Re: [ccp4bb] CCP4: scalepack2mtz problem
Hi, Eleanor: Thank you for your suggestions. In HKL, I integrated the data set with P222, and scaled it with P222 and P212121. From pointless, it says the data better fit with P212121. So I would like to process the data with P212121 as well. And you have NCS translation at 0,0,0.5 which makes it hard to be sure if the SG is P21212 or P212121 That gives the (probably incorrect) suggestion that the data might be twinned This might be the reason that import failed with P212121. Regard Uma On Tue, Dec 9, 2014 at 6:31 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I suspect this is an external problem - there is no failure message in the P212121 case.. Obviously the input data is different for the 2 sets, do you know why? The integration should be identical. And you have NCS translation at 0,0,0.5 which makes it hard to be sure if the SG is P21212 or P212121 That gives the (probably incorrect) suggestion that the data might be twinned Not sure why there are differences in the log files and the tasks - scale[pack2mtz and ctruncate should run more or less identically for these two cases. I suggest checking your input, and trying again Eleanor On 8 December 2014 at 21:51, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I try to import the .sca file from hkl2000. The data was scaled as P212121 from HKL2000. In ccp4, I used scalepack2mtz from Scalepack(Denzo) into MTZ format, run as Ctruncate. The import stopped at Twin fractione estimates exluding operators and didn't go any further. I scaled the data as P222, and imported it using scalepack2mtz. It went through without problem. I have the log files from both imports attached. Could you advice why the P212121 can't go through? Thank you Uma
[ccp4bb] MR: space group and cell units
Dear All: I try to solve a structure by using Phaser. The data set was collected at 3.9 A with 98.1% (95%) completeness. Based on pointless, the best space group for the data set is P21212. And the data can be processed with P21, P212121 as well. The I/sigma value for the data is 13.3 (1.9). There are some structure models available from PDB with space groups at P21, P212121 or P21212. I used these structures as templates to get initial models. I then use Refmac5 to refine the models. But the R-factors/R-free are around 0.5 /0.5. When I exam the .pdb and .mtz in coot, the models are not aligned with density. I then use the templates (PDB entries) against the processed .mtz files using Refmac5. Again, the R-factors are higher than 0.5. I notice that my data set has different cell units with templates. When the space group match to one template, the cell dimensions is differ from the template. When the cell dimension matches to one template, the space group is different. Is this the problem for modeling? Sequence identity between my protein and templates is 99%. Two residues were mutated in my protein. I use Phaser for molecular replacement, and Refmac5 for refinement. Data was processed using xds or mosfilm. Thank you for advice Uma On Wed, Aug 1, 2012 at 2:27 PM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I try to use Phaser to solve the structure by Molecular Replacement. The data set is collected @180 degree. I process the data using HKL, and have resonable good score: rejection (0.05), Linear R-factor (0.038), completeness (98.3), resolution (50-1.5). I then use Phaser to do MR. The parameter setting are: automated search components in asymmetric unit;number of residue 1332; number in asymmetric unit 1 perform search search using ensemble1 number of copies to search for 4 The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Thank you Uma
Re: [ccp4bb] MR: space group and cell units
HI, Mark: Thank you for your suggestions. try all possible spacegroups with PHASER Yes, this is done with all the modeling. make sure you are using a monomer as search model I also tried with monomer as search model. When run with refmac5, the R-value is above 0.5. I exam the model in coot. Some parts of model match with density. But some parts of model do not matched to the density. At what resolution are the templates in the PDB? 3.0 - 3.9 A At this stage, I would like to compare the structure at backbone level, not the side chain. May have to adjust model piece by piece to fit into the map. Thanks Uma On Wed, Jul 30, 2014 at 12:43 PM, Mark J van Raaij mjvanra...@cnb.csic.es wrote: try all possible spacegroups with PHASER, make sure you are using a monomer as search model, not a crystallographic multimer. In any case 3.9 A is not going to give you much information and you will be hard-pressed to see the mutation differences - so perhaps forget about this data and improve the crystals first to get higher-resolution data to 2.5A or preferably even better. At what resolution are the templates in the PDB? You should be able to get similar resolution to those. 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 Associate Editor of Virology Journal Academic Editor of PLoS One On 30 Jul 2014, at 18:38, Uma Ratu wrote: Dear All: I try to solve a structure by using Phaser. The data set was collected at 3.9 A with 98.1% (95%) completeness. Based on pointless, the best space group for the data set is P21212. And the data can be processed with P21, P212121 as well. The I/sigma value for the data is 13.3 (1.9). There are some structure models available from PDB with space groups at P21, P212121 or P21212. I used these structures as templates to get initial models. I then use Refmac5 to refine the models. But the R-factors/R-free are around 0.5 /0.5. When I exam the .pdb and .mtz in coot, the models are not aligned with density. I then use the templates (PDB entries) against the processed .mtz files using Refmac5. Again, the R-factors are higher than 0.5. I notice that my data set has different cell units with templates. When the space group match to one template, the cell dimensions is differ from the template. When the cell dimension matches to one template, the space group is different. Is this the problem for modeling? Sequence identity between my protein and templates is 99%. Two residues were mutated in my protein. I use Phaser for molecular replacement, and Refmac5 for refinement. Data was processed using xds or mosfilm. Thank you for advice Uma On Wed, Aug 1, 2012 at 2:27 PM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I try to use Phaser to solve the structure by Molecular Replacement. The data set is collected @180 degree. I process the data using HKL, and have resonable good score: rejection (0.05), Linear R-factor (0.038), completeness (98.3), resolution (50-1.5). I then use Phaser to do MR. The parameter setting are: automated search components in asymmetric unit;number of residue 1332; number in asymmetric unit 1 perform search search using ensemble1 number of copies to search for 4 The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Thank you Uma
Re: [ccp4bb] Set up ccp4 environment
