wednesday, January 22, 2014 3:50 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Two P1 xtals with same xtal contacts give 2 different asymmetric units Dear CCP4 Contributors, I have a bit of a mystery: Two co-crystals that I picked up from the same grid tray (the two conditions vary slightly in %PEG and [salt], both indexed as P1 (apo structure normally crystallizes in P3221). One dataset was indexed, integrated and scaled with HKL2000. The other was processed with MOSFILM (could not process in HKL2000). Downstream processing for both sets was done exactly the same in PHENIX. Though both asymmetric units contain two complete tetramers, the interesting thing is that the configuration of monomers is different between the solutions. One contains one complete tetramer, one trimer (with a void where the fourth monomer would be), and one monomer on off on its own. The asymmetric unit of the other dataset solution also contains a complete tetramer, but then has two dimers. Close analysis of contacts between symmetrically related molecules reveals that the crystal packing is exactly the same between the two solutions from the two datasets. Also, the Rwork and Rfree for both models are 0.18 and 0.20. Other quality indices are also comparable between the two sets. Here's my question: Does this phenomenon reveal anything important, or is this type of thing just seen sometimes with P1 solutions from crystals of the same protein and condition (more or less). I hope I have been clear. Thanks! Gabriel image001.png
Re: [ccp4bb] twinning fun
Dont forget that with twinning in apparent point group PG6/mmm the true SG may be P6i or P3i21 See the twinning notes: http://www.ccp4.ac.uk/dist/html/twinning.html Detecting twinning can be problematic - My rule of thumb, following the procedure od ctruncate:: 0) Check the matthews coefficient for likely number of molecules. Half a molecule must mean you are assigning too high a symmetry count. Lots of molecules means you need to check for non-crystallographic translation etc. 1) Look at the I^2/I^2 plot after correction for anisotropy If it isnt reasonably straight with resolution you probably have some data problems, and these can make all the tests pretty useless. 2) Is there a NC translation - truncate tells you that. If not, and the data is OK, you are unlikely to have twinning if I^2/I^2 for acentrics is ~ 2, and the L test looks OK. H test and Britten tests a bit more influenced by other NC symmetry considerations 3) If there IS NC translation I^2/I^2 for acentrics will probably be 2 but the L test is still pretty reliable. Good luck Eleanor experimental phasing is tricky with perfect twinning but it has been done. Sorry I have forgotten reference though.. Eleanor On 29 January 2014 09:17, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Dear Bert, as Dirk has pointed out, if P622 is the correct space group, then the twinning statistics printed out if you process in P6 are meaningless. Intensity statistics, like the ratio of I^2/I^2 , can be misleading if there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of NCS on the value of the ratio of I^2/I^2 is opposite to that of twinning. Thus if a crystal is twinned and has NCS, you might not notice any problem in the ratio of I^2/I^2 . The other statistics, like Britton and H-test, present the intensity statistics in a different way, but from my understanding do not give substantially different information. The L-test does look at a different kind of information and therefore gives additional insight. If your measurements suffer from high background, diffuse scatter, ice rings, smeared reflections, additional crystals in the beam, or any other pathology, then all these tests may give distorted answers. In other words, even if twinning is not really present, any test designed to convert the deviation of data from ideality into an estimate of the twinning fraction will give you an alpha 0. So my experience is: if your data are very good, then the tests give good answers; if the data are mediocre or bad, don't necessarily believe the numbers. Finally, it's not only twinning of P6 that would give you P622, it's also twinning of P3x21, P3x12 that gives P6y22. Hope this helps, Kay On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: Dear all, I recently collected several datasets for a protein that needs experimental phasing. The crystals are hexagonal plates, and (automatic) data processing suggests with high confidence that the space group is P622. This is where the fun begins. For some datasets (processed in P622), the intensity distributions are normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is no twinning (twinning fractions 0.05). However, for other datasets (same cell dimensions), the intensity distributions are not normal (eg Z-scores 10). Given that twinning is not possible in P622, this suggests to me that the real space group could be P6 with (near) perfect twinning. If I now process the normal L-test P622 datasets in P6, the twin-law based tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), suggesting all my data is twinned. Does this make sense (ie can one have twinning with normal intensity distributions)? If it does, would the normal L-test datasets have a higher probability of being solvable? Is there any strategy for experimental phasing of (near) perfect twins? SAD would be more suitable than SIR/MIR? (I also have potential heavy atom derivatives). Thanks for any insights! Bert
Re: [ccp4bb] Examples of multiple ASU copies with different conformations
Insulin can take very different structures for 20% of the residues in different conditions, and there are several examples of T3R3 hexamers, with one T and one R in the asymmetric unit. There are even observations of the transformation occurring within the crystal, which looked battered but still diffracted to some extent. Examples are: 2tci 3mth 1mpj Eleanor Dodson On 30 January 2014 11:06, Bernhard Rupp hofkristall...@gmail.com wrote: There is one additional point perhaps worth making: As already noted in the thread, if you have a NCS homo-oligomer, the different copies in general have different environment, and proper inspection of the contacts reveals the details. On multiple occasions during inspections and review I have noticed that such is not always interpreted or welcome as a functionally necessary manifestation of the plasticity of the protein in question. In case of binding sites, in contrast it occurs that the 'best' one where a ligand actually exists or assumes a pose/environment perceived as useful for the proposed hypothesis is picked as the representative (figure), and from there the story evolves. This misses the point, and somewhat reeks of confirmation bias (the neglect of negative or 'unsuitable' results) leading straight down the road to scientific serfdom in terms of becoming a slave of one's own (pre)conceptions Best regards, BR On Wed, Jan 29, 2014 at 10:24 AM, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Hi Shane, some crystal forms of trimeric AcrB (a multi-drug resistance secondary transporter) have 3 (or 6) monomers in the ASU and these are substantially different, which suggests how the protein functions. One reference is e.g. Seeger et al. (2006) Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism Science 313, 1295-1298 DOI: 10.1126/science.1131542 (sorry for the self-plug!) best, Kay On Mon, 27 Jan 2014 13:08:33 -0500, Shane Caldwell shane.caldwel...@gmail.com wrote: Hi ccp4bb, I'm putting together a talk for some peers that highlights strengths and weaknesses of structural models for the outsider. For one point, I'd like to find some examples of proteins that show very different conformations between different copies in the ASU. One example I know of is c-Abl (1OPL), which crystallizes with both autoinhibited and active forms in the ASU, with dramatically different domain organization. I'd like to find some additional examples - can anyone suggest some other structures that have multiple copies with large structural variations? Thanks in advance! Shane Caldwell McGill University -- - Bernhard Rupp (Hofkristallrat a. D) 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - The hard part about playing chicken is to know when to flinch -
Re: [ccp4bb] R factor from merged data
I am afraid there is no real solution except to read the paper! And even that doesnt help if you suspect the merging.. There is lots of discussion about the merit of depositing UNMERGED data - and that would certainly help developers. Add your voice to the request! Eleanor On 30 January 2014 08:26, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: I think the name says it all: Rmerge merged data So no, there wouldn't be. You'll find it in the header. On 30/01/2014 08:18, Fulvio Saccoccia wrote: Dear ccp4 users, does anyone know if there is a way to (re)calculate the Rmerge from a deposited .cif file in PDB? In this case is an already merged structure factor file. I know that the EDS from Uppsala makes it, providing the PDB entry, but I need a procedure in order to do it by myself. I see that ccp4i utilities only accept unmerged .mtz; have you got any advice? Thanks in advance Fulvio -- Fulvio Saccoccia, PhD Dept. of Biochemical Sciences Sapienza University of Rome 00185, Rome (Italy) Phone: +39 06 4991 0556
Re: [ccp4bb] high Rwork / Rfree after MR
Well - that depends on many things. Are you sure of the spacegroup? Those R factors sometimes indicate a wrong selection of SG P212121 instead of P21212 for example.. Are you sure the data is not twinned? Do the maps show features which are not in the model? etc etc Eleanor On 4 February 2014 09:36, Shanti Pal Gangwar gangwar...@gmail.com wrote: Dear All I have solved one structure by MR. The data data quality was poor so the Rmerge was high. The resolution of the data is 3.3 Angstrom.The refinement statistics are also very poor Rw/Rf= 40/42 %. After many efforts we are not able to get reasonable Rw/Rf. My question is can it be claimed that we have solved the structure? Thanks in advance -- regards Shanti Pal Gangwar School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.com
Re: [ccp4bb] Sister CCPs
Well - we ought to be! There is an awful lot of unpredictable slog goes on before we have the fun! Eleanor On 13 February 2014 12:50, Mike S mds...@gmail.com wrote: I'm sorry, but did you just use the words crystallographers and modest in the same sentence? :-) On Thu, Feb 13, 2014 at 6:41 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I agree with Frank - it keeps crystallographers modest to know how challenging wet lab stuff still is.. Eleanor On 12 February 2014 19:23, Robbie Joosten robbie_joos...@hotmail.com wrote: It's not an e-mail bulletin board, but Researchgate seems to be quite popular for wet lab questions. IMO the QA section of the social network is a bit messy. That said, the quality seems to improve gradually. Cheers, Robbie Sent from my Windows Phone Van: Paul Emsley Verzonden: 12-2-2014 19:23 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] Sister CCPs On 12/02/14 15:59, George Sheldrick wrote: It would be so nice to have a 'sister CCP' for questions aboud wet-lab problems that have nothing to do with CCP4 or crystallographic computing, The is clearly a big need for it, and those of us who try to keep out of wet-labs would not have to wade though it all. FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as: /The CCP4BB mailing list is for discussions on the use of the CCP4 suite, and macromolecular crystallography in general./ Thus wet-lab questions are not off-topic (not that anyone recently described them as such). Having said that, Jiscmail mailing lists are easy to set-up (providing that you can reasonably expect that the mailing list will improve knowledge sharing within the UK centered academic community) and relatively low maintenance. I, for one, would not be entirely unhappy to miss out on questions about lysis. Paul.
