[ccp4bb] references on application of broad specificity of enzyme
Dear All, I have a transferase, which is showing broad specificity for both the substrates (nucleotides) in our organism of interest, but is highly specific in other organisms. Are there any references showing the applications of (bi-substrate) proteins with such broad specificity towards nucleotides? Thanking you, regards,Sreetama
[ccp4bb] off-topic: different R-factor values shown in PDB page vs PDB file
Hi,Sorry for a slightly off-topic question. I just noticed that for some pdb files, the value of R-factor shown on the PDB web page is different from the value in the file obtained by clicking the link display file. Why is this so? examples where I noticed:4jzc , where the PDB page (http://www.rcsb.org/pdb/explore/explore.do?structureId=4JZC) shows | R-Value: | 0.990 (obs.) | | R-Free: | 0.248 | while the file shows :REMARK 3 R VALUE (WORKING SET) : 0.230 REMARK 3 FREE R VALUE : 0.248 3gkq, etc. Any idea why this is happening ? Thanks and regards,Sreetama
[ccp4bb] how to switch on probe clashes (disabled in coot)
Dear Users, The option ValidateProbe clashes is disabled in coot (version 0.8/CCP4-6.5, OS - ubuntu 12.04).How can this be enabled?Can I toggle between 'enable' and 'disable' according to need? Thanks in advance. SreetamaPhD student,IISc
[ccp4bb] software/web server to determine ligand volume
Dear all, Is there any software or web server available to calculate the volume of a ligand if the ligand coordinates are provided? Google seems to come up only with options to calculate protein cavity volume. Thanks in advance, Sreetama Das, phd student, Physics, IISc
[ccp4bb] RMSD between structures of homologous proteins
Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc
Re: [ccp4bb] Naccess
hi, I found the following reference in a paper- Hubbard S. PhD thesis. University College of London, Department of Biochemistry and Molecular Biology; 1992. NACCESS: a Program for Calculating Accessibilities. On Wednesday, 9 July 2014 8:20 PM, FOOS Nicolas nicolas.f...@synchrotron-soleil.fr wrote: Hello, i cited this program with : Hubbard, S.J., and Thornton, J.M. (1993). NACCESS (Computer Program, Department of Biochemistry and Molecular Biology, University College London.). I am not absolutly certain that's th best way. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Armando Albert [xalb...@iqfr.csic.es] Envoyé : mercredi 9 juillet 2014 17:02 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Naccess Dear all, Does anyone know how to properly cite Naccess for calculation of solvent accessible area (http://www.bioinf.manchester.ac.uk/naccess/)? Armando
[ccp4bb] R-merge,R-meas 1 in outermost resolution shell
Dear All, What are reasonable values of Rmerge in the outermost resolution shell? Some of the recent discussions suggest using data up to those shells where I/sig(I) ~2 and CC1/2 = 0.5. But I am getting Rmerge Rmeas 1 in the outermost shell for those values of I/sig(I) and CC1/2. Reducing the resolution cut-off while data reduction scaling (aimless) reduces the R-values, but I am not sure how much I should reduce the resolution (if at all). Following are the results from the aimless log file: At a resolution cut-off of 1.62A: Overall InnerShell OuterShell Low resolution limit 42.12 42.12 1.65 High resolution limit 1.62 8.87 1.62 Rmerge (within I+/I-) 0.071 0.019 1.325 Rmerge (all I+ and I-) 0.074 0.020 1.381 Rmeas (within I+/I-) 0.078 0.021 1.446 Rmeas (all I+ I-) 0.077 0.022 1.441 Rpim (within I+/I-) 0.031 0.009 0.575 Rpim (all I+ I-) 0.022 0.007 0.410 Rmerge in top intensity bin 0.031 - - Total number of observations 311022 1839 14876 Total number unique 25311 184 1212 Mean((I)/sd(I)) 19.2 52.7 2.0 Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.740 Completeness 100.0 99.4 100.0 Multiplicity 12.3 10.0 12.3 If I take improved resolution cut-off (1.6A, 1.57A), the I/sig(I) and CC1/2 in the outermost shell are lower, and the R-merge,meas,pim are higher. No resolution cut-off was used while integrating the data (imosflm), only a mask for the beam stop was used. looking forward to your suggestions, thanking you, sreetama