Dear Tim: With the commend of source /xtal/Suites/CCP4/ccp4-6.4.0/bin/ccp4.setup-sh, I am able to run the xdsstat and f2mtz now. Thank you very much for your help! Uma On Wed, Mar 12, 2014 at 5:42 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Uma, you have to run the command source /xtal/Suites/CCP4/ccp4-6.4.0/bin/ccp4.setup-sh in the terminal from which you start xdsstat (and f2mtz etc.pp). It is best to place the above command into your file ~/.bashrc so that you do not need to type it each time you want to use ccp4. (you will have to adjust the path /xtal/Suites/CCP4/ccp4-6.4.0 according to your installation). The file .bashrc resides in your home directory. Best, Tim On 03/11/2014 10:37 PM, Uma Ratu wrote: Dear All: I try to run xds in linux, but have some problems. With xdsconv, it complains: f2mtz: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory cad: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory With xdsstat, it complains: xdsstat: Cannot open environ.def It seems that one needs to set up a CCP4 environment in order to run xds in linux. I have ccp4 (the latest vision for linux) installed. And I use Ubuntu 12.04 LTS. Thank you for your advice Uma - -- - -- 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 Icedove - http://www.enigmail.net/ iD8DBQFTICwKUxlJ7aRr7hoRAj/tAJ0TvaJsyyqpAi3l/bg3oT4suzm/EACfV9KK 5IEXBIUDbUM2fbbEF3QY4PQ= =rTR6 -END PGP SIGNATURE-
[ccp4bb] Set up ccp4 environment
Dear All: I try to run xds in linux, but have some problems. With xdsconv, it complains: f2mtz: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory cad: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory With xdsstat, it complains: xdsstat: Cannot open environ.def It seems that one needs to set up a CCP4 environment in order to run xds in linux. I have ccp4 (the latest vision for linux) installed. And I use Ubuntu 12.04 LTS. Thank you for your advice Uma
Re: [ccp4bb] Water or ion
Dear All: Many thanks for your comments and inputs! Uma On Sat, Nov 23, 2013 at 7:14 PM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I use Coot to check water molecules in my model. Most of them are in good coordinates for water. Some of these waters have unusual coordinates. For example, one is in the H-bond distance with 2 nitrogen and 2 oxygen of protein residues, plus one oxygen from ligand (W_230.jpg), and the other is in the H-bond distance with 3 nitrogen, 1 oxygen of protein residue, plus one water(W_300.jpg). Would you advice if these molecules are water, or something else, ions? I tried Coot - highly coordinated water, and check/delete water. The program does not pick up anything unusual. Thank you Uma
[ccp4bb] Win-coot: replace residue
Hello, I try to replace one of cysteine residue to CSX using Win-coot. Here is how I did: Extensions Modelling Replace Residue... and enter the three letter code. The program place the CSX residue, but with break bond. The new residue is no longer linked with other residue and not in the chain. I then try to replace with other residues, such as ALA. The results are the same: the new residues all with break bond. How could I put the replaced residue back to the main-chain? I use Win-coot-0.7. Thank you Uma
Re: [ccp4bb] Win-coot: replace residue
Hi, Bernhard and Yury: I did change Cys to CSX or CSO with older version of Coot. And the program replace the residue inner the chain. With present Coot-0.7, it seems to be complicated to replace a residue. Thank you for your suggestions. Uma On Wed, Sep 11, 2013 at 4:40 PM, Bernhard Lechtenberg blechtenb...@sanfordburnham.org wrote: Hi Uma, when I do the same procedure (in OSX, but the problem seems to be the same), the new residue is stored in a new chain with residue number 1. So you would need to renumber the residue and change the chain ID back to the original chain ID. It's annoyingly complicated, but it works for me. Hope that helps, Bernhard On Sep 11, 2013, at 12:56 PM, Uma Ratu rosiso2...@gmail.com wrote: Hello, I try to replace one of cysteine residue to CSX using Win-coot. Here is how I did: Extensions Modelling Replace Residue... and enter the three letter code. The program place the CSX residue, but with break bond. The new residue is no longer linked with other residue and not in the chain. I then try to replace with other residues, such as ALA. The results are the same: the new residues all with break bond. How could I put the replaced residue back to the main-chain? I use Win-coot-0.7. Thank you Uma
Re: [ccp4bb] Unidentified blobs
Dear All: I thought that it is possible to be the PEG. The crystallization condition contains 3% of PEG. Thank you for your comments and input Uma On Thu, May 16, 2013 at 11:58 AM, Mario Sanches mario...@gmail.com wrote: It looks like structured PEG to me. Did you have PEG as a precipitant in you crystallization solution? This is a quite normal feature, you will find many examples in the PDB. You could try to model it and see how it fits the density, how your R-factors behave, etc. On Thu, May 16, 2013 at 8:45 AM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I collected a data set (1.6A) of one of my target proteins, and built a model by molecular replacement. I then exam the model by Coot and found two unidentified blobs. The two look the same and not in active sites. One is close to residues 269 TYR/B and 268 ASN/B, the other is close to 25 Gly/B and 67 Pro/B. The model contains a homo-dimer. The unidentified blobs are only observed in one monomer, not in the other one. I also looked other structures available in this protein family from PDB. None has this identified blobs in the position as I observed from my model. Here is my questions: 1. How to identify which compound of this electron density is? 2. Because the blobs are not in the active sites, one should just ignore? 3. Is it common for unidentified blobs shown in the structure models? Attached please find the images of the two blobs. Any clue what it is? Thank you for your comments and advices Uma -- Mario Sanches http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] S-nitrosylation protein
Dear All: Many thanks for your commoents and advice. I will keep them in mind. Uma On Thu, Feb 14, 2013 at 7:54 AM, Fischmann, Thierry thierry.fischm...@merck.com wrote: Collect small slices of data (instead of a complete data set) on several crystals then merge the data together to get a full data set. The slices of must be small enough so that the damage to the S-NO group is still very limited on each slice. You may have to play with beam attenuation a bit, depending on how fast the degradation occurs. ** ** Good luck ** ** Thierry ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Uma Ratu *Sent:* Wednesday, February 13, 2013 5:38 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] S-nitrosylation protein ** ** Dear All: I plan to use X-ray crystallography method to study the S-nitrosylated protein structure. The native protein crystals diffracted to 2A with synchrontron. I now have the crystals of S-ntrosylated protein. Since S-NO moiety appears to be unstable to synchrotron radiation, could you advice / comments on the stratage on the data collection of S-nitrosylated protein crystals? The protein crystals did not diffract well with in house X-ray. Thank you for your comments. Uma Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] S-nitrosylation protein
Dear All: I plan to use X-ray crystallography method to study the S-nitrosylated protein structure. The native protein crystals diffracted to 2A with synchrontron. I now have the crystals of S-ntrosylated protein. Since S-NO moiety appears to be unstable to synchrotron radiation, could you advice / comments on the stratage on the data collection of S-nitrosylated protein crystals? The protein crystals did not diffract well with in house X-ray. Thank you for your comments. Uma
Re: [ccp4bb] Find ligand with WinCoot