Re: [ccp4bb] How to find the unfound part of a big protein
The very best advice is: get better data if at all possible. Things are much easier and more informative at 3A than at 4.2A and even better at 2.5A. There are various tricks which might help improve diffraction.. But if you are stuck then: The partial MR phases are very useful for checking your derivatives - look at difference Fouriers and anomalous Fouriers to see if anything has bound.. Then if you do have substitution Phaser SAD + MR will help to improve phases. If there is NCS then you can use Parrot or DM to improve the phases substantially. Buccaneer is quite good at building missing parts - we are experimenting with different scenarios to optimise this.. Eleanor On 14 February 2014 09:41, Guenter Fritz guenter.fr...@uni-konstanz.de wrote: Dear Sun, I had a similar problem. If you have a good TaBr dataset this should give you good phases. You can combine the TaBr phases and Molrepl phases in SHARP. What worked in my hands very well and is easy to do: SAD using Phaser and the partial Mol Repl Model. You find here a input file for such a scenario: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Phenix#phenix.phaser_-_SAD_phasing_with_Phaser Or simply use the CCP4 gui Phaser and 'SAD with molecular replacement partial structure' After DM or parrot you might get some interpretable density even at 6.5 A. If you don't have a good Mol Repl Model for the missing part you might try http://toolkit.tuebingen.mpg.de/hhpred to look for structures that are not that closely related. SeMet can help a lot in this case too. You will get the SeMet positions at low resolution with Phaser as described above. The SeMet positions will guide you to place a model into a very crude density. HTH, Guenter Dear Crystallographers, I'm working on a ~90KDa membrane protein, with big extracellular part, probably function as dimer. Now we have dataset to ~4.2 Angstrom and using extracellular homolog structure we can find a solution for this part(~45% of the whole molecule MW) through molecular replacement, and the molecules are packed as layers, and the other part are presumably between these layers. However, we are having trouble to fit the rest of the protein even though there're some density between the solved part. Rfree is at 40% now. We're trying to do heavy atom soaking, such as TaBr. We collected data for MIR but it's not helping so far. (Can I combine these MIR data with the native dataset because the MIR set is only at ~6.5 Angstrom)? Other information: this protein is expressed in Sf9 cells (so very hard to do Se-Met derivatives). The crystals is nice and big and cubic. Any suggestions or examples? Thanks a lot. Bingfa
[ccp4bb]
One way is this: Align Domain1 of structure1 to Domain1 of structure 2 on domain 1 (using LSQKAB or whatever.) Then align domain2 of the output aligned structure 1 to domain 2 of structure 2 The polar Chi or Kappa or whatever output from that alignment is the angular shift required Eleanor On 22 Feb 2014, at 06:19, avinash singh wrote: Dear CCp4b users, I have a protein which has been crystallized in two different conditions. In one of those conditions, the structure shows the domain shifting. Is there any programme or online server which calculates the angular shift in domain when campared to the other condition crystallized structure which shows no such domain shift. Thanks in advance Avinash Singh
Re: [ccp4bb] Wilson plot of TRUNCATE
The Best column gives Wilson plot values obtained according to this reference: From the Arp/Warp page: We have implemented an expected Wilson plot derived by Popov Bourenkov (2003) On 26 February 2014 10:04, Tao Zhang zhang...@cryst.iphy.ac.cn wrote: Dear All, Recently, I have used TRANCATE to process some data. There is a Wilson plot table in its log file. The first column of table is resolution, the second is ln(I/I_th). And the third column is named Best. I want to know what mean the best. Is it the theoretical estimate of Wilson plot? Best wishes, Tao Zhang
Re: [ccp4bb] unusually low B-factors
I think it may well be a problem with the scaling. It is hard to get a reasonable estimate of the Wilson B at 3A - especially in a small cell. Look at the Log Graph plots - one associated with R factor gives you Fobs and Fcalc v resolution. They SHOULD overlap pretty well, but sometimes they dont and sometimes that is because the OVERALL B is badly estimated. Of course it is a function of the data but often hard to correct.. Eleanor On 27 February 2014 16:26, Paul Paukstelis shocksofmig...@gmail.com wrote: Greetings, We've been working on a number of related structures. Crystals are in P64 with fairly small unit cell (40,40,55) and only diffract to around 2.5-3.0, so we aren't working with a lot of reflections. For several of these data sets the maps look very reasonable but Refmac refinement gives us unusually low B-factors after either individual or grouped refinement (average of around 10 A2 with some residues around 2 A2) while others are more 'normal' (avg. 30-40) with very similar maps. I suspect something in data processing, but I was wondering if there are opinions about what I should look for in cases like this. Thanks in advance. --paul
Re: [ccp4bb] CCP4 lib file
There are lots of GLUCOSE type lib files for different sugars. One way to find what is available is: From the REfinement GUI click Monomer library sketcher You get a window with File in the top LH corner Click on that and ask for Read monomer from library Then enter a key word GLUCOSE and you get this list ADQ etc etc Select what you want and go from there.. Or do this crude method to get a complete list!: [ccp4@roo timm]$ grep -i glucose $CLIBD/monomers/*/* /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/a/ADQ.cif:ADQ ADQ 'ADENOSINE-5'-DIPHOSPHATE-GLUCOSE' non-polymer61 38 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/a/AGL.cif:AGL AGL '4,6-DIDEOXY-4-AMINO-ALPHA-D-GLUCOSE ' pyranose 24 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/b/BG6.cif:BG6 BG6 'BETA-D-GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/b/BGC.cif:BGC BGC 'BETA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/d/D6G.cif:D6G D6G '2-DEOXY-GLUCOSE-6-PHOSPHATE ' pyranose 26 15 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/d/DDA.cif:DDA DDA '2,6-DIDEOXY-BETA-D-GLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G16.cif:G16 G16 'ALPHA-D-GLUCOSE 1,6-BISPHOSPHATE' pyranose 30 20 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G1P.cif:G1P G1P 'ALPHA-D-GLUCOSE-1-PHOSPHATE ' pyranose 27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G2F.cif:G2F G2F '2-DEOXY-2FLUORO-GLUCOSE ' pyranose 23 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G4D.cif:G4D G4D '4-DEOXY-ALPHA-D-GLUCOSE ' pyranose 23 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6D.cif:G6D G6D '6-DEOXY-ALPHA-D-GLUCOSE ' non-polymer23 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6P.cif:G6P G6P 'ALPHA-D-GLUCOSE-6-PHOSPHATE ' pyranose 27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6Q.cif:G6Q G6Q 'GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GDA.cif:GDA GDA '4-DEOXY-4-AMINO-BETA-D-GLUCOSE ' non-polymer25 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GFP.cif:GFP GFP '2-DEOXY-2-FLUORO-ALPHA-D-GLUCOSE-1-P' pyranose 26 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLC-B-D.cif:GLC-b-D GLC 'beta_D_glucose ' D-pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLC.cif:GLC GLC 'ALPHA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLD.cif:GLD GLD '4,6-DIDEOXYGLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLF.cif:GLF GLF '1-FLUORO-ALPHA-1-DEOXY-D-GLUCOSE' pyranose 23 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLO.cif:GLO GLO 'D-GLUCOSE IN LINEAR FORM' non-polymer24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLT.cif:GLT GLT '5-DEOXY-5-THIO-ALPHA-D-GLUCOSE ' non-polymer24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GMM.cif:GMM GMM 'GLUCOSE MONOMYCOLATE' non-polymer 200 74 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GUD.cif:GUD GUD 'GLUCOSE-URIDINE-C1,5'-DIPHOSPHATE ' non-polymer58 36 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/i/ISX.cif:ISX ISX 'GLUCOSE BETA-1,3-ISOFAGAMINE' non-polymer44 21 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/k/KBG.cif:KBG KBG '2-KETO-BETA-D-GLUCOSE ' non-polymer22 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:ADQ ADQ 'ADENOSINE-5'-DIPHOSPHATE-GLUCOSE' non-polymer61 38 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:AGL AGL '4,6-DIDEOXY-4-AMINO-ALPHA-D-GLUCOSE ' pyranose 24 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:BG6 BG6 'BETA-D-GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:BGC BGC 'BETA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:D6G D6G '2-DEOXY-GLUCOSE-6-PHOSPHATE ' pyranose 26 15 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:DDA DDA '2,6-DIDEOXY-BETA-D-GLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:G16 G16 'ALPHA-D-GLUCOSE 1,6-BISPHOSPHATE' pyranose 30 20 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:G1P G1P 'ALPHA-D-GLUCOSE-1-PHOSPHATE ' pyranose 27 16 .
Re: [ccp4bb] CCP4 lib file
Have you run refmac with the Review restraints option - that generates LINKs if they seem necessary. Or can you use the SUGAR links already set upin the library? Eleanor On 7 March 2014 11:51, Remie remiefa...@gmail.com wrote: Thanks Eleanor, I ended up using a glucose ligand lib file from a different protein that my friend used and refinement Refmac5 worked out. However I need to add the LINK Command in PDB so CCP4 recognizes that glucoses connected. Can I get help please? Thank you, Remie On Mar 5, 2014, at 10:49 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: There are lots of GLUCOSE type lib files for different sugars. One way to find what is available is: From the REfinement GUI click Monomer library sketcher You get a window with File in the top LH corner Click on that and ask for Read monomer from library Then enter a key word GLUCOSE and you get this list ADQ etc etc Select what you want and go from there.. Or do this crude method to get a complete list!: [ccp4@roo timm]$ grep -i glucose $CLIBD/monomers/*/* /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/a/ADQ.cif:ADQ ADQ 'ADENOSINE-5'-DIPHOSPHATE-GLUCOSE' non-polymer61 38 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/a/AGL.cif:AGL AGL '4,6-DIDEOXY-4-AMINO-ALPHA-D-GLUCOSE ' pyranose 24 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/b/BG6.cif:BG6 BG6 'BETA-D-GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/b/BGC.cif:BGC BGC 'BETA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/d/D6G.cif:D6G D6G '2-DEOXY-GLUCOSE-6-PHOSPHATE ' pyranose 26 15 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/d/DDA.cif:DDA DDA '2,6-DIDEOXY-BETA-D-GLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G16.cif:G16 G16 'ALPHA-D-GLUCOSE 1,6-BISPHOSPHATE' pyranose 30 20 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G1P.cif:G1P G1P 'ALPHA-D-GLUCOSE-1-PHOSPHATE ' pyranose 27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G2F.cif:G2F G2F '2-DEOXY-2FLUORO-GLUCOSE ' pyranose 23 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G4D.cif:G4D G4D '4-DEOXY-ALPHA-D-GLUCOSE ' pyranose 23 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6D.cif:G6D G6D '6-DEOXY-ALPHA-D-GLUCOSE ' non-polymer23 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6P.cif:G6P G6P 'ALPHA-D-GLUCOSE-6-PHOSPHATE ' pyranose 27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6Q.cif:G6Q G6Q 'GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GDA.cif:GDA GDA '4-DEOXY-4-AMINO-BETA-D-GLUCOSE ' non-polymer25 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GFP.cif:GFP GFP '2-DEOXY-2-FLUORO-ALPHA-D-GLUCOSE-1-P' pyranose 26 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLC-B-D.cif:GLC-b-D GLC 'beta_D_glucose ' D-pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLC.cif:GLC GLC 'ALPHA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLD.cif:GLD GLD '4,6-DIDEOXYGLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLF.cif:GLF GLF '1-FLUORO-ALPHA-1-DEOXY-D-GLUCOSE' pyranose 23 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLO.cif:GLO GLO 'D-GLUCOSE IN LINEAR FORM' non-polymer24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLT.cif:GLT GLT '5-DEOXY-5-THIO-ALPHA-D-GLUCOSE ' non-polymer24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GMM.cif:GMM GMM 'GLUCOSE MONOMYCOLATE' non-polymer 200 74 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GUD.cif:GUD GUD 'GLUCOSE-URIDINE-C1,5'-DIPHOSPHATE ' non-polymer58 36 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/i/ISX.cif:ISX ISX 'GLUCOSE BETA-1,3-ISOFAGAMINE' non-polymer44 21 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/k/KBG.cif:KBG KBG '2-KETO-BETA-D-GLUCOSE ' non-polymer22 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:ADQ ADQ 'ADENOSINE-5'-DIPHOSPHATE-GLUCOSE' non-polymer61 38 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:AGL AGL '4,6-DIDEOXY-4-AMINO-ALPHA-D-GLUCOSE ' pyranose 24 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:BG6 BG6 'BETA-D-GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:BGC BGC 'BETA-D-GLUCOSE
Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
You dont say what resolution you are working at, or what the current R factor is. or how complete the model is. There are assumptions made in the refinement scaling algorithms and in their treatment of supposedly poorly ordered solvent which can generate false density (both positive and negative) on the boundaries of the model. And as well as Herman says the Sigma level is set globally whereas the actual density locally is affected by the B values. One of the strengths of COOT is that it is easy to adjust the sigma level at local regions. Eleanor On 11 March 2014 07:42, Amlan Roychowdhury amlan.iit...@gmail.com wrote: Dear All, Thank you very much for your reply. I have an another doubt and I want to discuss with you. Sometimes for some structure during refinement and model building we have found a green blob (Fo-Fc and 5 sigma). If I scroll down the blue 2Fo-Fc map to a very low sigma level nearly at 0.5 a really non convincing density will appear. And from the structural point of view, it will be very difficult to put anything within it due to its position within the structure. as an example once I have found an elongated green density without any trace of blue in between a helix and a beta sheet. Is it due to noise? The structure was well refined. What should we do in such cases? Regards amlan. On Mon, Mar 10, 2014 at 4:41 PM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Amlan Roychowdhury *Gesendet:* Montag, 10. März 2014 09:09 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
Re: [ccp4bb] twinning problem ?
