Re: [ccp4bb] Calculating water accessible volume in active site
Hi Yarrow, You can use the likes of areaimol to calculate the surface accessible area, with or without accounting for crystal symmetry: http://www.ccp4.ac.uk/html/areaimol.html There are other alternatives, but I hope that helps, Rob On 8 Sep 2012, at 00:37, Yarrow Madrona wrote: > I have a buried active site and would like to determine if there is room > for a water molecule in various mutants. Does anyone know of a good > program to calculate this? I have heard of GRID and VOID but have never > used them. > > Thanks. > > > -- > Yarrow Madrona > > Graduate Student > Molecular Biology and Biochemistry Dept. > University of California, Irvine > Natural Sciences I, Rm 2403 > Irvine, CA 92697
[ccp4bb] where to get jre 1.5_22 for mac osx 10.6
All: Sorry for the non-CCP4 question. But please help!! I have to install jre 1.5_22 on my mac to have the COEUS (MIT software premium). My computer already has java 1.6 How to install the old version. If this old is not on the computer, then the install commands do not work. Thanks a lot Subbu
[ccp4bb] Calculating water accessible volume in active site
I have a buried active site and would like to determine if there is room for a water molecule in various mutants. Does anyone know of a good program to calculate this? I have heard of GRID and VOID but have never used them. Thanks. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] Cryo-EM postdoctoral position: Rubinstein Laboratory, The Hospital for Sick Children and University of Toronto
Dear Colleagues, Please pass this ad on to any interested candidates. Thanks and best wishes, John -- John Rubinstein Molecular Structure and Function Program The Hospital for Sick Children 555 University Avenue, Rm. 3330 Toronto, ON Canada M5G 1X8 Tel: (+001) 416-813-7255 Fax: (+001) 416-813-5022 www.sickkids.ca/research/rubinstein * Project and laboratory:* A Postdoctoral position is available for the structural analysis of protein complexes by electron cryomicroscopy (cryo-EM). Projects relate to the analysis of protein complexes involved in endocytosis and vacuolar sorting and/or bacterial and viral proteins that interact with eukaryotic V-type ATPases. Candidates interested in studying other protein complexes, particularly membrane protein complexes, are also encouraged to apply. Applicants should have a PhD in biochemistry, biophysics, biological chemistry, or a related discipline. A background in structural biology is helpful but is not absolutely required. Please send applications, including a complete CV and contact information for three referees to: john.rubinst...@utoronto.ca The Rubinstein group is a small and interactive group comprised of members with diverse scientific backgrounds. We combine in-depth study of systems of biological and biomedical interest with expertise in cryo-EM technology and research into cryo-EM method development. By maintaining strong connections between biology and methodology, we ensure that our biological results are of the highest-possible quality and our methodological developments are useful for 'real world' problems. For more information, please visit www.sickkids.ca/research/rubinstein *The research environment and city of Toronto:* Located in the heart downtown Toronto, The Hospital for Sick Children's Research Institute includes approximately 225 Principal Investigators, 1,140 graduate students and postdoctoral fellows, and 800 other scientific staff. Research in the Institute spans from basic biology to clinical practices with seven research programs including Molecular Structure and Function, Cell Biology, Genetics and Genome Biology, and Developmental and Stem Cell Biology. The Research Institute is affiliated with the University of Toronto, which is ranked 1st in Canada and 19th worldwide in the Times Higher Education World University Rankings (9th worldwide out of public universities). Toronto is Canada's largest city and North America's 5th largest city. It is one of the world's most multicultural cities with vibrant arts and culture communities. Despite its size, it is also one of North America's safest cities and is ranked as the 4th most liveable city in the world by The Economist Intelligence Unit's Global Liveability Report. *Selected Recent publications:* Benlekbir, S., Bueler, S. A., Rubinstein, J. L. (2012). Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae. Nature Structural and Molecular Biology, In Press. Baker, L. A., Watt, I. N., Runswick, M. J., Walker, J. E., Rubinstein, J. L. (2012). The arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM. Proceedings of the National Academy of Sciences (USA) 109, 11675-80. (chosen by Faculty of 1000: f1000.com) Lau, W. C. Y., and Rubinstein, J. L. (2012). Sub-nanometer resolution structure of the intact T. thermophilus H+-driven ATP synthase. Nature 481, 215-8. (chosen by Faculty of 1000: f1000.com) Lau, W. C. Y., and Rubinstein, J. L. (2010). Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound Vo motor. Proceedings of the National Academy of Sciences (USA) 107, 1367-72. (chosen by Faculty of 1000: f1000.com)