[ccp4bb] (high) values of R-factors in outermost resolution shell
Dear All, What are reasonable values of Rmerge in the outermost resolution shell? Some of the recent discussions suggest going to those sheels where I/sig(I) ~2 and CC1/2 = 0.5. But I am getting Rmerge Rmeas 1 in the outermost shell for those values of I/sig(I) and CC1/2, and I don't think that makes any sense. Reducing the resolution cut-off while data reduction scaling (aimless) reduces the R-values, but I am not sure how much I should reduce the resolution (if at all). Following are the results from the aimless log file: At a resolution cut-off of 1.62A: Overall InnerShell OuterShell Low resolution limit 42.12 42.12 1.65 High resolution limit 1.62 8.87 1.62 Rmerge (within I+/I-) 0.071 0.019 1.325 Rmerge (all I+ and I-) 0.074 0.020 1.381 Rmeas (within I+/I-) 0.078 0.021 1.446 Rmeas (all I+ I-) 0.077 0.022 1.441 Rpim (within I+/I-) 0.031 0.009 0.575 Rpim (all I+ I-) 0.022 0.007 0.410 Rmerge in top intensity bin 0.031 - - Total number of observations 311022 1839 14876 Total number unique 25311 184 1212 Mean((I)/sd(I)) 19.2 52.7 2.0 Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.740 Completeness 100.0 99.4 100.0 Multiplicity 12.3 10.0 12.3 At 1.6A, the I/sig(I) and CC1/2 in the outermost shell are lower, and the R-merge,meas,pim are higher. looking forward to your suggestions, thanking you, sreetama
[ccp4bb] verifying protein crystal content by mass spectrometry or SDS-PAGE
Dear All, Is there any protocol for preparing a sample from (cleaning/dissolving) protein crystals for verifying their mass through mass spectrometry or SDS-PAGE? How many protein crystals are required? Should they all be from the same well, or can they be from different wells with slight changes in protein concentration/precipitant concentration/ pH? Should the crystals be cleaned with the reservoir solution and then dissolved in the protein buffer? I am getting very thin, needle-like crystals which are too tiny for mounting. Adding Izit dye has also not been conclusive. Similar looking crystals have appeared in 3 conditions. Changing precipitant concentration, protein concentration, pH , and seeding have made no difference so far. I wish to determine whether the full protein is crystallizing, or only some fragment. The protein molecular weight is ~22KDa, and the crystals take ~3 weeks to grow. Thanks in advance, sreetama
[ccp4bb] error in starting imosflm
Dear All, Whenever I open up imosflm GUI from the terminal, I get the following error: iMosflm version 7.1.1, 24th March 2014 ipmosflm is not compatible. Please configure iMosflm with the correct executable. clicking the Configure button at the bottom of the error message panel closes everything. the following is shown on the terminal: MOSFLM_EXEC set to ipmosflm testing MOSFLM_WISH (/home/*/Software/CCP4/ccp4-6.3.0/bin/wish) Tcl platform is unix i686 Linux 3.8.0-32-generic TclTk version from info patchlevel is 8.4.19 Tk windowing system is x11 I had run imosflm successfully just 2 hours before this instance. After that, I had enabled the older version of CCP4-6.3 (by sourcing it from the .bashrc file) for sometime to look at the options in molrep (in CCP4-6.3), and then re-enabled the newer CCP4-6.4. Enabling ccp4-6.3 from the .bashrc file typing imosflm opens up the older 7.0.1 version. The option settingsenvironment variables is not working in the newer imosflm version now, changing the value in the GUI of the older version of imosflm doesn't help either. I applied the latest CCP4 updates; that doesn't set things right. Please let me know how to re-configure iMosflm to run with ccp4-6.4. thanks regards, sreetama
Re: [ccp4bb] error in starting imosflm