Sorry - hadnt finished.. The twinning tests are distorted by NC translation - usually the L test is safe, but the others are all suspect.. On 11 March 2014 14:09, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: What is the NC translation? If there is a factor of 0.5 that makes SG determination complicated.. Eleanor On 11 March 2014 14:04, Stephen Cusack cus...@embl.fr wrote: Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions Stephen -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email: cus...@embl.fr Website: http://www.embl.fr Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 6 Rue Jules Horowitz, 38042 Grenoble, France **
Re: [ccp4bb] twinning problem ?
Zbyszek - do you have any measure of unintegrated streaks? It could be a help to at least have a rough score. Eleanor On 11 March 2014 20:04, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: Shape of the diffraction spots changes in the statistical disorder -- twinning continuum. At both ends spots shape is like in diffraction from crystals without such disorder. However, in the intermediate case, electron density autocorrelation function has additional component to one resulting from ordered crystal. This additional component of autocorrelation creates characteristic non-Bragg diffraction, e.g. streaks aligned with particular unit cell axis. In the absence of such diffraction pattern, the ambiguity is binary. The description of the problem indicates statistical disorder. Zbyszek Otwinowski Hi, If there's an NCS translation, recent versions of Phaser can account for it and give moment tests that can detect twinning even in the presence of tNCS. But I agree with Eleanor that the L test is generally a good choice in these cases. However, the fact that you see density suggests that your crystal might be more on the statistical disorder side of the statistical disorder -- twinning continuum, i.e. the crystal doesn't have mosaic blocks corresponding to one twin fraction that are large compared to the coherence length of the X-rays. So you might want to try refinement with the whole structure duplicated as alternate conformers. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 11 Mar 2014, at 14:10, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: Sorry - hadnt finished.. The twinning tests are distorted by NC translation - usually the L test is safe, but the others are all suspect.. On 11 March 2014 14:09, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: What is the NC translation? If there is a factor of 0.5 that makes SG determination complicated.. Eleanor On 11 March 2014 14:04, Stephen Cusack cus...@embl.fr wrote: Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions Stephen -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email: cus...@embl.fr Website: http://www.embl.fr Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 6 Rue Jules Horowitz, 38042 Grenoble, France ** Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390
Re: [ccp4bb] twin refinement
If the twin law is k,h,-l, then your a axis must almost equal the b axis? And if the twin fraction is 0.48 then you have additional symmetry I guess? How sure are you that the point group is P4/mmm? On 13 March 2014 20:41, Teresa Swanson teresa.m.swan...@gmail.com wrote: Dear collegues, I'm working with a drug complexed protein structure that is having major twinning issues. The drug has a single Br atom on a benzene ring, which I'd like to use for orienting the drug in the binding site. I have various anomalous data sets, ranging from 3.0A resolution, all scaled into P222 with a Rlin of .125. Using MR, the twin law (k, h, -l) and NCS restraints, I can confidently solve the structure without anomalous, and the drug density is clear in the Fo-Fc map, with Rw/Rf at ~.26/.29 and a space group of P21221. It might be important to note that any simulated annealing I've tried invariably increases the Rfree by 2-3%, so I've scraped it. As you can imagine, when using the twinned data, the anomalous maps are weak and random. I've used the Phenix detwin option in Xtriage to see if I can pull the anomalous signal out of it. If I use the .mtz file that is output for MR and calculate the anomalous maps, it looks promising. The twin fraction for the one particular dataset I've been using is estimated at approx .48. Is this too close to 50% to do the detwinning? Now I'm wondering how to properly refine this further. I'm assuming that since I've detwinned the data, I do refinement without the twin law. But that gives an initial Rf of .38 when using it gives .31. Since I've already solved the structure without using the anomalous flag, can I just use the detwinned reflections and the refined structure to calculate an anomalous map (without having to redo the refinement)? Mainly, my main question is about how to tease out and properly refine the anomalous data from a twinned structure. Also, how much of a difference will it make to scale into P222 versus P21212. And, if I have quite high redundancy, should I scale anomalous in HKL2000 or just use the anomalous flag? Any help on refining this twinned structure would be greatly appreciated! Thanks, Teresa PhD Student
[ccp4bb]
I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: For estimated molecular weight 44000. Nmol/asym Matthews Coeff %solvent P(3.20) P(tot) 1 6.8482.03 0.00 0.00 2 3.4264.07 0.18 0.13 3 2.2846.10 0.81 0.86 4 1.7128.13 0.01 0.01 5 1.3710.17 0.00 0.00 Phaser Stats: Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. My belief is that there really is only two molecules in the ASU and that there just happens to be a very large solvent channel giving a 65% solvent content. I would like help in determining whether this is likely or if I have missed something. Thank you for your help in advance! -Yarrow Post Doctoral Scholar UCSF Genentech Hall, Rm N551 600 16th St., San Francisco, CA 94158-2517
[ccp4bb]
How pretty - I love circular molecules! Eleanor On 19 March 2014 15:58, Yarrow Madrona amadr...@uci.edu wrote: Thank you to everyone for their input. I am posting a picture to some of the symmetry related molecules shortly. There are six dimers related by symmetry (60 degrees) with a donut hole in the middle. This was troubling to me as I have solved mostly tighter packing structures (monoclinic or orthorhombic) in the past. If expanded further there are a bunch of tightly packed donut holes (though I didn't show these). I want to know if this is really a viable solution. The crystals are huge (300microns X 300microns) and this would maybe explain why they are only diffracting to 3.2 angstroms. Thank you! https://www.dropbox.com/s/r01u37owbkz9pon/donut.png -Yarrow On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona amadr...@uci.edu wrote: Yes in the first couple of rounds of refinement it refines very well for a 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs contiguously except for a donut hole in between six dimers that are related by symmetry. Trying to put a molecule there disrupts the symmetry and leads to clashes. I have a synchrotron trip next week, hopefully this should help clear things up a bit. On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. *My belief is that there really is only two molecules in the ASU and that there just happens to be a very large solvent channel giving a 65% solvent content.* *I would like help in determining whether this is likely or if I have missed something. Thank you for your help in advance!* *-Yarrow* Post Doctoral Scholar UCSF Genentech Hall, Rm N551 600 16th St., San Francisco, CA 94158-2517
Re: [ccp4bb] ccp4i molrep errors
This is a problem for the ccp4 team at STFC. Eleanor On 20 March 2014 12:11, Ming Sun ms4...@columbia.edu wrote: Hi All I'm running molrep on a large complex, while I have met the following two problems 1) If the pdb model is smaller than the map, is it possible that molrep could not find the right positions? It keeps giving me the same error messages: #CCP4I TERMINATION STATUS 0 At line 12546 of file c:/mb/msys/home/ccp4/ccp4-src/checkout/molrep/molrep_prog.f Fortran runtime error: Bad value during integer read #CCP4I TERMINATION TIME 20 Mar 2014 07:48:02 #CCP4I MESSAGE Task failed 2) Meanwhile, can Molrep write out the results in mmCIF format? Thanks again Thanks -Ming -- Ming Sun Columbia University (E): ms4...@columbia.edu
Re: [ccp4bb] Error in ccp4lib?
You don't say how you are doing the transformation? I would simply input the file to cad cad hklin1 thisfile.mtz hklout newfile.mtz labi file 1 allin end I think (and hope) that the data and phases will be converted correctly to the CCP4 asymmetric unit. Eleanor On 25 Mar 2014, at 09:16, Zbyszek Otwinowski wrote: I am reading an external file, which contains phases and ABCDs in the space group P43212. My file has an asymmetric unit with k= h. Since CCP4 uses a different asymmetric unit with h=k, this requires phase and ABCD coefficients transformation. The transformation seems to be correct for reflections with initial h not equal to zero, but gives wrong result for 0 k l reflections. Zbyszek Otwinowski Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
Re: [ccp4bb] Problem with making a continuous nucleic acid chain
Why dont you try CCP4MG ? Eleanor On 22 April 2014 22:37, Xiaoming Ren xiaomingre...@gmail.com wrote: Dear all: I am facing a display issue in pymol. In my structure, there is a nucleic acid chain with some monomers which are modified nucleotides built in scketcher of ccp4 suite. However, When I showed the structure in pymol as cartoon, the nucleic acid chain was discontinuous where the monomers are placed. I could not get a intact nucleic acid chain except setting the cartoon_nucleic_acid_mode value to 1, with the backbone passing through ribose C3' atoms. I tried to add SEQRES lines into the pdb file according to the pdb instruction, but it doesn't work if I want the backbone to pass through phosphorus atoms. May I get some suggestions from you? Although it's not a big problem, it's really painful. Thanks a lot and best, Xiaoming
Re: [ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps
A technecality - the Dano only contains information about anomalous scatterers and should therefore show these more precisely. The 2Fo-Fc map will tend to show anything which is included in the phasing at the position given. For safety you can set all the atom occupancies in the vicinity of the site to 0.00 and then use phases calculated from the edited structure to check again. ... This sort of thing sometimes happens with the Se of MSE, and the explanation there is that the MSE is in more than one conformation.. Eleanor On 24 April 2014 10:39, Petr Leiman petr.lei...@epfl.ch wrote: Dear All, We are looking for an explanation for a very strange observation. *Problem*: We have *two fully independent data sets* (two different crystals), in which the Bijvoet Difference Fourier map peak of one particular metal site is shifted by 0.47 A from its position in the 2Fo-Fc map. *Relevant information*: The resolution of both data sets is 1.5-1.6 A. The 2Fo-Fc and BDP maps are calculated using the same phases. The metal ion is water hydrated and all the details are crystal clear in both 2Fo-Fc maps. The crystals are grown in the presence of Sr and the data sets are collected at the Sr K-edge. There are many other Sr sites and all strong peaks in the BDF map overlap with 2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but it is well above the noise level of the BDF map. *Additional information*: The weird site is actually fully buried inside an internal cavity (it is surrounded by protein atoms from all sides), but a Sr atom is able to diffuse into this cavity somehow. All other Sr sites are on the surface of the protein. Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc map peak are welcome! Thank you very much, Petr -- Petr Leiman EPFL BSP 415 CH-1015 Lausanne Switzerland Office: +41 21 69 30 441 Mobile: +41 79 538 7647 Fax: +41 21 69 30 422
Re: [ccp4bb] AW: [ccp4bb] rigid body refinement or molecular replacement.