Dear Giovanna and Bosch: Thank you for your advices. I tried the way as you suggested, but not work. Here is how I did: 1. Center on the ligand density, and Find ligand Right here. 2. Increase Fo-Fc at 3 sigma or reduce 2Fo-Fc maps to 0.8 sigma. Coot failed to recognize the density. You do specify the ligand you are looking for (as PDB file) right? The ligand that I put into protein is NADH. I use NAD to do the search. It found two but failed on the other two. Thanks for advice Uma On Wed, Oct 24, 2012 at 10:44 AM, Bosch, Juergen jubo...@jhsph.edu wrote: If you show symmetry mates I'm sure the ones Coot found outside your protein are actually in the sites you are missing. Nothing to worry, just safe the symmetry mate and read in those coordinates merge with your current model. Jürgen .. 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 On Oct 24, 2012, at 10:16 AM, Uma Ratu wrote: Hello, I have problems to find the ligand using WinCoot. The protein was purified with NADH, and crystallized. Data diffraction is bellow 2A. Structure is solved using molecular replacement. The protein is homo-tetramer. I then exam the model. I can identify two ligand positions inside two of the monomers. When I take a close look at the other two monomer, it is very clear that there are big molecular there. But Coot failed to find them. Here is how I did: Method 1: Load NAD. Then Calculator - Other Modelling Tools - Find Ligand Coot find 4 ligands. Two inside the tetramer, two outside the tetramer. Method 2: Validate - Unmodelled blobs Coot returns 4 blobs, two inside the tetramer, two outside the tetramer Both methods failed to identify the other two ligands inside the tetramer. Attached is the electronic density map of one of the questioned ligands. I use WinCoot - 0.7 - pre-1 Thank you for advice Uma blob-1.jpg
Re: [ccp4bb] MR with Phaser
Thank you very much for your comments. I re-do the data process with P21, and use the output for Phaser. This time, Phaser finishes with good resolution, the TFZ is 28. And the model is tetramer. Cheers Uma On Thu, Aug 2, 2012 at 9:45 PM, Roger Rowlett rrowl...@colgate.edu wrote: A few thoughts: 1. Search for all possible space groups (e.g., P2 and P21 in this case). Be happy it isn't C222, which means 8 possible combinations of screw axes to search! As mentioned already, P21 is far more common than P2. I think P21 is one of the most common space groups in protein crystallography. 2. How are you determining how many copies of the search model go in the ASU? It is not necessarily one biological unit, or an integral number of biological units. Run a cell content analysis in Phaser (e.g. Matthews probability calculator) and start there, but consider the results a suggestion only. For larger ASUs, the predicted number is not very accurate. Six might actually be 4 or 8 chains. 3. Look at the crystal packing in Pymol, Coot, or in you favorite tool. You can do this by enabling a large symmetry molecule radius. If you see a regular lattice of proteins with nice solvent channels and protein-protein contacts, things are looking up. (But you can be fooled into a premature victory at times.) 4. Partial Phaser solutions may provide a big hint about how many molecules are in the ASU when packing is examined. Often the placement of the missing molecules is quite obvious, as it completes a solvent channel or fills in symmetrical protein-protein contacts. 5. Finally, look at the maps. Crappy maps probably mean the wrong space group, especially if chains don't pack well. Good maps with good packing usually mean you are on the right track. Cheers and good luck, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/1/2012 2:27 PM, Uma Ratu wrote: Dear All: I try to use Phaser to solve the structure by Molecular Replacement. The data set is collected @180 degree. I process the data using HKL, and have resonable good score: rejection (0.05), Linear R-factor (0.038), completeness (98.3), resolution (50-1.5). I then use Phaser to do MR. The parameter setting are: automated search components in asymmetric unit;number of residue 1332; number in asymmetric unit 1 perform search search using ensemble1 number of copies to search for 4 The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Thank you Uma
Re: [ccp4bb] Process multiple data sets
Dear All: Thank you very for your comments and advices. ' I am getting to know why are the problems. And will try again. I appreciate you all for your inputs regards Uma On Thu, Aug 2, 2012 at 4:11 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: An earlier post said the point-group is P2, and these reported cells do not quote the beta angles: what are these angles?. In the monoclinic system it is possible to have two closely-similar alternative cells in certain special cases, and if the crystals have been indexed differently this could prevent their merging. The program Pointless would sort that out for you. Phil On 2 Aug 2012, at 00:45, Edwin Pozharski wrote: The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. This is very substantial difference and with unit cell expanding by ~20% one would expect scaling problems. Try using the same unit cell/orientation on all datasets, it's easy to do if you abandon gui and feed input file directly to denzo. -- Edwin Pozharski, PhD University of Maryland, Baltimore
[ccp4bb] Process multiple data sets
Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
Re: [ccp4bb] Process multiple data sets
The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non-isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
Re: [ccp4bb] Process multiple data sets
Please correct me if I am wrong: The HKL is not good to combine multiple data sets, even they are come from the same crystal? With HKL, I also tried this way: Index, integrate each data set individually, they all have the same space group. Then scale them together. Still, the graph from scale of the whole set look very wired compared with those of individuals. Uma On Wed, Aug 1, 2012 at 9:55 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: Note that neither Aimless nor Scala will do a particularly good job at scaling data from Denzo or Scalepack, since the output files from Scalepack are missing essential geometrical information. They work well with data from Mosflm or XDS (or Saint) (although AFAIK the XDS Saint scaling programs work perfectly well) However, Pointless may still be useful to check that you have indexed them consistently Phil On 1 Aug 2012, at 14:36, Harry Powell wrote: Hi I'd process (i.e. index, refine, integrate) each data set individually, check that (at least) they all have the same crystal system, then combine the datasets using Pointless. Then scale with Aimless. Of course, I'd use Mosflm/Pointless/Aimless rather than HKL, but that's another question (pace, ZO, WM, MM!) On 1 Aug 2012, at 14:08, Uma Ratu wrote: The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non-isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] MR with Phaser