Dear Sir, If I sorce ccp4-6.3, and then change in the bashrc file source ccp4-6.4, (and source the modified .bashrc file in either case) but don't close the previous terminal, then opening imosflm gives the aforementioned error. After closing the imosflm GUI, if I type at the terminal, I get the following: sreetama@sreetama-laptop:~/data $ $MOSFLM_EXEC BFONT COLOR=#FF!--SUMMARY_BEGIN-- html !-- CCP4 HTML LOGFILE -- hr !--SUMMARY_END--/FONT/B Mosflm version 7.0.9 for Image plate and CCD data 14th May 2012 *** Andrew Leslie and Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK E-mails: and...@mrc-lmb.cam.ac.uk, ha...@mrc-lmb.cam.ac.uk References: Mosflm: A.G.W. Leslie and H.R. Powell (2007), Evolving Methods for Macromolecular Crystallography, 245, 41-51 ISBN 978-1-4020-6314-5 New auto-indexing based on DPS: I. Steller R. Bolotovsky and M.G. Rossmann (1997) J. Appl. Cryst. 30, 1036-1040 New iMosflm GUI: T.G.G. Battye, L. Kontogiannis, O. Johnson, H.R. Powell and A.G.W. Leslie.(2011) Acta Cryst. D67, 271-281) How much of this information is useful in diagnosing user problems? Mosflm run on Monday, April 28 2014 at 19:01 by sreetama Compiler command: gfortran Compiler version: GNU Fortran (GCC) 4.4.7 Copyright (C) 2010 Free Software Foundation, Inc. GNU Fortran comes with NO WARRANTY, to the extent permitted by law. You may redistribute copies of GNU Fortran under the terms of the GNU General Public License. For more informatio Executable type: ELF 32-bit LSB executable, Intel 80386, version 1 (SYSV), dynamically linked (uses shared libs), not stripped This executable was built by ccb on Thursday, June 28 2012 at 09:33 MOSFLM = Also the terminal refuses to close if I type exit. Forcibly closing the terminal , and typing imosflm in a new terminal solves the problem. regards, sreetama On Monday, 28 April 2014 6:52 PM, Harry Powell ha...@mrc-lmb.cam.ac.uk wrote: Hi When you get this error, and everything has been closed, what do you get if you type (in the same terminal window that you tried to start iMosflm) - $MOSFLM_EXEC ? It's plain from the message that ipmosflm is not compatible' that iMosflm is trying to run with an incompatible MOSFLM_EXEC (maybe from the old ccp4 6.3 distribution, but there could be other reasons). On 28 Apr 2014, at 13:05, sreetama das wrote: Dear All, Whenever I open up imosflm GUI from the terminal, I get the following error: iMosflm version 7.1.1, 24th March 2014 ipmosflm is not compatible. Please configure iMosflm with the correct executable. clicking the Configure button at the bottom of the error message panel closes everything. the following is shown on the terminal: MOSFLM_EXEC set to ipmosflm testing MOSFLM_WISH (/home/*/Software/CCP4/ccp4-6.3.0/bin/wish) Tcl platform is unix i686 Linux 3.8.0-32-generic TclTk version from info patchlevel is 8.4.19 Tk windowing system is x11 I had run imosflm successfully just 2 hours before this instance. After that, I had enabled the older version of CCP4-6.3 (by sourcing it from the .bashrc file) for sometime to look at the options in molrep (in CCP4-6.3), and then re-enabled the newer CCP4-6.4. Enabling ccp4-6.3 from the .bashrc file typing imosflm opens up the older 7.0.1 version. The option settingsenvironment variables is not working in the newer imosflm version now, changing the value in the GUI of the older version of imosflm doesn't help either. I applied the latest CCP4 updates; that doesn't set things right. Please let me know how to re-configure iMosflm to run with ccp4-6.4. thanks regards, sreetama Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
[ccp4bb] superposing multiple structural segments between two PDBs
Dear All, Is there a way to superpose multiple, discontinuous stretches of residues between two protein chains ? Dali output shows that there are discontinuous stretches between the PDBs that are being aligned. But I can not find any option to download the superposed PDB. The output generated from PDBefold (which gives link to the transformed PDB) is different. regards, sreetama
Re: [ccp4bb] preparation of citrate buffer pH3-6.5
Dear All, Thank you for your replies: 2 different methods were suggested for the citrate buffer preparation over a small range of pH: 1. mixing citric acid with sodium citrate salts 2. mixing citric acid with NaOH we found the latter method easier for our purpose.