[ccp4bb] Cryo-EM postdoctoral position: Rubinstein Laboratory, The Hospital for Sick Children and University of Toronto
Dear Colleagues, Please pass this ad on to any interested candidates. Thanks and best wishes, John -- John Rubinstein Molecular Structure and Function Program The Hospital for Sick Children 555 University Avenue, Rm. 3330 Toronto, ON Canada M5G 1X8 Tel: (+001) 416-813-7255 Fax: (+001) 416-813-5022 www.sickkids.ca/research/rubinstein * Project and laboratory:* A Postdoctoral position is available for the structural analysis of protein complexes by electron cryomicroscopy (cryo-EM). Projects relate to the analysis of protein complexes involved in endocytosis and vacuolar sorting and/or bacterial and viral proteins that interact with eukaryotic V-type ATPases. Candidates interested in studying other protein complexes, particularly membrane protein complexes, are also encouraged to apply. Applicants should have a PhD in biochemistry, biophysics, biological chemistry, or a related discipline. A background in structural biology is helpful but is not absolutely required. Please send applications, including a complete CV and contact information for three referees to: john.rubinst...@utoronto.ca The Rubinstein group is a small and interactive group comprised of members with diverse scientific backgrounds. We combine in-depth study of systems of biological and biomedical interest with expertise in cryo-EM technology and research into cryo-EM method development. By maintaining strong connections between biology and methodology, we ensure that our biological results are of the highest-possible quality and our methodological developments are useful for 'real world' problems. For more information, please visit www.sickkids.ca/research/rubinstein *The research environment and city of Toronto:* Located in the heart downtown Toronto, The Hospital for Sick Children's Research Institute includes approximately 225 Principal Investigators, 1,140 graduate students and postdoctoral fellows, and 800 other scientific staff. Research in the Institute spans from basic biology to clinical practices with seven research programs including Molecular Structure and Function, Cell Biology, Genetics and Genome Biology, and Developmental and Stem Cell Biology. The Research Institute is affiliated with the University of Toronto, which is ranked 1st in Canada and 19th worldwide in the Times Higher Education World University Rankings (9th worldwide out of public universities). Toronto is Canada's largest city and North America's 5th largest city. It is one of the world's most multicultural cities with vibrant arts and culture communities. Despite its size, it is also one of North America's safest cities and is ranked as the 4th most liveable city in the world by The Economist Intelligence Unit's Global Liveability Report. *Selected Recent publications:* Benlekbir, S., Bueler, S. A., Rubinstein, J. L. (2012). Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae. Nature Structural and Molecular Biology, In Press. Baker, L. A., Watt, I. N., Runswick, M. J., Walker, J. E., Rubinstein, J. L. (2012). The arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM. Proceedings of the National Academy of Sciences (USA) 109, 11675-80. (chosen by Faculty of 1000: f1000.com) Lau, W. C. Y., and Rubinstein, J. L. (2012). Sub-nanometer resolution structure of the intact T. thermophilus H+-driven ATP synthase. Nature 481, 215-8. (chosen by Faculty of 1000: f1000.com) Lau, W. C. Y., and Rubinstein, J. L. (2010). Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound Vo motor. Proceedings of the National Academy of Sciences (USA) 107, 1367-72. (chosen by Faculty of 1000: f1000.com)
[ccp4bb] Postdoc position at EMBL Hamburg + EMBL Grenoble