PLEASE do NOT do MR - You will most likely finish up with a different origin for your SG, and it will make comparisons a pain in the neck! Just do a rigid body refinement starting with the native structure. If you like you can change the cell to the substrate one pdbset xyzin native.pdb xyzout substrate.pdb CELL 39.6 63.9 117.8 90 90 90 end but the refinement will sort that out too. Eleanor On 24 April 2014 13:11, herman.schreu...@sanofi.com wrote: I would do a rigid body refinement, rebuild the structure and then go on with refinement. To compare refined structures, you still need to superimpose them first. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Faisal Tarique *Gesendet:* Donnerstag, 24. April 2014 14:01 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] rigid body refinement or molecular replacement. Dear all I have a high resolution (2A) native structure of a protein and structure factors for a relatively low resolution (2.6A) of the same protein bound with its substrate (complex) having same space group and cell parameters (P212121 is the space group and cell parameters are a,b,c =39.4,64.5,117.2. and a,b,c = 39.6,63.9,117.8 for native and complex)..My question is ..what is the best way to solve the complex structure ??..whether it should be done through molecular replacement with the native structure or simply by doing rigid body refinement through mtz from complex and coordinates of the native structures..in summary...what should be the ideal method employed for the structure solution if the aim is to compare between the native and complex structure of the same protein ??. -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] anomalous signal
Look at the aimless plot of CCanom . That is the best indicator I think and very sensitive when you have such high redundancy Eleanor On 25 Apr 2014, at 22:13, Jim Pflugrath wrote: d/sig should be above 0.80 There seems to be plenty of signal there with all values above 1.02. We have solved structures with less multiplicity and lower d/sig. There is a different criteria of signal for when you know the positions of the anomalous substructure atoms and when you need to find the positions of the anomalous substructure atoms. As for no signal, I think I am on record that there is always an anomalous signal. :) But can you detect it? Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique [faisaltari...@gmail.com] Sent: Friday, April 25, 2014 4:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] anomalous signal Dear all sorry about my previous mail where i forgot to mention that the data was collected on home source at Cuk alpha and at 1.54A. written below is the log file of an anomalous data processed through SHELXC..my question is ..what is the strength of anomalous signal ?? as it is said For zero signal d'/sig and d/sig should be about 0.80. Then in the present case is there really a signal or can be assumed no signal..we are expecting one Ca atom bound to the protein at its active site..the redundancy of the data is 11.6..with this signal strength can we assume Ca to be present there or whatever little anomalous if present is due to something elseor there is no signal at all ??... Resl. Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 2.60 N(data) 375 493 580 1319 450 538 679 866 1081 1414 1709 I/sig58.8 38.6 32.6 38.3 27.7 27.2 21.9 18.4 12.6 9.5 6.1 %Complete 94.7 99.0 99.3 99.5 100.0 99.6 99.7 99.8 99.6 99.6 90.9 d/sig 1.65 1.27 1.18 1.25 1.19 1.12 1.11 1.11 0.97 1.02 1.05 -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Problem with Rfactor
We need more information. First if the pointgroup is hexagonal are you searching al likely SPACEgroup - eg PG P6, SGs might P6 P61 P62P63 P64 P65.. There is a GUI option to do this for both MOLREP Phaser. You can guess the likely no of molecules using matthews In the GUI - that is in the MOL REp task - option analyse. Eleanor On 29 April 2014 10:21, Dom Bellini dom.bell...@diamond.ac.uk wrote: Dear Sudarshan, I have the feeling the your R factor after Molrep is really high because the program has failed to produce a correct/not-partial solution, which could be due to many things, but usually the main problem is how good your search model is. The difference in spacegroups is not a problem as in MR the model will be rotated and translated inside the new cell. The number of molecules that you need to search for is a tricky question. You need to make some educated guesses with help from analysis of, for example, crystal solvent content (e.g., Matthews program from CCP4) and/or self-rotation maps (Molrep or Polarrfn programs, also available from CCP4 suite). If you have never before used molecular replacement, however, I would advise that you get some help/guidance from some more experienced colleague/friend in the Department, since you may encounter quite a few more other problems that would perhaps be a bit too long to go through by email. Some of these problems could also be probably self-solved by looking at some tutorials on Molecular replacements. Best wishes, D From: Sudarshan Murthy [mailto:sudarshan.murthy...@gmail.com] Sent: 29 April 2014 05:11 To: ccp4bb Subject: [ccp4bb] Problem with Rfactor Dear All, I am very new to the field of crystallography, I have a few questions which are very basic and getting input from this forum would help me a lot. Firstly I am trying to solve a structure using MR Molrep. In the result the Rfactors are really high how do I reduce the same?? ..When I tried with Phaser I didnt get any solution at all.. Will der be a problem with the space group?? like the model is in monoclinic and the data which I have is processed in hexagonal. Secondly while using hexagonal space group should I search for only one monomer in Molrep?? These are very basic questions if anybody could help me out with this , I would be very happy for the same. Sudarshan .N. Murthy Crystallography Division Bangalore
Re: [ccp4bb] anomalous signal
Well - this is pretty common, and really doesnt matter. I just let SHELXC/D give an occupancy to its anom scatterer solutions and assume that the stronger one is Ca. But in fact it wont matter at all for the phasing, and IF the experiment works it is easy to sort out S from Ca in the final map. On 30 April 2014 13:21, Tobias Weinert tobias.wein...@psi.ch wrote: Dear Faisal, you could use Phenix to do an f’’ refinement against the data like described here: Liu, Q., Liu, Q. Hendrickson, W. A. (2013). Acta Cryst. D69, 1314-1332. best regards, Tobias On 30 Apr 2014, at 14:01, Faisal Tarique faisaltari...@gmail.com wrote: Dear all I am working on a metalloprotein which probably contains Ca at its active site..The sulfur containing amino acid constitutes almost 5.4% of the total amino acid residues of this protein..I have collected the data at home source (CuKalpha=1.54A)..Since f'' of Sulfur is 0.56 and that of Ca is 1.28 we can always expect some anomalous signal out of the data..My question is ..how we will know if the anomalous signal is coming out of Sulfur or from Calcium ?? is there any method through which we can get to know the identity of the scattering molecule through the data..Can FFT anomalous map from CCP4 is of any help in this direction, if yes then please tell me how to interpret the output from this.. -- Regards Faisal School of Life Sciences JNU
[ccp4bb] odd phaser failure
I should be sending thids to the phaser team I guess, but maybe there is an easy fix.. This is the end of the log file.. Sg I222 or I212121 Scoring 500 randomly sampled orientations and translations Spreading calculation onto 2 threads. Generating Statistics for TF SET #1 of 2 0% 100% |===| DONE Mean Score (Sigma): -18.84 (17.72) SET #1 of 2 TRIAL #1 of 245 --- Search Euler = 99.1 82.9 249.8, Ensemble = ensemble1 Doing Fast Translation Function FFT... Done Helix is perpendicular to two-fold: point exclusions applied BFONT COLOR=#FF8800 $TEXT:Warning: $$ Baubles Markup $$ - UNHANDLED ERROR: cctbx Error: site_symmetry: min_distance_sym_equiv too large. - $$ EXIT STATUS: FAILURE CPU Time: 0 days 0 hrs 0 mins 28.27 secs ( 28.27 secs) Finished: Mon May 19 11:44:52 2014 /pre
Re: [ccp4bb] problem with refmac and xloggraph
This is fixed now if you re-install updates and
Armando - this is crude but if you edit the log file and replace
Rfactor v. resln :N:1,6,7 11 12with Rfactor v. resln :N:1,6,7,11,12
it will work.
On 21 May 2014 10:20, Armando Albert xalb...@iqfr.csic.es wrote:
Dear all,
does anyone experience a problem viewing refmac results with Log Graph?
The window appears and disappears in less than one second and this is the
output on the terminal window:
Error in startup script: syntax error in expression 10 11 12 + 12:
extra tokens at end of expression
while executing
expr [string trim $ele] + $data(NCOLUMNS)
(procedure extract_tables_from_GRAPH line 44)
invoked from within
extract_tables_from_$filetype $input $arrayname
(procedure extract_tables_from_file line 31)
invoked from within
extract_tables_from_file $system(SCRIPT) $system(FORMAT) data
invoked from within
if { $system(SCRIPT) != } {
if { ![ElementExists system FORMAT] || $system(FORMAT) == } {
set system(FORMAT) [GetFileFormat $system(SCRI...
(file /Applications/ccp4-6.4.0/share/ccp4i/loggraph/loggraph.tcl
line 2324)
invoked from within
source [file join $env(CCP4I_TOP) loggraph loggraph.tcl]
(file /Applications/ccp4-6.4.0/share/ccp4i/bin/loggraph.tcl line 83)
Armando
Re: [ccp4bb] Reprocess data with new resolution cutoff?
An obvious point - remember refinement exists to give you the best possible model so you need to look at the maps. And I guess that has to be a subjective assessment - if you SEE anything more clearly at the higher resolution - I would use that data, but if the maps do not improve discard it.. This is purely anecdotal, but I find there isnt much gained by using very incomplete higher resolution bits of data. However complete weak data can improve the scaling and temperature factor discrimination a good deal.. Eleanor On 21 May 2014 11:50, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Hi Thomas, I too would use the data out to 2.55A, as you did. The main point is that the 2.45A model produces a worse Rfree (30.16%) at 2.55A than the 2.55A model does (30.03%). And this tendency is confirmed for the 2.35A model, so there's no point in going beyond 2.55A. best, Kay
[ccp4bb] Potentially serious if unusual bug in handling symmetry
Someone here has seen his Rfactors leap from 18% to 42% and was naturally distressed! We have tracked down the problem to this: Steps he took were: 1) Integrate with XDS and feed output through pointless/aimless etc. He used a phenix refined mtz to Match Laue and indexing The space group there is given by PHENIX as P 2 21 21 but the spacegroup NUMBER is given as 18 (ie P21 21 2 in CCP4 symmetry library) . pointless then output an mtz file with SPACEGROUP number as 18 and SG name as P 2 21 21. The symmetry operators are correctly listed for P 2 21 21 This is the version number: CCP4 6.4: POINTLESS version 1.9.8 : 02/05/14 2) When the refinement was repeated with REFMAC the spacegroup was taken as P21 21 2 - ie the SG number 18 overrode the listed symmetry operators in the mtz file, and the SG name P 2 21 21 given on the CRYST1 card was ignored. The refmac version used is CCP4 6.4: Refmac_5.8.0071 version 5.8.0071 : 04/04/14 The user thinks that in REFMAC 5.8.0033 this problem did not occur, but I havent checked that.. Anyway - it does emphasise the importance of checking symmetry operators for consistency with both the SG name and number (Yes, George, I know you have always said this is the only correct policy!) Maybe an extra subroutine could be added to the symmetry library and called from other programs.. And ideally checking all sources of symmetry information for consistency and stopping if there is a serious disagreement. In the short term we simply took the processed mtz file and ran mtzutils hklin1 corrupted.mtz hklout fixed.mtz SYMM 3018 (or symm P2 21 21 ) and the fixed file was OK Eleanor Dodson
Re: [ccp4bb] Scalepack2mtz keeps running
Do check for any differences in format.. Or attach the sca file and I can check why/what (maybe!) Eleanor On 25 May 2014 13:02, chen chenc...@gmail.com wrote: hi all, I have an .sca file processed by HKL2000 about 4 years ago, I just used the Scalepack2mtz program to change it into .mtz file. However, the program keeps running. To ensure that the Scalepack2mtz program is ok, I use another new .sca file for verification and it turns out that this file is changed into .mtz file in seconds. Has anyone encountered such case ? best regards chen
Re: [ccp4bb] AW: [ccp4bb] Refmac5