Dear All: I try to use Phaser to solve the structure by Molecular Replacement. The data set is collected @180 degree. I process the data using HKL, and have resonable good score: rejection (0.05), Linear R-factor (0.038), completeness (98.3), resolution (50-1.5). I then use Phaser to do MR. The parameter setting are: automated search components in asymmetric unit;number of residue 1332; number in asymmetric unit 1 perform search search using ensemble1 number of copies to search for 4 The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Thank you Uma Crystal - Global Refinement *Space Group* P2 *Resolution Processing* 50.00 - 1.52 *Resolution Scaling*50.00 - 1.52 *a* 79.70 ± 0.01 *b* 125.27 ± 0.01 *c* 83.60 ± 0.01 *alpha* 90.000 ± *beta* 118.02 ± 0.05 *gamma* 90.000 ± *Volume*736873.6 *Mosaicity Range* 0.20 - 0.34 Global Statistics *Crystal* crystal1 *Date* Aug 01, 2012 *Experimenter* KP *Rawdata directory* /home/KP/Desktop/BY/0712/process-0712/data/CD6_7 *Processing directory* /home/KP *Data files*CD6_7_1_0001.cbf[1-180] *Wavelength*0.97920 *Total Number of Reflections* 728105 *Number of Unique Reflections* 220394 *Number of Reflections Marked for Rejection*346 *Percentage of Reflections Marked for Rejection*0.05 *Percentage of Reflections Rejected*0.00 *Linear R-factor in shell 50.00 - 4.13* 0.038 *Change of B-factor*-0.40 - 8.33 *Completeness* 98.3 *Completeness in shell 1.55 - 1.52* 91.9 *Mean I/sigma* 10.5 *Mean I/sigma in shell 1.55 - 1.52* 0.7 *Total Linear R-Merge* 0.094 Site Information *Detector* CCD pilatusCBF *Polarization* 0.99 *X Beam Position* 217.5 *Y Beam Position* 219.5 *Cassette Rotation X* 0.00 *Cassette Rotation Y* 0.00 *Cassette Rotation Z* 0.00 *Yscale*1.0 *Skew* 0.0 *Crossfire X* 0.00 *Crossfire Y* 0.00 *Crossfire XY* 0.00 Scale and B vs. Frame Chi^2 and R-Factor vs. Frame *Frame Number* *Scale* *B* *Chi^2* *R-factor* 1 10.91589-0.40 1.185 0.082 2 10.77627-0.38 1.234 0.082 3 10.53368-0.33 1.162 0.083 4 10.36566-0.27 1.162 0.079 5 10.32651-0.19 1.064 0.077 6 10.18485-0.10 1.030 0.075 7 10.00.001.076 0.076 8 10.051110.101.013 0.078 9 9.92706 0.210.995 0.076 10 9.83898 0.310.952 0.075 11 9.64515 0.420.907 0.077 12 9.61982 0.530.880 0.076 13 9.61376 0.630.885 0.069 14 9.44380 0.740.834 0.073 15 9.44114 0.850.765 0.068 16 9.29782 0.960.834 0.069 17 9.21481 1.080.750 0.067 18 9.12255 1.180.737 0.071 19 9.15504 1.280.798 0.073 20 9.06806 1.380.795 0.076 21 9.03864 1.470.740 0.074 22 8.96390 1.570.793 0.070 23 8.87753 1.660.759 0.073 24 8.81280 1.760.774 0.076 25 8.80572 1.840.777 0.073 26 8.64398 1.930.825 0.079 27 8.56313 2.020.796 0.074 28 8.58220 2.110.720 0.073 29 8.45429 2.190.763 0.074 30 8.43629 2.270.746 0.072 31 8.37449 2.340.821 0.081 32 8.32575 2.410.767 0.069 33 8.23116 2.470.848 0.080 34 8.25275 2.530.717 0.080 35 8.20440 2.570.734 0.073 36 8.12370 2.620.788 0.079 37 8.10550 2.660.777 0.077 38 8.04424 2.700.726 0.077 39 7.98665 2.740.760 0.077 40 7.92014 2.770.692 0.079 41 7.94125 2.810.711 0.079 42 7.89751 2.850.760 0.078 43 7.91178 2.900.650 0.079 44 7.84116 2.940.692 0.078 45 7.80008 2.990.737 0.077 46 7.78514 3.030.658 0.076 47 7.79019 3.070.656 0.077 48 7.77994 3.100.651 0.081 49 7.76673 3.140.567 0.077 50 7.71487 3.180.630 0.079 51 7.64531 3.220.631 0.082 52 7.65363 3.260.586 0.082 53 7.69821 3.310.604 0.080 54 7.67023 3.360.589 0.083 55 7.66701 3.420.550 0.082 56 7.67157 3.470.544 0.080 57 7.66821 3.520.609 0.087 58 7.67582 3.570.550 0.084 59 7.70985 3.630.572 0.086 60 7.69346 3.680.553 0.084 61 7.75456 3.730.550 0.083 62 7.73917 3.790.546 0.082 63 7.76342 3.840.553 0.081 64
Re: [ccp4bb] Process multiple data sets
I notice one thing with my data sets. The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. Is this the reason that I can't index both with same parameter in HKL? And subsequently, can't integrate and scala together. If so, is there a way that I can fix it? Thank you for your advice Uma On Wed, Aug 1, 2012 at 8:50 AM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
Re: [ccp4bb] Serine
Thank you All for you inputs. Uma On Mon, May 21, 2012 at 5:06 PM, Van Den Berg, Bert lambertus.vandenb...@umassmed.edu wrote: Yes, as Jacob says, alternative conformation of the serine. Quite common. Bert -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Uma Ratu [rosiso2...@gmail.com] *Sent:* Monday, May 21, 2012 4:57 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Serine Dear All: Some of serine residues in my model have extra positive Fo-Fc density at the edge of side chain. Some don't have. It is not like from phosphates. I am wondered what is the cause for these extra density. Could these serines be post-translational modified? I have the images attached. P289ser-0512-1 does not have the extra green, where P140ser-0512-1 has. Thank you for you advice and comment Uma
Re: [ccp4bb] CSX
Dear All; Thank you very much for your comments and advices. My protein is homo-tetramer. Based on difference density, only one cysteine (among the four catalytic cysteins) has observed two positive density along SG-group. This one could be modeled into OCS, while the rest will be modeled as non-oxidized. I very much appreciate for all your inputs regards Uma On Fri, May 4, 2012 at 6:09 PM, Savvas Savvides savvas.savvi...@ugent.bewrote: Dear Uma Your modeling of a Cysteine sulfenic group (SOH) is reasonable given the observed difference density but do keep in mind that sulfenic groups are very susceptible to further oxidation to sulfinic and sulfonic acids. Stabilization of sulfenic and sulfinic groups in enzyme active sites is possible as shown crystallographically by Becker et al Nat Struct Biol 5:267-271 (1998). Also make sure that you flip the Histidine in the active site that faces the SOH group to account for a possible hydrogen bond. Best regards Savvas On 04 May 2012, at 20:24, Uma Ratu rosiso2...@gmail.com wrote: Dear All: Thank you very much for your advices and comments. Following your instructions, I am able to change the CYS to CSX. Both Get Monomer and Replace Residue work well. I have the refined conformation CSX (by real space refinment ) attached (named as M-CSX-1). The Fo-Fc map is shown with sigma @2.0. Is this conformation reasonable? Why there are two bond conformation there? Thank you for comments. Uma On Fri, May 4, 2012 at 12:10 PM, Hugo Correia h.corr...@campus.fct.unl.pt wrote: Dear Uma, You can do this using coot. Go to Extensions Modelling Replace Residue... and enter the three letter code. Cheers Hugo Correia 2012/5/4 Uma Ratu rosiso2...@gmail.com Dear All: My protein has a key cysteine residue involved in catalytic activity. The template structure used for the modeling has the same key cysteine. In the template structure, this key cysteine residue is assigned as CSX based on the observation from its electronic density. I compared the electron density from the template as well as my model. I can't tell if the cysteine in my model is oxidized or not. The ones from the template also looks different from each other, although both assigned as CSX. I have the snapshots of these cysteines attached. The ones from my model named as M-, and the ones from the template named as T-. Plus, how to change the residue label from Cys to CSX if the cystein is oxidized? In coot, I could not find such function. Thank you very much for your advice Uma M-CSX-1.tif
[ccp4bb] Covert Structure Factor to mtz
Dear All: I try to convert the .cif files (the structure factor files from PDB) to mtz file. From ccp4i, I chose convert to/modify/extend mtz for this purpose. But program keep complanining: no cell information in keywords or files I open the .cif file in text, and could not find any information about cell information. Is there a easy way to convert the .cif file from PDB to mtz? Thank you for advice Uma
Re: [ccp4bb] Covert Structure Factor to mtz
It works! Thank you very much Uma On Thu, May 17, 2012 at 3:16 PM, martyn.w...@stfc.ac.uk wrote: Reflection cif files from the PDB do not always have cell and symmetry information in them, particularly the older ones, and it sounds like this is your case. In that case, you need to manually copy the cell information from the PDB web page into the ccp4i interface before running. HTH Martyn From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Uma Ratu Sent: 17 May 2012 20:04 To: ccp4bb Subject: [ccp4bb] Covert Structure Factor to mtz Dear All: I try to convert the .cif files (the structure factor files from PDB) to mtz file. From ccp4i, I chose convert to/modify/extend mtz for this purpose. But program keep complanining: no cell information in keywords or files I open the .cif file in text, and could not find any information about cell information. Is there a easy way to convert the .cif file from PDB to mtz? Thank you for advice Uma