[ccp4bb] preparation of citrate buffer pH3-6.5
Dear All, We have obtained many tiny protein crystals in a condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too small for mounting in loops. We intend to vary the salt concentration pH to obtain larger crystals. Could anyone direct us to some links, or provide us with a method (with calculations) to calculate the amounts of citric acid trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? I have come across online buffer calculators and links where the amounts of the components required are mentioned in grams, but none explaining how those values were arrived at. Thanks regards, sreetama
Re: [ccp4bb] residues forming the surface of a cavity
Dear All, Sorry for the late reply. I wish to compare binding pockets of some homologous proteins, a couple of which have a ligand bound, while the rest don't. I wish to check if there is any difference in the nature of the cavity in the homologous proteins, e.g. properties like hydrophobicity, electrostatics, etc. But I also wish to determine exactly which residues are surfacing/lining the cavity. Thanks and regards, sreetama From: Francois Berenger beren...@riken.jp To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, 4 October 2012 12:56 PM Subject: Re: [ccp4bb] residues forming the surface of a cavity On 10/04/2012 04:09 PM, sreetama das wrote: Dear all, Is there any CCP4 module/ other software/ web server which can determine which particular residues form the surface of a pocket/ binding-site in a protein? Do you mean residues not far from any ligand atom? And you know where is the ligand. Or do you mean residues surfacing a pocket or cavity? Regards, F.
[ccp4bb] residues forming the surface of a cavity
Dear all, Is there any CCP4 module/ other software/ web server which can determine which particular residues form the surface of a pocket/ binding-site in a protein? Thanks in advance, sreetama
[ccp4bb] suggestions for good yet cost-effective pipettes
Dear all, We are planning to buy electronic dispensing pipette (single channel) and mechanical multi-channel pipette (8 channel) in the range 0.5 - 10 ul in our lab. Currently, we are considering IKA PRECISION pipettes. It will be helpful if we can get feedback on how these pipettes work from labs which have used them, or any other relevant comments. We are also open to looking at other options, provided they cost about 600 USD or less. Thanks, sreetama
[ccp4bb] to determine missing atoms and residues in a PDB file
Dear All, I have a PDB file which does not have the REMARKS cards 465 (for missing residues) and 470 (for missing atoms). This is not a deposited PDB file. Is there any program to figure out the missing residues and atoms (some programs complain about missing atoms) ? Or do I have to check in any particular file generated during the processing of the diffraction data? The S2C program from Prof. Dunbrack's Lab does not show any option of uploading pdb files (this solution was mentioned to a previous query on the BB). Thanks in advance, sreetama
[ccp4bb] residuewise rmsd for multiple chain superposition
Dear all, Is there any software which will print the RMSDs (residue wise, and not chain wise) for more-than-2 chains (having similar/same sequences) superposed together? Thanks in advance, sreetama
[ccp4bb] pH optimisation for crystallisation
Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama
[ccp4bb] LSQKAB error: you have failed to find any atoms to fit
Dear All, I am trying to superpose 2 chains from the same pdb file. I have generated 2 separate pdb files, each with one chain, using PDBSET. When I run LSQKAB with the 2 generated files (on UBUNTU 10.04, ccp4 6.2) it fails to run. The 2 input files CONTAIN the columns for occupancies and B-factors. ( some old mails in the BB mention checking if these columns are present) details of the error are: OPEN FILES AS REQUESTED opening coordinate file of model to be moved Logical name: XYZIN2 File name :/abc/def/../../*2.pdb pdb file is being opened on unit 1 for input Matrices derived from CRYST1 CARD in COORDINATE FILE RF RO matrices . logical name: XYZOUT filename: /abc/def/../../*12.pdb pdb file is being opened on unit 2 for output opening coordinate file of fixed model logical name:XYZIN1 filename:/abc/def/../../*1.pdb pdb file is being opened on unit 3 for input MAtrices derived from cryst1 card in coord. file RF RO matrices... FORMATTED UNKNOWN file opened on unit 7 logical name: RMSTAB, filename :/abc/def/../../*_rmstab.graph FORMATTED UNKNOWN file opened on unit 8 logical name: DELTAS, filename: /abc/def/../../*_deltas.log ** ZERO OCCUPANCIES IN WORKING SET** 0.0 ** ZERO OCCUPANCIES IN REFERENCE SET** 0.0 LSQKAB: ***ERROR - YOU HAVE FAILED TO FIND ANY ATOMS TO FIT *** ## Any idea how to fix the problem? Thanks in advance, regards, sreetama