There are differences between the LINK pdb definition and that in REFMAC - hence the different name LINK and LINKR LINKR allows you to do this: ie cross reference to a dictionary link which is much more informative than a simple LINK record which can only define a link distance. However for reasons I do not know this addition has never been adopted as standard PDB information so there is confusion between different programs.. Eleanor. Definition of LINK with LINKR part in red. The LINK records specify connectivity between residues that is not implied by the primary structure. Connectivity is expressed in terms of the atom names. This record supplements information given in CONECT records and is provided here for convenience in searching. Record Format COLUMNSDATA TYPE FIELD DEFINITION 1 - 6Record name LINK 13 - 16Atomname1 Atom name. 17 Character altLoc1 Alternate location indicator. 18 - 20Residue nameresName1Residue name. 22 Character chainID1Chain identifier. 23 - 26Integer resSeq1 Residue sequence number. 27 AChar iCode1 Insertion code. 43 - 46Atomname2 Atom name. 47 Character altLoc2 Alternate location indicator. 48 - 50Residue nameresName2Residue name. 52 Character chainID2Chain identifier. 53 - 56Integer resSeq2 Residue sequence number. 57 AChar iCode2 Insertion code. 60 - 65SymOP sym1Symmetry operator for 1st atom. 67 - 72SymOP sym2Symmetry operator for 2nd atom. 73 - 80LinkID linkid Cross-reference to LINK definition in CCP4 libraries On 28 May 2014 16:10, herman.schreu...@sanofi.com wrote: Dear Remie, For reasons that probably only Garib understands, Refmac still uses LINKR instead of LINK to link atoms. However (at least for Refmac) both LINK and LINKR should work. After a lot of complaining in the past (also from my side), the handling of carbohydrates in Refmac is ok. I just did some tests with N-linked carbohydrates (NAG, MAN): after using the Make Link option in coot and saving the file, there were regular LINK records, created by coot. After running refmac from within coot and saving the files, the LINK records were converted (perverted?) to LINKR records. However, they did not disappear and after restarting coot and loading the file again, they were still there. Also my version of pymol recognizes both LINK and LINKR records, since the connections are shown when I load the files before and after running refmac. Some suggestions: check that you are using recent versions of coot, refmac and pymol. Older versions may have problems. I don't know how you construct your carbohydrates, but if you get the monomers, you have to delete an oxygen atom in the link (in my case the O1 atom). Make also sure that your starting geometry is acceptable. Just doing a real space refinement usually works well, but sometimes the carbohydrate crashes into the protein density and you will have to manually fit the sugar as good as possible. Some of the older files in the PDB may have distorted carbohydrates, so here it is probably best to build the carbohydrates again from scratch (get monomer, delete linking oxygen) etc. Good luck! Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Remie Fawaz-Touma Gesendet: Mittwoch, 28. Mai 2014 16:13 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Refmac5 Hello everyone, When I refine in CCP4 a structure with Glc units attached every few of them as a ligand and there are several of them in the same file, I start off with a PDB that shows the LINK records between the GLC units as LINKR because I add the links by Extensions Modelling Make Link (2 atoms) in COOT. What does LINKR mean? why not just LINK? More importantly, when I refine, the output PDB file does not contain the LINK records, why do they go away? and in PyMol I see only GLC that are close enough to make a covalent bond stay connected, the others become detached, in CCP4 they all show detached, but if I do a real space refinement for those sugars of the same ligand in the output file, they come close together and attached? Any suggestions? Thank you very much in advance. Remie
Re: [ccp4bb] lsqkab problem to be reported to developer
Can you send more details? the log file? the pdb On 30 May 2014 22:54, Carter, Charlie car...@med.unc.edu wrote: This is a bizarre problem. I'm trying to superimpose multiple conformations of the same protein using segments I expect not to change. LSQKAB bails with this error each time: *** RWBROOK error: point code unitfunction ***1 -1022MMDB_F_Atom *** file : 4ARC_rot.pdb *** reason : internal error #2 -- report to developer *** Execution stopped. It also appears very likely that the superposition is wrong. I expect an RMSD for the superimposed atoms (main chain only) of less than 2 Å, yet the program reports that the RMSD is 7 Å. Thanks to anyone for a diagnosis and prescription. Charlie
Re: [ccp4bb] negative density around disulfide bond
I have seen similar features fairly often when the data collection has been pushed to the limit. The theory is that it is due to radiation damage - you could check by only merging say the first 50% of your data, then seeing if the di-sulphide is intact in those maps. ( wouldnt re-refine much - just set CB-SG occs to 0.00 - recalculate SFs and check the maps) Eleanor On 2 June 2014 07:44, Marjolein Thunnissen marjolein.thunnis...@biochemistry.lu.se wrote: Hi, I would guess radiation damage, see Weik et al., 2000, PNAS 97, 623-628 or for a more recent discussion and thorough overview Sutton et al., 2013, Acta Cryst D69, 2381-2394. best regards Marjolein On 02 Jun 2014, at 07:08, Eze Chivi ezech...@outlook.com.ar wrote: Hello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel disulfide.jpgdisulfide2.jpg *Marjolein Thunnissen* Science Coordinator MX MAX IV Laboratory Lund University P.O. Box 118, SE-221 00 Lund, Sweden Visiting address: Ole Römers väg 1, 223 63 Lund Telephone: +46 766 32 04 17 www.maxlab.lu.se
Re: [ccp4bb] baverage: no tables were found in this file
I thought the addition of ... around file names got round this problem eg This_is_Mine.pdb and That is Yours.pdb Eleanor On 3 June 2014 13:15, Eugene Krissinel eugene.krissi...@stfc.ac.uk wrote: I am afraid that it is us who will need to conform eventually :), same to say about Program Files (x86) and the overall culture in Windows, the system where some 40% () of CCP4 users have chosen to work. Eugene On 3 Jun 2014, at 13:03, Mark J van Raaij wrote: completely agree with avoiding spaces in paths and file names, but Google Drive is very handy for sharing files, so every once in a while I forget to move a file someone shares with me before running Unix programs on it and get strange errors. Depending on how awake one is at the time finding out the source can be time-consuming...Google should really remove the space! Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 3 Jun 2014, at 13:47, Eugene Krissinel wrote: I take this chance to to confirm once more, as publicly as possible, that file paths with spaces are discouraged in today's CCP4. This inconvenience originates from ancient times in computing when good half of CCP4 was written and when spaces were disallowed on file system nodes. Please take a notice of this fact as CCP4 core still receives (albeit infrequent) bug reports, where surprising behaviour is due to using file paths with white spaces. Fixing this has proved to be a hard problem, purely because of technical choices made quite a number of years ago. But good news are that this limitation will be removed in new CCP4 Gui under development. Eugene On 3 Jun 2014, at 08:23, Mark J van Raaij wrote: This also occurred to me once where the file path had a space,(/Google Drive/), when I moved the file somewhere else it worked. I was using baverage from the CCP4i GUI. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 3 Jun 2014, at 09:20, Tim Gruene wrote: Dear Bing, can you post the exact command you were using, please? Also please check with a different PDB file. In case you are using baverage from the command line, can you make sure you are actually using the program from ccp4 by typing 'which baverage' at the command prompt? Regards. Tim On 06/02/2014 10:16 PM, Wang, Bing wrote: Hi CCP4, Recently when I input my pdb file and run the baverage in the ccp4 suit to check the temperature factor, it always tell me No tables were fund in this file. Could you tell me how to fix this problem? Or is there another software instead of baverage I could use to check the temperature factor? Thanks! Bing -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Scanned by iCritical. -- Scanned by iCritical.
Re: [ccp4bb] AW: [ccp4bb] possible twinning issue in P4212 / I422
It helps to look at the output from the truncate step quite critically. First is there a non cryst translation of 1/2,1/2,1/2 indicated in the P4 2i2 data set? If so then the I centring at lower resolution might just be approximate.. If there is NC translation then other twinning statistics are distorted and I find the only semi-reliable one is the L test. But if you say there is no room for your protein with that translation and 4/mmm symmetry then there must be twinning or you have crystallised something else! Eleanor On 4 June 2014 08:48, herman.schreu...@sanofi.com wrote: Dear Bjørn, I guess the first step to enlightment is to recognize that we as mere mortals are not able to deduce the space group from diffraction data alone. All Aimless, XDS etc. can produce are educated guesses what the space group might be. Especially when twinning is involved, the crystal packing may not heed the rules and classifications that we humans try to impose. In many cases, one might have to go down to P1 and solve the structure in P1 to find out what the true space group is. Here are some comments to your questions: -the same protein under the same crystallization conditions and even in the same drop may produce crystals with very different crystal packings, even with the same unit cell, so your 4 and 7.5Å crystals may be different. -If there is no way to fit the protein in the asymmetric unit that is a very strong indication that you do have twinning. -There have been some discussions in the CCP4BB, but I do not believe that twinning can generate body centering. -You might be barking at the wrong tree and the twinning axis might be parallel to the 4-fold axis, or even generating the 4-fold. You may even have 4-fold twinning. -You may have pseudo body centering, which is perfect at low resolution, but breaks down at higher resolution. As a test, you could process your 4Å data only to 7.5Å and see what the statistics would look like. What I would do: If you have more crystals, collect data on them all, maybe there is one which is not or not perfectly twinned. If there is a model which could be used for molecular replacement: process the data in P4, I4, P222 and P1 and run molecular replacement with all possible space groups for both crystals. However, at 4Å with unclear twinning, solving your structure will be tough. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Bjørn Panyella Pedersen Gesendet: Dienstag, 3. Juni 2014 21:01 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] possible twinning issue in P4212 / I422 Dear All, I have a strange potential twinning issue that I cannot understand. I've searched high and low on all the internets to find an answer but have come up empty-handed, so I look to the wisdom of The Board to enlighten me. I have a 4'ish Å dataset that processes nicely in P 4 21 2 (#90). However intensity distributions indicate possible almost perfect twinning (eg. I^2/I^2 : 1.592 ). So I speculate that the real space group might be P 4 (#75). Recently we collected a new fairly low resolution (7.5Å) dataset, from the same type of crystals (same purification, same conditions). But the space group in XDS and aimless now comes out very clearly as either I422 (#97) or I4212 (#98) (screw-axis is unclear given the data). The unit-cell parameters are exactly the same as in sg #90, which btw. means that in the body-centered lattice there is no way the protein can fit in the asym. unit. So I guess what I don't understand is: Is it possible to go from a primitive lattice to a body-centered lattice by twinning. Is this just a low-resolution artifact? Or is this a P4 unitcell that can appear like P4212 or I422 depending on small variations (weak dehydration or similar). Has anyone experienced something similar? Am I missing a basic facet of how twinning works, or is something else at play here? Thanks for any insights or suggestions! All the best, /Bjørn -- Bjørn Panyella Pedersen Macromolecular Structure Group University of California, San Francisco
Re: [ccp4bb] AW: [ccp4bb] possible twinning issue in P4212 / I422
There are cases of different crystal forms of the same thing turning up with similar lattice dimensions... But if if the cell dimensions are the same, and one form is P4i with 4 molecules in the cell, and just enough space for one mol/asymm unit, and the 2nd form is I422 then isnt the asymm volume much too small in that form? Eleanor On 4 June 2014 20:23, Bjørn Panyella Pedersen bj...@msg.ucsf.edu wrote: Dear All, Thanks for all the suggestions, on and off the board! Summary: Some have asked for the L-statistic in P 4 21 2: Mean |L| :0.397 (untwinned: 0.500; perfect twin: 0.375) All programs tried (xtriage, truncate, pointless) agree that the 4Å data is likely twinned P4 with an estimated twin-fraction ranging from 0.38 to 0.48. People seem to agree (as was my initial understanding) that twinning by itself cannot change a primitive lattice to a body-centered lattice. Thus this change in my low-resolution dataset must be caused by something else. Pseudo body centering has been suggested as the likely explanation for the primitive to body-centered lattice change in the low-resolution dataset. - As suggested, I have looked at the self-patterson for the 4Å dataset and see no peaks, at 1/2,1/2,1/2 or elsewhere. - As suggested, I have looked at the truncate output of the table Analysis of mean intensity by parity for reflection classes in the h+k+l column, but see no differences from h+k+l=2n to h+k+l=2n+1 in the 4Å dataset. - As suggested I have processed the 4Å data at 8Å to see if pseudo body centering was breaking down at higher resolution. At 8Å there was still not sign of pseudo body centering. Thus the 4Å data does not not support pseudo body centering, as far as I can tell. Some has suggested that the crystals are simply two different things. This might be, but since the low-resolution dataset has exactly the same unit-cell parameters as the 4Å dataset, it seems unlikely to me that the crystal packing is significantly different, or that the content of the crystals differ. It seem likely that both crystals contain the same thing in approx. the same type of packing. So no clear explanation for the observed behavior so far, but most likely the low resolution is obscuring the real problem in this particular instance. As is almost always the case, the way forward is to get more and better data to understand the problem. As one suggested offboard (tongue-in-cheek): Why not find crystals that are not twinned, probably with higher resolution? I will do that, and thanks again for your input! All the best, -Bjørn On 06/04/2014 03:09 AM, Eleanor Dodson wrote: It helps to look at the output from the truncate step quite critically. First is there a non cryst translation of 1/2,1/2,1/2 indicated in the P4 2i2 data set? If so then the I centring at lower resolution might just be approximate.. If there is NC translation then other twinning statistics are distorted and I find the only semi-reliable one is the L test. But if you say there is no room for your protein with that translation and 4/mmm symmetry then there must be twinning or you have crystallised something else! Eleanor On 4 June 2014 08:48, herman.schreu...@sanofi.com mailto:herman.schreu...@sanofi.com wrote: Dear Bjørn, I guess the first step to enlightment is to recognize that we as mere mortals are not able to deduce the space group from diffraction data alone. All Aimless, XDS etc. can produce are educated guesses what the space group might be. Especially when twinning is involved, the crystal packing may not heed the rules and classifications that we humans try to impose. In many cases, one might have to go down to P1 and solve the structure in P1 to find out what the true space group is. Here are some comments to your questions: -the same protein under the same crystallization conditions and even in the same drop may produce crystals with very different crystal packings, even with the same unit cell, so your 4 and 7.5Å crystals may be different. -If there is no way to fit the protein in the asymmetric unit that is a very strong indication that you do have twinning. -There have been some discussions in the CCP4BB, but I do not believe that twinning can generate body centering. -You might be barking at the wrong tree and the twinning axis might be parallel to the 4-fold axis, or even generating the 4-fold. You may even have 4-fold twinning. -You may have pseudo body centering, which is perfect at low resolution, but breaks down at higher resolution. As a test, you could process your 4Å data only to 7.5Å and see what the statistics would look like. What I would do: If you have more crystals, collect data on them all, maybe there is one which is not or not perfectly twinned. If there is a model which could be used for molecular