[ccp4bb] Ligand geometry
Dear All: I use Refmac5 to refine my model. After the run, I check the model quality by Coot. Here is the problem: In Coot, the ligand - NAD, has bad geometry as indicated by a big red bar. While the geometry of NAD fit nicely with the electron density. If I use refine tools (i.e. regularize Zone or real space refine zone), the geometry of NAD turns to perfec with bond, angle and so on. But the ligand slightly turn away from the electron density map. If I run Refmac5 again with this modified model, the NAD turns back, fit nice to electron density, but gives red bar in coot geometry. The Refinment Parameters in Refmac5 is set @ use automatic weight and use experinmental sigmals to weight X-ray terms. Thank you for advice and comments Ros
Re: [ccp4bb] Refmac and sigma value
Dear All: Thank you very much for you comments and advices. Now I have a better understanding on this issue. regards Ros On Fri, Apr 27, 2012 at 9:25 AM, Eleanor Dodson eleanor.dod...@york.ac.ukwrote: Two points. 1) the fit to ideal geometry as flagged in coot validation does not guarantee a correct model - the best model should be the one that fits the experimental data best, without having unlikely geometry. You could easily get a model with perfect geometry which was incorrectly placed in the unit cell.. 2) the AUTO weighting in REFMAC tries to take into account resolution of the data,and Rfree Have you used that? It isn't infallible of course.. Eleanor On 27 April 2012 10:57, Robbie Joosten robbie_joos...@hotmail.com wrote: Hi Uma, The optimal weight is indeed resolution dependent, but hard to predict. In Refmac you can follow LLfree when you optimize the restraint weight and also keep an eye on the gap between R and R-free (it should not be too wide). Like Rob said, your geometry should be 'reasonable'. This may be a bit vague, but there is no clear target for bond/angle rmsd at a given resolution (some referees will disagree). If you look at the rmsZ values Refmac gives, the target is a bit clearer: rmsZ 1.000. The average rmsZ does go down with resolution (i.e. lower resolution gives lower rmsZ), but an ideal value cannot be given easily (or at all). Tightening the restraints improves the effective data/parameter ratio of your model. You can also improve it by adding additional restrains (e.g. NCS restraints) or by removing parameters (e.g. changing the complexity of your B-factor model). Note that the absence of geometric outliers does not prove that your model is optimal. If you use too tight restraints you can end up hiding genuine fitting errors. Cheers, Robbie -- Date: Fri, 27 Apr 2012 10:04:11 +0200 From: herman.schreu...@sanofi.com Subject: Re: [ccp4bb] Refmac and sigma value To: CCP4BB@JISCMAIL.AC.UK It all will depend on the resolution. At low resolution, relaxing the geometric restraints will allow the refinement program to tweak the model such that the difference between Fobs and Fcalc is minimized, but not that the model gets closer to the truth. I once struggled for a long time with a 3.5Åish data set with a protein where the most important feature was a rather flexible loop. It was before maximum likelyhood methods and Rfrees and the only way I could get rid of the model bias was to use extremely tight geometric restraints. The Rfactor would go up, but suddenly the electron density maps would no longer accept incorrectly placed side chains and new features, not present in the model, would appear. So my advice: at low resolution use as tight restraints as possible and monitor with Rfree if you are going in the right direction. At high or very high resolution, you can follow what your diffraction data tells you. In fact many very high resolution structures ( 1.5 Å) have higher rmsd's for bond lenghts and angles as medium resolution structures. However, at medium or low resolution there is not enough data to justify to relax the geometric restraints too much. Best regards, Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Robert Nicholls *Sent:* Friday, April 27, 2012 9:25 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Refmac and sigma value Hi Uma, Altering sigma affects the strength of geometry restraints throughout the model - bonds, angles, etc. Choosing a very low sigma will cause geometry to be more tightly restrained towards ideal values, which is why you observe improvements in Coot validation. Note that strengthening the geometry weight causes the observations (data) to be less influential in refinement. The risk of this is that your model may no longer appropriately/optimally describe your data. You can assess this locally by manual inspection of the electron density, and globally by considering overall refinement statistics (as reported at the bottom of the Refmac5 log file). Ideally, you want your model to both describe the data and have reasonable geometry. Regards Rob On 26 Apr 2012, at 21:26, Uma Ratu wrote: Hi, Alex: Which sigma do you mean? The one for automatic weight, not for Jelly-body refinement. I did not turn the Jelly-body refinement on. Thanks Ros On Thu, Apr 26, 2012 at 4:08 PM, aaleshin aales...@burnham.org wrote: Hi Uma, Which sigma do you mean? The one for Jelly-body refinement? J-B sigma=0.01 means very small fraction of the gradient will be used in each step. It is used usually with very low resolution (less then 3A) Alex On Apr 26, 2012, at 11:38 AM, Uma Ratu wrote: Dear All: I use Refmac5 to refine my structure model. When I set the sigma value to 0.3 (as recommended from tutorial), the resulted model has many
[ccp4bb] Refmac and sigma value
Dear All: I use Refmac5 to refine my structure model. When I set the sigma value to 0.3 (as recommended from tutorial), the resulted model has many red-bars by coot validation (geometry, rotamer, especially, Temp Facotr). I then lower the sigma value to 0.1, the resulted model is much improved by coot validation. I then lower the sigma value to 0.01, the resulted model is almost perfect, by coot validation and Molprobity. My question is: what is the risk for very low value sigma value? Thank you for your advice Ros
Re: [ccp4bb] Refmac and sigma value
Hi, Alex: Which sigma do you mean? The one for automatic weight, not for Jelly-body refinement. I did not turn the Jelly-body refinement on. Thanks Ros On Thu, Apr 26, 2012 at 4:08 PM, aaleshin aales...@burnham.org wrote: Hi Uma, Which sigma do you mean? The one for Jelly-body refinement? J-B sigma=0.01 means very small fraction of the gradient will be used in each step. It is used usually with very low resolution (less then 3A) Alex On Apr 26, 2012, at 11:38 AM, Uma Ratu wrote: Dear All: I use Refmac5 to refine my structure model. When I set the sigma value to 0.3 (as recommended from tutorial), the resulted model has many red-bars by coot validation (geometry, rotamer, especially, Temp Facotr). I then lower the sigma value to 0.1, the resulted model is much improved by coot validation. I then lower the sigma value to 0.01, the resulted model is almost perfect, by coot validation and Molprobity. My question is: what is the risk for very low value sigma value? Thank you for your advice Ros
Re: [ccp4bb] Molecular Replacement