Dear CCP4BBers we have an opening for an EMBL Interdisciplinary Postdoc (EIPOD) position to develop data collection procedures for the new high-precision diffactrometer on the P14 beamline at EMBL Hamburg. Beamline P14 will be fully tunable and have a sub-10-micron focus and is equipped with a PILATUS 6M detector. The diffractometer features a high precision vertical spindle axis with a mini-kappa goniostat with a sphere confusion << 500 nm and allows to collect crystallographic data with enormous mechanical precision. The project will be a collaboration between the groups of Thomas Schneider at EMBL-Hamburg and Florent Cipriani at EMBL- Grenoble and will allow you to learn about high-precision mechanics and diffraction data collection on ultimately small and difficult crystals. The position is fully funded for three years. Please note that the position is also suitable for people without a PhD who have at least 4 years of full-time equivalent research experience. To apply, please go to: http://www.embl.org/eipod For informal enquiries, please contact: thomas.schnei...@embl-hamburg.de --- Dr. Thomas R. Schneider Project Coordinator EMBL@PETRA3 EMBL c/o DESY Notkestr. 85 fax: 0049-(0)40-89902-149 22603 Hamburg phone: 0049-(0)40-89902-190 Germany email:thomas.schnei...@embl-hamburg.de --- EMBL@Petra3: http://www.embl-hamburg.de/services/petra/index.html ---
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Dear Ed - I agree with you, and I felt a little funny giving that advice, since I know that Rmerge is rather outdated as a resolution cutoff. What I was really reacting to was the slope of I versus resolution; if you look at Qing's Scalepack table, you can see that average I plateaus in the low 4 A range, and I usually get worried about the data when I don't see a steady decrease in I with increasing resolution. Having said that, I don't usually work with datasets at this resolution, and it's possible (even likely?) that I was fooled by the bump in the Wilson plot around 4. Perhaps this data does extend to 4.1. - Matt On 9/7/12 11:39 AM, Edwin Pozharski wrote: Matt, On 09/07/2012 09:56 AM, Matthew Franklin wrote: I'm also a bit dubious about the 4.3 A limit; your useful data may be ending around 4.6 instead, despite the high I/sigma numbers. Why? I would rather suggest Qing extends resolution to where I/sigma~1. Other than Rmerge, I don't see what else you may be looking at, and that is "traditional but inferior" way to determine resolution cutoff. Cheers, Ed. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Matt, On 09/07/2012 09:56 AM, Matthew Franklin wrote: I'm also a bit dubious about the 4.3 A limit; your useful data may be ending around 4.6 instead, despite the high I/sigma numbers. Why? I would rather suggest Qing extends resolution to where I/sigma~1. Other than Rmerge, I don't see what else you may be looking at, and that is "traditional but inferior" way to determine resolution cutoff. Cheers, Ed.
[ccp4bb] Post-doc position available
Dear All, The Ho lab at the Institute of Biological Chemistry of Academia Sinca in Taiwan seeks a motivated structural biologist at post-doctoral level with interest in structure and activity studies of pharmaceutically important enzymes. The laboratory currently works on the structure/function studies of protein arginine methyltransferases and inhibitor design for glucuronidase and caspase-7. Applicant should have a strong background in protein expression and purification. Some experience in protein crystallography is required. Candidate possessing experience in high-throughput protein production, protein thermal assay, or fragment-based screening are preferred. This position provides the opportunity or broad training in protein expression, crystallization, small angle X-ray scattering, NMR, enzymology and protein-protein interaction. Applicants should submit the curriculum vitae and the names and contact information of three professional references via email to: sbddintai...@gmail.com For more information about the lab, please visit http://ho.ibc.sinica.edu.tw/ We are looking forward to receiving your application! Joseph Ho
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Just a comment - Your space group is almost certainly P31 2 1. Your scaling statistics results point to this - if your true space group was P31, then your statistics would be noticeably (even dramatically) worse in the higher symmetry space group. I've seen a "dramatic" counter-example to this. Very reasonable scaling statistics in a higher symmetry group but R-free stuck at >50%. Problem solved with lower symmetry. The resolution in my case was beyond 2Å. Thierry 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.