Re: [ccp4bb] Help in Cell content analysis
That is puzzling. 18% solvent is not common and you would expect very strong diffraction with such a low solvent content. one possibility is that the NC symmetry is parallel to a crystal axis and is making monoclinic data appear to have an extra 2-fold axis. (There is a case I have heard of wherethe SG turned out to be P21 and not P2i2i2i - done by Michael Isupov. You could probably find a reference to it) I would look carefully at the pointless summary of the quality of the symmetry operators. If one 2-fold is better than the other two maybe process the data with that 2 fold only.. Of course another possibility is that the protein has been chewed up in crystallisation! Do you have an MR model? Eleanor
Re: [ccp4bb] seeking help about running SCALEPACK2mtz
It is very hard to diagnose this sort of error - I didnt think scalepack2mtz cared about data quality! There is more likely some format problem.. Is a sca file meant to have some sort of terminator? But best to attach the offending sca file so that experts can check it.. Eleanor On 26 June 2014 19:41, chen c chenc...@gmail.com wrote: Dear CCP4BBers: I'e got a problem about data processing when running SCALEPACK2mtz. Hope you can give me some advice on that. Here's my problem: I have an .sca file processed by HKL2000 about 4 years ago, I just ran the Scalepack2mtz program in order to transform it into .mtz file. However, the program keeps running. To ensure that the Scalepack2mtz program is ok, I use another .sca file for verification and it turns out that this file for verification is successfully transformed into .mtz file within seconds. I checked the .log file, the program keeps running when there are the following messeages: Mean acentric moments I from input data: I^2/I^2 = 2.360 (Expected = 2.000, Perfect Twin = 1.500) I^3/I^3 = 9.187 (Expected value = 6.000, Perfect Twin = 3.000) I^4/I^4 = 52.348 (Expected value = 24.000, Perfect Twin = 7.500) Mean acentric moments I from anisotropically corrected data: I^2/I^2 = 3.121 (Expected = 2.000, Perfect Twin = 1.500) I^3/I^3 = 12.788 (Expected value = 6.000, Perfect Twin = 3.000) I^4/I^4 = 57.952 (Expected value = 24.000, Perfect Twin = 7.500) does this means that the .sca file isn't processed properly? I also checked the scale.log file, and there are the following messages: Shell Summary of observation redundancies: Lower Upper % of reflections with given No. of observations limit limit 0 1 2 3 4 5-6 7-8 9-12 13-19 19 total 50.00 5.60 5.9 6.1 18.0 15.1 26.9 19.7 8.3 0.0 0.0 0.0 94.1 5.60 4.45 7.9 9.4 13.0 12.9 23.2 27.5 6.1 0.0 0.0 0.0 92.1 4.45 3.88 19.2 5.6 13.3 13.5 20.4 21.9 6.0 0.0 0.0 0.0 80.8 3.88 3.53 31.9 6.5 11.6 11.3 14.9 17.9 5.8 0.0 0.0 0.0 68.1 3.53 3.28 2.4 4.7 11.3 15.3 23.5 34.1 8.7 0.0 0.0 0.0 97.6 3.28 3.08 0.2 3.6 7.0 15.4 23.7 40.1 9.9 0.0 0.0 0.0 99.8 3.08 2.93 0.4 4.4 8.7 16.8 21.1 41.3 7.2 0.0 0.0 0.0 99.6 2.93 2.80 4.2 8.0 12.7 15.2 20.3 33.7 6.0 0.0 0.0 0.0 95.8 2.80 2.69 15.4 9.1 14.0 13.8 19.2 25.2 3.3 0.0 0.0 0.0 84.6 2.69 2.60 31.6 16.2 11.7 14.2 13.7 10.6 2.0 0.0 0.0 0.0 68.4 All hkl 11.8 7.3 12.2 14.4 20.8 27.1 6.4 0.0 0.0 0.0 88.2 This clearly indicates an ice ring problem in the data-collection process. However, the ice ring problem shouldn't have caused the error during the .sca to .mtz process. In fact, I have use it for structure-solvement, and currently the two R factor is around 30%. However, the large deviation of I^3/I^3 or I^4/I^4 do means something wrong. Would you tell me what exact mistakes did I make? Can I use this .sca file for subsequent structure solving and refinement? Great thanks for your help best regards chen --
Re: [ccp4bb] Input reflection file for refmac runs
Keep on using the master file. Refmac corrects the observations in a few ways The overall anisotropic B correction is always applied In the worst case scenario where you are refining considering twinning the 2FP2 output is no longer the observed FP but one after a detwinning correction.. So always stick with the master file..Eleanor On 1 July 2014 13:40, Mark J van Raaij mjvanra...@cnb.csic.es wrote: Using the master mtz file (as you call it) is the safe thing to do. If you use the previous refmac output file, you have to careful the right structure factor columns are selected. They should be non-modified by the previous refmac run, otherwise you are refining both the data and the model. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 1 Jul 2014, at 14:35, Dilip Badjugar wrote: Dear CCP4bb users and Refinement experts, As input for (n+1)st run of refmac, should I use as input reflection file the output produced by refmac in the nth run? Currently I have been giving the master mtz file as input for all runs. I would appreciate the clarification from the community. Thanking you Dilip Badgujar Graduate Student ACTREC
Re: [ccp4bb] Metal ion differentiation - reg
It is difficult! Ni Zn are rather interchangable.. You dont say what resolution you have: There will be a small difference in the number of electrons you expect to see at the metal site, depending on whether it is Zn Zn2+ etc etc, and you can correct that to take account of the f' as well. So you would expect a slightly smaller peak for Ni than Zn but it will be hard to give a definitive answer unless you have high resolution data. First Q - was there Zn or Ni or both around in the crystallisation mix? Second Q: consider the coordination: This article discusses examples. Journal of Inorganic Biochemistry http://www.sciencedirect.com/science/journal/01620134 Volume 71, Issues 3–4 http://www.sciencedirect.com/science/journal/01620134/71/3, September 1998, Pages 115–127 Eleanor On 1 July 2014 16:10, Dhanasekaran Varudharasu dhana...@gmail.com wrote: Dear all, I have solved a structure (using molecular replacement) of metallo-enzyme which may have Zn or Ni at its active site. I collected data at in-house CuKa radiation. Now, I am able to locate the active site metal ion preciously but I am not able to differentiate whether it is Zn or Ni. I computed anomalous difference map and I got good anomalous map also. But still there is ambiguity, since Zn and Ni have closest F values at CuKa radiation ( For Zn = 0.68 and For Ni = 0.51). But both, Zn and Ni have different F' values at CuKa radiation ( For Zn = -1.61 and For Ni = -3.07). My question is that, *Can we used F' value information to differentiate metal ions?.Is it possible to find whether I have Zn or Ni at active site of my enzyme using crystallographic technique?. * Thanks in advance -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA
Re: [ccp4bb] twin or untwinned
To answer the original question. The indicators are that it is not twinned, If the Mean s are close to the untwinned values - you can probably believe it. Why are you worried? Eleanor Determining possible twin laws. 0 merohedral twin operators found 0 pseudo-merohedral twin operators found In total, 0 twin operator were found Mean |L| :0.378 (untwinned: 0.500; perfect twin: 0.375) Mean L^2 :0.205 (untwinned: 0.333; perfect twin: 0.200) On 3 July 2014 15:50, Nat Echols nathaniel.ech...@gmail.com wrote: On Thu, Jul 3, 2014 at 6:53 AM, Dirk Kostrewa kostr...@genzentrum.lmu.de wrote: yes - unfortunately, in my hands, phenix.xtriage reads the XDS_ASCII.HKL intensities as amplitudes, producing very different output statistics, compared both to the XDS statistics and to an mtz file with amplitudes created from that XDS file. This is incorrect. It does read it correctly as intensities - the confusion probably arises from the fact that Xtriage internally converts everything to amplitudes immediately, so that when it reports the summary of file information, it will say xray.amplitude no matter what the input type was (the same will also be true for Scalepack and MTZ formats). However, the data will be converted back to intensities as needed for the individual analyses. Obviously this isn't quite ideal either since the original intensities are preferable but for the purpose of detecting twinning I hope it will be okay. In any case the incorrect feedback confused several other users so it's gone as of a few weeks ago, and the current nightly builds will report the true input data type. (The actual results are unchanged.) Tim: I have no reason to think we handle unmerged data poorly; I'm not sure who would have told you that. In most cases they will be merged as needed upon reading the file. I'm a little concerned that you're getting such different results from Xtriage and pointless/aimless, however. Could you please send me the input and log files off-list? Dirk, same thing: if you have an example where XDS and Xtriage are significantly in disagreement, the inputs (and logs) would be very helpful. In both cases, I suspect the difference is in the use of resolution cutoffs and absolute-scaled intensities in Xtriage versus other programs, but I'd like to be certain that there's not something broken. thanks, Nat
Re: [ccp4bb] twin or untwinned
Sorry - the stats DO indicate perfect twinning - I misread the first Email.. Please ignore that comment! Eleanor On 4 July 2014 13:16, Philip Kiser p...@case.edu wrote: Hi Eleanor, If I'm not mistaken, the mean I stats are indicating perfect twinning. Philip On Fri, Jul 4, 2014 at 4:49 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: To answer the original question. The indicators are that it is not twinned, If the Mean s are close to the untwinned values - you can probably believe it. Why are you worried? Eleanor Determining possible twin laws. 0 merohedral twin operators found 0 pseudo-merohedral twin operators found In total, 0 twin operator were found Mean |L| :0.378 (untwinned: 0.500; perfect twin: 0.375) Mean L^2 :0.205 (untwinned: 0.333; perfect twin: 0.200) On 3 July 2014 15:50, Nat Echols nathaniel.ech...@gmail.com wrote: On Thu, Jul 3, 2014 at 6:53 AM, Dirk Kostrewa kostr...@genzentrum.lmu.de wrote: yes - unfortunately, in my hands, phenix.xtriage reads the XDS_ASCII.HKL intensities as amplitudes, producing very different output statistics, compared both to the XDS statistics and to an mtz file with amplitudes created from that XDS file. This is incorrect. It does read it correctly as intensities - the confusion probably arises from the fact that Xtriage internally converts everything to amplitudes immediately, so that when it reports the summary of file information, it will say xray.amplitude no matter what the input type was (the same will also be true for Scalepack and MTZ formats). However, the data will be converted back to intensities as needed for the individual analyses. Obviously this isn't quite ideal either since the original intensities are preferable but for the purpose of detecting twinning I hope it will be okay. In any case the incorrect feedback confused several other users so it's gone as of a few weeks ago, and the current nightly builds will report the true input data type. (The actual results are unchanged.) Tim: I have no reason to think we handle unmerged data poorly; I'm not sure who would have told you that. In most cases they will be merged as needed upon reading the file. I'm a little concerned that you're getting such different results from Xtriage and pointless/aimless, however. Could you please send me the input and log files off-list? Dirk, same thing: if you have an example where XDS and Xtriage are significantly in disagreement, the inputs (and logs) would be very helpful. In both cases, I suspect the difference is in the use of resolution cutoffs and absolute-scaled intensities in Xtriage versus other programs, but I'd like to be certain that there's not something broken. thanks, Nat
Re: [ccp4bb] AW: [ccp4bb] Weired Crystal packing
I suggest submitting your coordinates to the PDBe (EBI) web site to run PISA or using it via tthe CCP4I interface. This analysing contacts, and suggests the most favoured packing. It is not fool proof but worth checking. Eleanor On 15 July 2014 09:53, herman.schreu...@sanofi.com wrote: Dear Appu, What is your problem? To me this crystal packing looks completely normal. If you look for tetramers in space groups with 422 symmetry I am sure you will find examples with a similar packing. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Appu kumar *Gesendet:* Dienstag, 15. Juli 2014 00:57 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Weired Crystal packing Dear All, I need your valuable suggestion in defining the arrangement of tetrameric protein in crystal upon symmetry mates information. I have attached a ppt for your evaluation. The monomer protein is composed of two domain (Domain 1 and Domain 2). In space group I422, the arrangement of tetramer upon symmetry mate information look weired because there are two kind of crystal contacts. The tetramer1 form crystal contact with tetramer2 through domain1 but on other side tetramer1 form crystal contact with tetramer3 through domain 2. Things will be clear if you will have look at the attached ppt. Therefore I request you to please look at the attached ppt. I have looked at the crystal packing in many tetrameric proteins which are either arranged in head to head or tail to tail in crystal but not in both head to head and tail to tail contacts in same crystal. Is this kind of crystal packing is possible? I need your valuable suggestion. Crystal is solved as monomer which upon symmetry mate information gives correct tetramer.