[ccp4bb] LSQKAB error: you have failed to find any atoms to fit
Dear All, I am trying to superpose 2 chains from the same pdb file. I have generated 2 separate pdb files, each with one chain, using PDBSET. When I run LSQKAB with the 2 generated files (on UBUNTU 10.04, ccp4 6.2) it fails to run. The 2 input files CONTAIN the columns for occupancies and B-factors. ( some old mails in the BB mention checking if these columns are present) details of the error are: OPEN FILES AS REQUESTED opening coordinate file of model to be moved Logical name: XYZIN2 File name :/abc/def/../../*2.pdb pdb file is being opened on unit 1 for input Matrices derived from CRYST1 CARD in COORDINATE FILE RF RO matrices . logical name: XYZOUT filename: /abc/def/../../*12.pdb pdb file is being opened on unit 2 for output opening coordinate file of fixed model logical name:XYZIN1 filename:/abc/def/../../*1.pdb pdb file is being opened on unit 3 for input MAtrices derived from cryst1 card in coord. file RF RO matrices... FORMATTED UNKNOWN file opened on unit 7 logical name: RMSTAB, filename :/abc/def/../../*_rmstab.graph FORMATTED UNKNOWN file opened on unit 8 logical name: DELTAS, filename: /abc/def/../../*_deltas.log ** ZERO OCCUPANCIES IN WORKING SET** 0.0 ** ZERO OCCUPANCIES IN REFERENCE SET** 0.0 LSQKAB: ***ERROR - YOU HAVE FAILED TO FIND ANY ATOMS TO FIT *** ## Any idea how to fix the problem? Thanks in advance, regards, sreetama
[ccp4bb] LSQKAB error: you have failed to find any atoms to fit
Dear All, I am trying to superpose 2 chains from the same pdb file. I have generated 2 separate pdb files, each with one chain, using PDBSET. When I run LSQKAB with the 2 generated files (on UBUNTU 10.04, ccp4 6.2) it fails to run. The 2 input files CONTAIN the columns for occupancies and B-factors. ( some old mails in the BB mention checking if these columns are present) details of the error are: OPEN FILES AS REQUESTED opening coordinate file of model to be moved Logical name: XYZIN2 File name :/abc/def/../../*2.pdb pdb file is being opened on unit 1 for input Matrices derived from CRYST1 CARD in COORDINATE FILE RF RO matrices . logical name: XYZOUT filename: /abc/def/../../*12.pdb pdb file is being opened on unit 2 for output opening coordinate file of fixed model logical name:XYZIN1 filename:/abc/def/../../*1.pdb pdb file is being opened on unit 3 for input MAtrices derived from cryst1 card in coord. file RF RO matrices... FORMATTED UNKNOWN file opened on unit 7 logical name: RMSTAB, filename :/abc/def/../../*_rmstab.graph FORMATTED UNKNOWN file opened on unit 8 logical name: DELTAS, filename: /abc/def/../../*_deltas.log ** ZERO OCCUPANCIES IN WORKING SET** 0.0 ** ZERO OCCUPANCIES IN REFERENCE SET** 0.0 LSQKAB: ***ERROR - YOU HAVE FAILED TO FIND ANY ATOMS TO FIT *** ## Any idea how to fix the problem? Thanks in advance, regards, sreetama
[ccp4bb] to show multiple sequence alignment with sec. str.
Dear All, Is there any module in CCP4/ other related software/servers which can show a multiple alignment of homologous sequences from a protein family, together with their secondary structures? Thanks in advance, regards, sreetama
[ccp4bb] check
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[ccp4bb] use of beta-mercaptoethanol
Dear all, I have a 20 KDa protein that has a single cysteine , about 42 residues from the C - terminal . The protein aggregates during crystallisation setup (hanging drop, vapour diffusion ) . What ratio of protein and mercapto-ethanol should be used during trials to prevent aggregation and help in crystallisation ? Thanking in advance,
[ccp4bb] use of beta-mercaptoethanol in crystallization
Dear all, I have a 20 KDa protein that has a single cys residue , about 42 residues from the C-terminal . The protein aggregates during crystallization setup ( hanging drop vapour diffusion) . What ratio of protein and mercaptoethanol is to be used to avoid aggregation and help in crystallization ? Thanking in advance, Sreetama