Ed: Thank you very much for your advice and inputs regards Ros On Wed, Apr 18, 2012 at 8:44 AM, Ed Pozharski epozh...@umaryland.eduwrote: On Tue, 2012-04-17 at 17:49 -0400, Uma Ratu wrote: In order to have my target .pdb, I need to mutate the residues using coot? Others already recommended CHAINSAW to prepare the model. Note that coot has a nice feature under Extensions-All molecule... called [Post MR] Fill partial residues By default, CHAINSAW will truncate non-conserved residues to gamma atom, so you will end up with some weird-looking tryptophans. See http://www.biop.ox.ac.uk/coot/doc/coot/Fill-Partial-Residues.html for details. IMPORTANT! You still have to inspect the model manually, adjust some side chains, build alternate conformers, fix the backbone where needed, fill gaps etc. But you already know this. Cheers, Ed. -- Coot verendus est
[ccp4bb] Molecular Replacement
Hello, I have a question about molecular replacement. I use Phaser or AutoMR to generate models of my target protein. Input .mtz is from X-ray diffraction. Template is from a known structure. I also set up seq file using my target protein. The sequence identity between template and my target protein is quite high, over 90%. When I exam the ouputs, I found the sequence of the output .pdb is exactly same as the template. Is this normal for Molecular replacement? In order to have my target .pdb, I need to mutate the residues using coot? Thank you for advice Ros
Re: [ccp4bb] Molecular Replacement
Thank you very much for your inputs and comments. I am getting understand what is going on now. If your resolution is high (2.2 A or better?) and you have ARP/wARP Yes, the resolution is about 2A. I have ARP/wARP. Will give a try. One more question about Molecular Replacement. With Phaser, I got tetramer conformation. With autoMR, it gave me dimer conformation. Each molecuale was trucated into two chains. The scores from both methods were high, as expected from 90% sequence identity between template and target. Thank you for advice Ros On 4/18/12, Edward A. Berry ber...@upstate.edu wrote: I would say yes. In any case you need to examine each mutated residue to be sure the correct conformation is chosen, so you might as well do the mutagenesis in coot or O. The ccp4 chainsaw program is sometimes used to prepare models for MR, but it truncates the mutated residues to variable extent rather than mutating- the purpose is to improve chances of success, not to arrive at a model with the correct sequence. If your resolution is high (2.2 A or better?) and you have ARP/wARP installed, run A/W in the mode to improve an existing model, giving it your current model and the correct sequence. Not only will it build most of the mutated residues correctly, but in its role as a model bias remover it will fix or remove incorrect parts of the structure that may not be obvious in the initial maps. Uma Ratu wrote: Hello, I have a question about molecular replacement. I use Phaser or AutoMR to generate models of my target protein. Input .mtz is from X-ray diffraction. Template is from a known structure. I also set up seq file using my target protein. The sequence identity between template and my target protein is quite high, over 90%. When I exam the ouputs, I found the sequence of the output .pdb is exactly same as the template. Is this normal for Molecular replacement? In order to have my target .pdb, I need to mutate the residues using coot? Thank you for advice Ros
Re: [ccp4bb] Molecular Replacement
Does that mean a different number of molecules in the asymmetric was found, With Phaser, 4 monomers. With AutoMR, 2 monomers. did you divide the molecule and find each part separately? Each monomer (by AutoMR) is composed of two chains. One chain is part of my target. The other chain matchs to the rest of my target. The original template .pdb is a tetramer with four monomers. I used one of the monomer as the template for the molecualr replacement in both Phaser and AutoMR. The seq file is the whole of my target. Provide more details like space group Space group is P21. whether the tetramer is crystallograhic or all in the asymmetric unit, The tetramer is crystallograhic Thank you Ros On 4/18/12, Edward A. Berry ber...@upstate.edu wrote: One more question about Molecular Replacement. With Phaser, I got tetramer conformation. With autoMR, it gave me dimer conformation. Each molecuale was trucated into two chains. The scores from both Does that mean a different number of molecules in the asymmetric was found, or just that they were arranged differently? If the latter, try generating symmetry-mates around one dimer and see if the tetramer is created. Ideally if the solutions are right they should be the same, within an arbitrary choice of asymmetric unit and origin. What does it mean, each molecule was . . two chains? did you divide the molecule and find each part separately? Is you dimer a heterodimer? Provide more details like space group and whether the tetramer is crystallograhic or all in the asymmetric unit, and some expert may be able to provide suggestions. Uma Ratu wrote: Thank you very much for your inputs and comments. I am getting understand what is going on now. If your resolution is high (2.2 A or better?) and you have ARP/wARP Yes, the resolution is about 2A. I have ARP/wARP. Will give a try. One more question about Molecular Replacement. With Phaser, I got tetramer conformation. With autoMR, it gave me dimer conformation. Each molecuale was trucated into two chains. The scores from both methods were high, as expected from 90% sequence identity between template and target. Thank you for advice Ros On 4/18/12, Edward A. Berryber...@upstate.edu wrote: I would say yes. In any case you need to examine each mutated residue to be sure the correct conformation is chosen, so you might as well do the mutagenesis in coot or O. The ccp4 chainsaw program is sometimes used to prepare models for MR, but it truncates the mutated residues to variable extent rather than mutating- the purpose is to improve chances of success, not to arrive at a model with the correct sequence. If your resolution is high (2.2 A or better?) and you have ARP/wARP installed, run A/W in the mode to improve an existing model, giving it your current model and the correct sequence. Not only will it build most of the mutated residues correctly, but in its role as a model bias remover it will fix or remove incorrect parts of the structure that may not be obvious in the initial maps. Uma Ratu wrote: Hello, I have a question about molecular replacement. I use Phaser or AutoMR to generate models of my target protein. Input .mtz is from X-ray diffraction. Template is from a known structure. I also set up seq file using my target protein. The sequence identity between template and my target protein is quite high, over 90%. When I exam the ouputs, I found the sequence of the output .pdb is exactly same as the template. Is this normal for Molecular replacement? In order to have my target .pdb, I need to mutate the residues using coot? Thank you for advice Ros
Re: [ccp4bb] Water