Re: [ccp4bb] Translational NCS and molecular replacement.
No need to reindex - just do this to change the space group in the scala header. mtzutils hklin1 scala.mtz hklout scala-P22121.mtz symm P22121 end There are other ways of course.. Eleanor On 16 July 2014 13:28, Vajdos, Felix felix.vaj...@pfizer.com wrote: Dear Sudipta— Herman is correct, you just need to reindex your scala.mtz file to the correct space group. Regarding translational NCS, it has been many years since I’ve dealt with this, but it is my impression that modern refinement methods treat this much better, especially with maximum likelihood targets, as the low(er) intensity reflections will have systematically lower sig/noise than the other parity groups. *Felix F. Vajdos* * Associate Research Fellow Structural Biology Biophysics* *Pfizer Inc* *Eastern Point Road * *MS 8220-3273* *Groton, CT 06334* *860-715-6504 860-715-6504* *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of * herman.schreu...@sanofi.com *Sent:* Wednesday, July 16, 2014 3:08 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] AW: [ccp4bb] Translational NCS and molecular replacement. Dear Sudipta, you are correct, your original scala.mtz has the wrong space group in it, resulting in very high Rfactors (and presumably bad electron density). In these cases, I usually reprocess (remerge) the data in the correct space group to get the statistics right (and gain probably a few extra h00 reflections that got rejected in P212121). If you use the same a, b and c axes as before, you do not need to rerun Phaser, otherwise you have to. If you have translational NCS, you have to live with it. The only way to get rid of it is to find another crystal with a different crystal packing. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Sudipta Bhattacharyya *Gesendet:* Dienstag, 15. Juli 2014 20:32 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Translational NCS and molecular replacement. Dear Community, I have some doubts to clarify. In a way to solve a structure by Phaser-MR, I found phaser ended up with a potentially good MR solution (with good statistics, packing and electron density, and as we know the homolog structure so in a reasonable biological assembly also). However, the space group where phaser found the potential solution (P22121) is different what we got in pointless (P212121), and phaser also indicated the presence of translational NCS (NCS translation vector = 0.500, 0.494 0.391). Now when we try to refine the structure with its original scala.mtz file (which is indexed and sclaled in P212121) and phaser generated pdb file, the R/Rfree is very high (around 0.5) but when I tried refining with mtz file generated by phaser, it was reasonable, at least for first cycle of refinement (R/Rfree, 0.41/0.46). Now my question is, can I use the phaser generated mtz file instead of the original scala.mtz for further refinement? Or I have to reindex my original data into phaser suggested spacegroup and run the MR again? Translational NCS are generally associated with high R values, is there any way to get rid of that problem? Best regards, Sudipta.
Re: [ccp4bb] Hi
Yes - is it rot mat? at home so can't check but look at the documentation - I remember it turned any rotation definition into all the other likely ones… Eleanor On 21 Jul 2014, at 05:01, vijay srivastava wrote: Dear All, Is there is any program to convert euler angles into theta, phi and kappa? regards Vijay
Re: [ccp4bb] AW: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site
Well yes, The bulk solvent model can distort the density, especially if the ligand is large. But it usually isnt too serious - try doing SIMPLE scaling and see if that has any effect on the appearance of the density Eleanor On 22 July 2014 10:16, herman.schreu...@sanofi.com wrote: Dear Armando, Is 1.9Å really the diffraction limit of your crystals, or do they diffract further and is 1.9Å just a convenient resolution cutoff? In the latter case you might be looking at truncation effects. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Armando Albert Gesendet: Freitag, 18. Juli 2014 18:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site Dear all, I am screening a small library of ligands against my protein crystals. Following a soaking with different ligands, I collect datasets to 1.9A resolution and refine them against an empty model without any problem. What is the meaning of a rather large negative electron density in the Fo-Fc map at the binding site?. Could it be related to an incorrect bulk solvent model? Thank you in advance Armando
Re: [ccp4bb] core rmsd in coot
I wish Paul, that at least SOME of the great info that coot prints to the screen then scrolls out of sight could be directed to a very-useful-things-to-remember box.. Eleanor On Apr 4 2012, Paul Emsley wrote: On 03/04/12 21:51, Ursula Schulze-Gahmen wrote: When superimposing 2 structures in coot, I get a core rmsd in the output. What does this mean? Which residues are included in the core rmsd? Are these all the residues that have equivalent residues in the moving and reference molecule? It is the r.m.s.d. (after superposition) of the aligned C-alphas - yes they have equivalent residues. The residues are tabulated in the output after: Moving Reference Distance Krissinel E, Henrick K Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 60, 2256-2268, 2004. http://journals.iucr.org/d/issues/2004/12/01/ba5056/ba5056.pdf (Open Access) HTH, Paul. -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
This could well be due to radiation damage - S are often affected, also Glu and Asp side chains. It is hard to know what to do since the effects are time related. If you have high redundancy maybe you could not use he later batches? Otherwise maybe just relax the B factor restraints and let them show the loss of atoms.. The trouble with that is that you have to relax all side-chain B restraints which may not be so appropriate for ILE say... Eleanor On Apr 4 2012, Chris Meier wrote: MessageDear all, I am refining the X-ray structure of a protein:Data to ~2A were collected at a latest-generation synchrotron.The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms(e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks,Chris -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] problem in scaling the Zn-MAD data
You know that point group has two indexing conventions (h k l and -k,-h,-l if I remember - see the CCP4 documentation on alternate indexing and use pointless to sort it out.. Eleanor . Use pointless to check On Apr 4 2012, Deepthi wrote: Hello everyone I have a problem scaling the MAD data which was collected a week ago.The data was collected at 1.5A resolution using three wavelengths for Zn-MAD experiments. Scaling the data for MAD experiments, the number of rejections and chi2 values were very high even after adjusting the error-scale factor and error model. The space group i used was p312 which i obtained by running a self-rotation function in MOLREP. When i scale my data using p312 spacegroup the chi2 and rejections were huge. But he data was scaling well in p321 spacegroup. can anyone explain whats going on? Thank you very much Deepthi -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] problem in scaling the Zn-MAD data
I don't see how a self rotation function can determine SG. If you processed data as P1 then found the SR maps showed 3 folds and 2 folds you might suspect that POINT GROUP!!! (Remember space groups are guessed on the basis of systematic absences - you don't really know that till you have a structure solution) At the integration stage it is important to choose a crystal class - e.g. trigonal with a=b and gamma = 120 The point groups then may be P3 P312 P321 P6 P6/mmm After integration a program like pointless checks for symmetry agreement. eg do h k l k (-h-k),l and -h-k,h,l agree well? In that case you probably have 3 fold symmetry. And so on for other possible symmetry operators.. After that you can probably be confident of your point group. But as Clemens pointed out - there are different equally possible indexing conventions for some of these choices. You need to check pass 2 against pass 1, etc - see pointless GUI Eleanor On Apr 5 2012, Deepthi wrote: Hello I arrived at the p312 space group by running a self rotation function using MOLREP. The maps show the space group as p312. I was scaling the data individually for each wavelength. None of the three wavelengths are scaling are scaling in p312 space group. On Thu, Apr 5, 2012 at 2:17 AM, Clemens Vonrhein vonrh...@globalphasing.com wrote: Hi, On Wed, Apr 04, 2012 at 02:07:58PM -0700, Deepthi wrote: Hello everyone I have a problem scaling the MAD data which was collected a week ago.The data was collected at 1.5A resolution using three wavelengths for Zn-MAD experiments. Scaling the data for MAD experiments, the number of rejections and chi2 values were very high even after adjusting the error-scale factor and error model. The space group i used was p312 which i obtained by running a self-rotation function in MOLREP. When i scale my data using p312 spacegroup the chi2 and rejections were huge. But he data was scaling well in p321 spacegroup. can anyone explain whats going on? When you say 'Scaling the data for MAD experiments': do you mean scaling the various scans for your 3-wvl MAD data in a single scaling job? Unless you already took care of this during data integration, remember that your separate scans could have been indexed differently and therefore don't match up. See eg. http://www.ccp4.ac.uk/html/reindexing.html for some lookup-tables in P312 and P321. You can use the CCP4 program 'reindex' on MTZ files if needed. But I guess most modern data-processing and scaling programs will take care of that automatically anyway? Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] unsubscribe
I think the community should thank you Wolfram for all you have contributed - I do hope this message catches you or someone passes it on! All the best for your retirement eleanor Dodson On 11 April 2012 11:21, Wolfram Saenger saen...@chemie.fu-berlin.de wrote: Dear CCP4 bulletin board, since I am retired, I would like to unsubscribe. It was fun reading the problems others have and to help them out. Best wishes Wolfram Saenger. -- Prof. Dr. Wolfram Saenger Freie Universität Berlin Fachbereich Biologie, Chemie, Pharmazie Institut fuer Chemie und Biochemie/Kristallographie Takustrasse 6 14195 Berlin Tel.: 030 - 838 534 12 Fax: 030 - 838 567 02 E-Mail : saen...@chemie.fu-berlin.de
Re: [ccp4bb] Expanding p4212 coordinates to p1
Cad will do this correctly Reflection utilitied - merge mtz ( rather confusing job title - sorry..) mtz in - the P4121.mtz data Output - P41212-ext.mtz Defne mtz output Select define limit for refl;action by Laue code select P1 then you will get a list of all P1 reflections with phases correctly modified. If you really want to work in P1 you will need to change the space group in P41212-ext.mtz Easiest way is mtzutils hklin1 P4121-ext.mtz hklout P41212-ext-symP1.mtz SYMM P1 end DONT change the G of P41212.mtz before doing the extension - the program needs the P41212 sym ops to change the phases correctly.. If you want the coordinates extended too use pdbset to get a P1 set of cds.. (Coordinate utilities Edit pdb select pdbset and generate chains by symmetry operators eleanor Eleanor On 12 April 2012 06:51, Henning Stahlberg henning.stahlb...@unibas.chwrote: Hi Everybody, I am working in a P4212 plane group, which is a p4 symmetric structure with a screw axis in addition. My map has in the unit cell two 4-fold symmetric structures, one if which is upside-down with respect to the other one. If using expand in sftools, then the resulting map is p4 symmetrized in the center between two p4-symmetric structures, which is wrong. This is probably the phase origin problem referred to here: http://www.ccp4.ac.uk/dist/html/sftools.html#expand Would anybody have a suggestion how I can expand the p4212 MTZ file to full p1, but with properly respecting the symmetry phase origin? My illiterate guess would be to either 1) shift the phase origin in the p4212 MTZ file by (180.0;0.0); then use sftools with expand 1 to create the full reflection sets in p1; then shift back by (180.0;0.0); or 2) use sftools with the command expand 1, and then modify (invert?) the phases of the reflections in the quadrants that have wrong phases. In option 1) I am not sure if it is possible to shift phase origin, while staying in p4212 symmetry. How could I do that? In option 2) I would probably get into deep water the next time I work with another symmetry, like p6212, or p2121, where I would have to deal not with quadrants but with triangles of 60deg angle? Any suggestions would be highly appreciated. All the best, Henning. Henning Stahlberg, PhD Prof. for Structural Biology, C-CINA, Biozentrum, University Basel Mattenstrasse 26 | D-BSSE | WRO-1058 | CH-4058 Basel | Switzerland http://c-cina.org | Tel. +41-61-387 32 62
Re: [ccp4bb] Expanding p4212 coordinates to p1
Apologies - I didn't notice the plane group.. Although I think if the sym ops are correctly listed in the P4121 file they will just be applied as given.. Eleanor On 12 April 2012 11:58, Ian Tickle ianj...@gmail.com wrote: I didn't realise the CCP4 suite could handle the plane groups: where are they listed (they're not in symop.lib or syminfo.lib)? Or are they doing some clever projections of the space groups? Cheers -- Ian On 12 April 2012 11:48, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: Cad will do this correctly Reflection utilitied - merge mtz ( rather confusing job title - sorry..) mtz in - the P4121.mtz data Output - P41212-ext.mtz Defne mtz output Select define limit for refl;action by Laue code select P1 then you will get a list of all P1 reflections with phases correctly modified. If you really want to work in P1 you will need to change the space group in P41212-ext.mtz Easiest way is mtzutils hklin1 P4121-ext.mtz hklout P41212-ext-symP1.mtz SYMM P1 end DONT change the G of P41212.mtz before doing the extension - the program needs the P41212 sym ops to change the phases correctly.. If you want the coordinates extended too use pdbset to get a P1 set of cds.. (Coordinate utilities Edit pdb select pdbset and generate chains by symmetry operators eleanor Eleanor On 12 April 2012 06:51, Henning Stahlberg henning.stahlb...@unibas.ch wrote: Hi Everybody, I am working in a P4212 plane group, which is a p4 symmetric structure with a screw axis in addition. My map has in the unit cell two 4-fold symmetric structures, one if which is upside-down with respect to the other one. If using expand in sftools, then the resulting map is p4 symmetrized in the center between two p4-symmetric structures, which is wrong. This is probably the phase origin problem referred to here: http://www.ccp4.ac.uk/dist/html/sftools.html#expand Would anybody have a suggestion how I can expand the p4212 MTZ file to full p1, but with properly respecting the symmetry phase origin? My illiterate guess would be to either 1) shift the phase origin in the p4212 MTZ file by (180.0;0.0); then use sftools with expand 1 to create the full reflection sets in p1; then shift back by (180.0;0.0); or 2) use sftools with the command expand 1, and then modify (invert?) the phases of the reflections in the quadrants that have wrong phases. In option 1) I am not sure if it is possible to shift phase origin, while staying in p4212 symmetry. How could I do that? In option 2) I would probably get into deep water the next time I work with another symmetry, like p6212, or p2121, where I would have to deal not with quadrants but with triangles of 60deg angle? Any suggestions would be highly appreciated. All the best, Henning. Henning Stahlberg, PhD Prof. for Structural Biology, C-CINA, Biozentrum, University Basel Mattenstrasse 26 | D-BSSE | WRO-1058 | CH-4058 Basel | Switzerland http://c-cina.org | Tel. +41-61-387 32 62
Re: [ccp4bb] Coot chain ID
Edit, or use coot to reorder chains If you read that edited pdb into pdbset pdbset xyzin edited.pdb xyzout edited-and-renumbered.pdb end I think the renumbering happens automatically.. Eleanor On 14 April 2012 09:46, Bernhard Lechtenberg bc...@cam.ac.uk wrote: In Coot you can use Extensions - Modelling - Reorder Chains Bernahrd -- Bernhard Lechtenberg PhD student Huntington lab University of Cambridge Department of Haematology Cambridge Institute for Medical Research Wellcome Trust/MRC Building, Hills Road Cambridge, CB2 0XY United Kingdom -Original Message- From: Dipankar Manna dipanka...@aurigene.com Reply-to: Dipankar Manna dipanka...@aurigene.com To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Coot chain ID Date: Sat, 14 Apr 2012 06:55:10 + Dear All, I fitted a ligand into a structure along with SO4 and waters (COOT). Then I did the “Merge Molecules” and did the refinement. In output PDB file the chain ID sequence is showing B, A, C( ligand, protein and SO4 respectively) and the ATOM numbering is starting from ligand (Chain B instead of Chain A). How can I make the sequence A, B and C (Protein, ligand and SO4). Best wishes Dipankar This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
Re: [ccp4bb] Good way to check ion sites on Coot
And Marjorie Hardings results are nicely tabulated on a web site.. Google for it Eleanor On 13 April 2012 22:08, Marc Kvansakul m.kvansa...@latrobe.edu.au wrote: Hi Andre, Majorie Harding wrote a few nice papers on metal ion binding sites and their associated coordination geometry, examples are: Harding MM (2006) Acta D vol. 62 pp. 678-82 or Harding MM (2004) Acta D vol. 60 pp. 849-59 Best Marc Dr. Marc Kvansakul Laboratory Head, NHMRC CDA Fellow Dept. of Biochemistry| La Trobe University | Bundoora Rm 218, Phys Sci Bld 4, Kingsbury Drive, Melbourne, 3086, Australia T: 03 9479 2263 | F: 03 9479 2467 | E: m.kvansa...@latrobe.edu.au | From: Zhijie Li zhijie...@utoronto.ca Reply-To: Zhijie Li zhijie...@utoronto.ca Date: Fri, 13 Apr 2012 16:37:43 -0400 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Good way to check ion sites on Coot Hi This is what I do: ValidateHighly coordinated waters *From:* Andre Godoy andre_go...@yahoo.com.br *Sent:* Friday, April 13, 2012 3:57 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Good way to check ion sites on Coot Dear all. can someone tell me what is the best way to check for ion binding sites on my structure? I mean, a text with coordination examples, or maybe a tool on coot for it ... best wishes Andre
Re: [ccp4bb] Disorder or poor phases?
Nothing profound to add to this interesting discussion, but I too would like to plug FobsA - FobsB type maps - when A and B are similar but not quite the same.. It is prudent to omit the interesting parts of model A (or B) - whichever you use to calculate the PHIC and FOM - but the peaks and pits often clear up ambiguity brilliantly. You need to CAD the two data sets together, and make sure both Fobs are on the same scale.. Eleanor On 13 April 2012 19:50, Gloria Borgstahl gborgst...@gmail.com wrote: a recent experience in our lab with molecular replacement (wt and disordered point mutant; same space group and unit cell) was solved with a combination of two methods. 1. We made omit maps in the disordered region at several lower resolutions. The region became interpretable after suffereing through these maps, building residue by residue and refinement. 2. Then we had the bright idea to make Fwt-Fmutant maps to confirm our interpretation. Happily this map did confirm the unexpected large structural changed caused by a point mutant. On Fri, Apr 13, 2012 at 1:31 PM, James Holton jmhol...@lbl.gov wrote: Francis, I think in the cases you describe the region in question is disordered. Time and time again I have users coming to my beamline wanting to clean up a questionable region by getting experimental phases. Ahh! If only I had a nickle for each one. Oh wait, I suppose I kind of do? I take that back! Go MAD everyone! Much as I hate to discourage people from using my favorite technique, Tim is right: phases are not region-specific in electron density maps. Dale does make a good point that there is such a thing as model bias and one can argue that experimental phases don't have it. But, this is only true if you have not yet applied solvent flattening. How long has it been since you looked at a raw experimentally-phased map (before solvent flattening)? I'm willing to bet a while. With very few exceptions, raw experimental phases are lousy. We have actually become quite dependent on density modification to clean them up. In fact, solvent flattening is the only reason why SAD works at all. However, you CAN use anomalous differences to clear up disordered regions in a different way. Something I started calling SeMet scanning a number of years ago. A few of my users have done this, and a good example of it is Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate residues in the disordered region one at a time to SeMet, and look at phased anomalous difference Fourier (PADF) maps. These maps are surprisingly clear, even when the anomalous difference signal is so weak as to make experimental phasing hopeless. Yes, the best phases to use for PADF maps are model phases, but, as always, it is prudent to refine the model after omitting the thing you are looking for before calculating such phases. Another way to get residue-specific labeling for low-resolution chain tracing is radiation damage. If you expose for the right amount of time, Asp and Glu side chains will be specifically burnt off, but not Asn and Gln. You will also see Met loosing its head, etc. So, as long as you have read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to guide sequence assignment, even at 4.5 A and worse. Anyway, when it comes to the question of is it disordered or is it model bias?, I think it is usually the former. It is very difficult to make model bias suppress a region that is actually well-ordered. Try it! After all, this is the whole reason why we bother looking at fo-fc maps. Then again, it is always possible to have a model so bad that the phase error is enough to squash anything. An excellent example of this can be found in the Book of Fourier. Taking amplitudes from the image of a cat, you can see what happens when you use the phases of a duck: http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif as opposed to what happens if you use the phases of a manx: http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif A manx is a species of cat that doesn't have a tail, so no animals were harmed in obtaining these phases. My point here is that the cat's tail can be seen quite readily in the 2fo-fc map if most of the structure is already right, but if your model is completely unrelated to the true structure (fitting a duck into a cat-shaped hole), then everything is in the noise. Real structures are usually somewhere between these two extremes, and I think an important shortcoming in modern crystallography is that we don't have a good quantitative description of this middle-ground. We all like to think we know what model bias is, but we don't exactly have units for it. Should we be using a scale of 0 to 1? Or perhaps duck to cat? Yes, I know we have figure of merit, but FOM is not region-specific. In
Re: [ccp4bb] wwPDB and CCDC
A jolly good thing - congratulations and thanks to the instigators... Eleanor On 13 April 2012 15:40, Gerard DVD Kleywegt ger...@xray.bmc.uu.se wrote: Hi Paul, You saw the wwPDB/CCDC JPG in my PPT at GSK :-) Yes, wwPDB and CCDC have signed an MoU. In pounds and pennies it means, amongst a number of other things, that wwPDB will be allowed to use Mogul in its validation pipeline and that wwPDB will be allowed to incorporate and redistribute CSD coordinates of small molecules that occur in both PDB and CSD. --Gerard (K) On Fri, 13 Apr 2012, Paul Emsley wrote: On 13/04/12 14:30, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Paul, you are not referring to Gerard Bricogne's announcement for the grade-server, are you? http://www.mail-archive.com/**ccp4bb@jiscmail.ac.uk/**msg25770.htmlhttp://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg25770.html :) No. If not - what type of agreement do you have in mind? IIRC, I remember a picture of Gerard (K), Helen, Haruki and a smiling Colin Groom sitting along a table signing something. I was wondering if more details are available on the web. Paul. Best wishes, --Gerard ** Gerard J. Kleywegt http://xray.bmc.uu.se/gerard mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. ** Little known gastromathematical curiosity: let z be the radius and a the thickness of a pizza. Then the volume of that pizza is equal to pi*z*z*a ! **
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Oh dear - this is the version of Refmac in the latest ccp4 release - can this be updated on the web site as soon as possible ? Eleanor On 16 April 2012 12:02, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/ refmac5.7_linux.tar.gzhttp://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk jen...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