Dear All: I appreciate for your comments and inputs. Thank you Uma On Thu, Mar 8, 2012 at 12:16 AM, Shekhar Mande shekhar.ma...@gmail.comwrote: Welljust to add, it has been our contention that many of the metal ions have been modelled as waters in several structures- due perhaps to the lack of sufficiently high resolution data. We published some of the potential metal binding sites in many structures a few years ago: Proteins. 2008 Mar;70(4):1206-18. Shekhar On Thu, Mar 8, 2012 at 9:42 AM, Parthasarathy Sampathkumar spart...@gmail.com wrote: Dear Uma, The water pictured in W12-1.jpg: could this be a potential metal ion? If you flip the side chain on Asn at 3.08Angstrom, then this has 3 or 4 coordination with oxygen atoms. So, provided your crystallization condition or buffer contains metal ion(s), you could attempt to see if it fits better with a refinement cycle. May be a similar situation with the water described in W11-1.jpg as well? Difficult to say from these figures. COOT within the validate wizard has an option to search for hihgly-coordinated waters like the one you have pictured. Hope this helps, Partha On Wed, Mar 7, 2012 at 4:21 PM, Uma Ratu rosiso2...@gmail.com wrote: Dear Roger: Thank you very much for your comments. I use them as guideline and remove many 'false waters. Still, I am not clear of some of these 'waters' are real or not. I have the pic attached. In Pic-W11-1, the 'water' is connected to the adjust residues with 4 contacts, which are 'N' or 'O' atoms. I would consider this 'water' is false. My question is: if these 4 contacts include C from residues, will it be a polar contact or not? In Pic-W12-1, the 'water' is connected to the adjust residues with 3 contacts. The 4th is to another 'water'. Will this 'water' is true or not? Similar case is seen in Pic-W190-1 In Pic-W109-1, some 'waters' are connected to adjust residues, some not. Are these 'water' true or not? Further more, and the b-factors are not way out of line, I am not clear on how to define out of line. How to find b-factor of individual residue in Coot? I search the web, but find no answer. Thank you for advice Uma On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett rrowl...@colgate.eduwrote: Uma, Remember that your structure, ultimately, is a model. A model is your best judgment of the true representation of the protein structure in your crystal. Your model should make chemical sense. Coot is pretty good at placing waters, but it cannot substitute entirely for the experimentalist. Coot will miss some waters, and mis-assign others into weak, unmodeled or alternate side- or main-chain density, or into density that might be attributable to cations and anions or other crystallization materials. Your waters should be subjected to inspection and verification. It is really helpful to turn on environment distances in Coot when you do this. Even in a large protein model, it is possible to inspect all waters for reasonableness pretty quickly. If you have no significant positive or negative difference density, and the b-factors are not way out of line, and hydrogen bonding partners are reasonable, then modeling a water is probably a good call. Waters should have hydrogen bonding partners with side chains or main-chain polar atoms, within reasonable distances, or be withing hydrogen bonding distance of other waters that are (chains of waters). If a water has strong electron density and more than 4 polar contacts, you might consider anion or cation occupancy. Most anions and cations will have higher electron density, and appropriately different types of polar contacts. (e.g. you might find sulfates near a cluster of basic residues). Low occupancy anions can often look a lot like water. PEGs can create ugly snakes of variable density that may be challenging to model. Modeling non-protein structural bits is endlessly entertaining for the protein crystallographer. ;) Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 3/7/2012 11:20 AM, Uma Ratu wrote: Dear All: I try to add water to my model. Here is how I did: Coot: Find Wates Map: FWT PHWT; 1.8 rmsd; Distances to protein atoms: 2.4 min/3.2 max Coot found 270 water molecules. I then examed these waters. Most of them had ball shape. Some had two or more balls together. Some had irregular shape (not glabol shape). I run Water Check. The program did not find any mis-matched water. Here is my question: how could I tell the waters are real? Or something else? Thank you for advice Ros -- Shekhar C. Mande (शेखर चिं मांडे) Director, National Centre for Cell Science Ganeshkhind, Pune 411 007 Email: shek
[ccp4bb] Water
Dear All: I try to add water to my model. Here is how I did: Coot: Find Wates Map: FWT PHWT; 1.8 rmsd; Distances to protein atoms: 2.4 min/3.2 max Coot found 270 water molecules. I then examed these waters. Most of them had ball shape. Some had two or more balls together. Some had irregular shape (not glabol shape). I run Water Check. The program did not find any mis-matched water. Here is my question: how could I tell the waters are real? Or something else? Thank you for advice Ros
Re: [ccp4bb] Water
Hi, Joel: Thank you for your comments. Some of these waters have 4 contacts all with O from adjacent residues. As O can be doner as well as acceptor, I would consider them as 'real' water. Some of these 'water' have more than 4 contacts, I would consider them as 'false'. I lower the H-bond seeting to 2 - 3.2. This helps to reduce the noise. The B-factor is displayed in Coot along the bottom (left) when you middle click on an atom. You can also see the B-factor when you read the pdb file as text I found the B-factor using both ways. Thank you very much! Uma On Wed, Mar 7, 2012 at 5:39 PM, Joel Tyndall joel.tynd...@otago.ac.nzwrote: Hi Uma, ** ** Water has the capability of making 4 h-bonds, 2 from the two non-bonding pairs of electrons (h-bond acceptors - expect an N-H from an amide for example) as well as the two hydrogens (h-bond donors). I would refine all those waters and assume they are waters. If the distance to the other atoms is between 2.5-3.2 then you can assume the water to be correct. In many cases waters will h-bond (only) to other water molecules. ** ** The B-factor is displayed in Coot along the bottom (left) when you middle click on an atom. You can also see the B-factor when you read the pdb file as text ** ** Hope this helps. ** ** _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034 ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Uma Ratu *Sent:* Thursday, 8 March 2012 10:22 a.m. *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Water ** ** Dear Roger: Thank you very much for your comments. I use them as guideline and remove many 'false waters. Still, I am not clear of some of these 'waters' are real or not. I have the pic attached. In Pic-W11-1, the 'water' is connected to the adjust residues with 4 contacts, which are 'N' or 'O' atoms. I would consider this 'water' is false. My question is: if these 4 contacts include C from residues, will it be a polar contact or not? In Pic-W12-1, the 'water' is connected to the adjust residues with 3 contacts. The 4th is to another 'water'. Will this 'water' is true or not? Similar case is seen in Pic-W190-1 In Pic-W109-1, some 'waters' are connected to adjust residues, some not. Are these 'water' true or not? Further more, and the b-factors are not way out of line, I am not clear on how to define out of line. How to find b-factor of individual residue in Coot? I search the web, but find no answer. Thank you for advice Uma On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett rrowl...@colgate.edu wrote: Uma, Remember that your structure, ultimately, is a model. A model is your best judgment of the true representation of the protein structure in your crystal. Your model should make chemical sense. Coot is pretty good at placing waters, but it cannot substitute entirely for the experimentalist. Coot will miss some waters, and mis-assign others into weak, unmodeled or alternate side- or main-chain density, or into density that might be attributable to cations and anions or other crystallization materials. Your waters should be subjected to inspection and verification. It is really helpful to turn on environment distances in Coot when you do this. Even in a large protein model, it is possible to inspect all waters for reasonableness pretty quickly. If you have no significant positive or negative difference density, and the b-factors are not way out of line, and hydrogen bonding partners are reasonable, then modeling a water is probably a good call. Waters should have hydrogen bonding partners with side chains or main-chain polar atoms, within reasonable distances, or be withing hydrogen bonding distance of other waters that are (chains of waters). If a water has strong electron density and more than 4 polar contacts, you might consider anion or cation occupancy. Most anions and cations will have higher electron density, and appropriately different types of polar contacts. (e.g. you might find sulfates near a cluster of basic residues). Low occupancy anions can often look a lot like water. PEGs can create ugly snakes of variable density that may be challenging to model. Modeling non-protein structural bits is endlessly entertaining for the protein crystallographer. ;) Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline
Re: [ccp4bb] MTZ file
Many thnaks for your input. regards Ros On Wed, Feb 29, 2012 at 4:51 PM, Eleanor Dodson eleanor.dod...@york.ac.ukwrote: mtz(1) will contain h k l F SIGF I SIGI and optionally F+ SIGF+ and F- SIGF- This is your master file, providing the space group is correct It may NOT have the correct space group however - MR searches may select one space group from several possible ones. If the final SG is different from the one specified after scalepack2mtz just run a jiffy to change it. e.g. mtzutils hklin1 scal1.mtz hklout scal2.mtz symm SGname end then use scal2.mtz as your master.. The output from phaser will have the same F but after anisotropic scaling. There will be other columns suitable for input to coot the output from REFMAC will also give a scaled F plus extra columns . Always start each new refinement with your master mtz as input.. coot will automatically select the best output from PHASER or REFMAC to calculate maps. The columns FWT and PHWT are used to generate a 2mFobs-DFcalc map The columns DELFWT and PHDELWT generate a mFobs-DFcalc map Eleanor On Feb 29 2012, Uma Ratu wrote: Hello, I have a question about .mtz files used in model building. Here is how I did: Diffraction data - HKL 2000: .sca CCp4i: scalepack2mtz: .mtz (1) Phaser: In: template pdb .mtz(1) Out: model .pdb(1) .mtz(2) Refmac5: model .pdb(2) .mtz(3) Here is the question: 1. Coot check and refinment: which mtz file shoudl I use? 2. With further refinemnt by refmac5, which mtz file should I use? 3. When I deposit data, which mtz file to use? 4. What is the difference between .mtz(1) and the .mtz files generated from phaser and refmac? Thank you for advice Ros -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
[ccp4bb] Temp Fact Variance Analysis
Hello, I run my model in Coot to do Temp Fact Variance Analysis. There are red bars from the B-factor Variance graphy. I click each red bar to exam the residues in Coot. Many of these residues do not have the electronic density on their side chains, especially Lys residues. Here is my questions: 1. The lack of electronic density is the cause of these red-bars? 2. How do I fix them? delete the side chains? The diffraction data is 2A, and data completness is 98.8%. Thank you for comments Ros
Re: [ccp4bb] Temp Fact Variance Analysis
Dear All: Thank you very much for your comments. I did not notice that this issue has been dicussed lately. Thank you for your inputs regards Ros On Thu, Mar 1, 2012 at 9:38 AM, Kelly Daughtry kddau...@bu.edu wrote: Ros, I haven't used the Temp Fact Variance Analysis in Coot, but can guess that the red bars indicate increased b-factor compared to the average of your protein model? If so, then: *1. The lack of electronic density is the cause of these red-bars?* Likely yes. If there is no density to support the position of the side chain, then you will see an increase in B-factor. *2. How do I fix them? delete the side chains?* This question has been under debate in our community recently. If I recall correctly (and my opinion) is you should leave the side chains as they are and let the b-factor be an indication of the uncertainty of their position. I'm willing to bet these side chains are surface residues, or in a mobile part of our protein. A good thing to compare is the average b-factor of your entire model compared to these residues, as well as the b-factor of these specific side chains compared to main chain atoms. Hope this helps clarify. Sincerely, Kelly Daughtry *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Thu, Mar 1, 2012 at 9:26 AM, Uma Ratu rosiso2...@gmail.com wrote: Hello, I run my model in Coot to do Temp Fact Variance Analysis. There are red bars from the B-factor Variance graphy. I click each red bar to exam the residues in Coot. Many of these residues do not have the electronic density on their side chains, especially Lys residues. Here is my questions: 1. The lack of electronic density is the cause of these red-bars? 2. How do I fix them? delete the side chains? The diffraction data is 2A, and data completness is 98.8%. Thank you for comments Ros
Re: [ccp4bb] MTZ file
Thank you very much! It is clear for me now. Cheers Ros On Wed, Feb 29, 2012 at 9:18 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi, The refinement program generates an mtz file with map coefficients (including difference Fourier coefficients) so you should use that one for model rebuilding in coot; for the next refinement rounds, at the beginning of each round you should provide the initial mtz file coming from data processing and post processing (1 in your terminology I suppose). This is because several refinement programs scale the Fobs (and sigmaFobs). Refmac is one of them I think; for data deposition, you deposit the same mtz file (1), and if you also wish to deposit map coefficients you can also extract the map coefficients from the final refinement and map calculation round mtz (using, say, sftools) and deposit those. Phaser does not carry out model refinement per se (adjusting atom positions and temperature factors). But the map coefficients present in the mtz file are generated in the same way (Sigmaa coefficients) with Phaser and with Refmac. There might be small difference in terms of the program code used internally but that shouldn't make much of a difference. Since a model that has seen refmac is (normally) improved, the electron density maps generated using the refmac mtz should be improved wrt the maps coming directly from Phaser (molecular replacement). Normally, the model you refine will have had the differences in the sequence between molecular replacement search model and 'your' structure corrected (gradually?), the solvent model is introduced and improved, ligands, ions etc are being introduced... Thus the maps improve seen that your model should reflect more and more what is present in the crystal as you build and refine. HTH, Fred. Uma Ratu wrote: Hello, I have a question about .mtz files used in model building. Here is how I did: Diffraction data - HKL 2000: .sca CCp4i: scalepack2mtz: .mtz (1) Phaser: In: template pdb .mtz(1) Out: model .pdb(1) .mtz(2) Refmac5: model .pdb(2) .mtz(3) Here is the question: 1. Coot check and refinment: which mtz file shoudl I use? 2. With further refinemnt by refmac5, which mtz file should I use? 3. When I deposit data, which mtz file to use? 4. What is the difference between .mtz(1) and the .mtz files generated from phaser and refmac? Thank you for advice Ros
[ccp4bb] Coot Crashed
Dear All: I am having trouble with Coot. The program keeps crashing when I click on rotamer analysis. Other functions, sych as geometry analysis all worked fine. It runs normal before, and only happened when I added the ligands into the model. I am using WinCoot_0.7_pre-1-revision-3772, and window vista. Thank you for advices. Ros