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Hi Matt, You may be right, but how do you distuinguish you between a crystallographic 2-fold and perfect twinning? A solution that refines to an Rfree of 55.6% is not exactly a textbook example of a correct solution. Poor data, low-resolution and twinning/pseudosymmetry can cause all kinds of artefacts. I would really recommend to keep all options open until a solution is found that really refines. Best regards, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew Franklin Sent: Friday, September 07, 2012 3:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] poorly diffracting and twinned trigonal crystal Hi Qing - Just a couple of observations on your story, without a real solution: - Your space group is almost certainly P31 2 1. Your scaling statistics and Phaser results point to this - if your true space group was P31, then your statistics would be noticeably (even dramatically) worse in the higher symmetry space group. - Your twinning result does not mean that your crystal is twinned. In space group P31, you see a two-fold rotation axis along the ab unit cell edge; the twin fraction of 0.475 means that this is nearly perfect. Adding that two-fold to P31 gives you the P3121 space group. - I suspect that your molecular replacement solution is correct, although I've had Z scores that high before and not gotten a usable solution. I believe your data is simply too weak to permit any sort of refinement. I'm also a bit dubious about the 4.3 A limit; your useful data may be ending around 4.6 instead, despite the high I/sigma numbers. I'm guessing that you have very high redundancy in this dataset? Sadly, I have no advice for you other than to pursue larger crystals, with better X-rays. (I'm assuming the data you show us is from a synchrotron; if not, take these crystals there immediately!) How radiation sensitive are these crystals? Perhaps a microfocus synchrotron beamline would help; even if your crystals are large, you could shoot small parts of the crystal until they burned out, then move to a new region. You also have to consider whether a 3.5 A structure (probably the best you could hope for, barring a breakthrough in crystal quality) will answer the questions you have regarding this system, and be publishable. Best of luck, Matt On 9/6/12 7:48 PM, Qing Luan wrote: > I have a ~4.3 angstrom data set of a trigonal crystal of a seven subunit protein complex which I can scale in P3, P31, P32, P321, P3121 and P3221 with similar statistics: > > P3 > Shell Lower Upper Average Average Norm. Linear Square > limitAngstrom I error stat. Chi**2 R-fac R-fac >50.00 9.25 1296.889.223.5 1.233 0.064 0.077 > 9.25 7.35 356.318.5 9.7 1.512 0.065 0.066 > 7.35 6.4297.1 8.2 7.5 1.584 0.143 0.140 > 6.42 5.8355.2 8.3 8.1 1.503 0.247 0.241 > 5.83 5.4251.4 9.4 9.3 1.438 0.297 0.284 > 5.42 5.1047.010.510.5 1.469 0.374 0.345 > 5.10 4.8448.311.811.9 1.421 0.398 0.383 > 4.84 4.6343.612.913.1 1.474 0.488 0.449 > 4.63 4.4540.314.114.2 1.530 0.546 0.477 > 4.45 4.3030.814.715.0 1.601 0.732 0.631 >All reflections203.819.612.3 1.477 0.125 0.085 > > > P3121: > > Shell Lower Upper Average Average Norm. Linear Square > limitAngstrom I error stat. Chi**2 R-fac R-fac >50.00 9.14 1242.951.818.3 1.200 0.057 0.068 > 9.14 7.26 314.011.2 6.5 1.454 0.070 0.069 > 7.26 6.3586.9 5.3 5.0 1.499 0.158 0.152 > 6.35 5.7751.9 5.5 5.3 1.248 0.264 0.252 > 5.77 5.3546.9 6.1 6.0 1.213 0.330 0.305 > 5.35 5.0444.3 6.9 6.7 1.137 0.393 0.363 > 5.04 4.7943.4 7.7 7.4 1.109 0.434 0.407 > 4.79 4.5839.2 8.5 8.1 1.128 0.533 0.478 > 4.58 4.4034.2 9.1 8.6 1.115 0.634 0.549 > 4.40 4.2524.9 9.9 9.3 1.064 0.872 0.766 >All reflections199.012.4 8.1 1.216 0.127 0.080 > > Unit cell parameters: 129.653 129.653 358.28090.00090.000 120.000 > > The systematic absences are consistent with either P31, P32, P3121, or > P3221. Analyzing the cell contents in P3121 suggests either 1 > (Matthews coefficient of 3.86, 68.2% solvent) or 2 mol/ASU (Matthews > coefficient of 1.93, 36.38% solvent) > > > I built a molecular replacement model (a polyala model containing about 2/3 of the protein complex) and ran phaser in multiple space groups with one (for P3121 or P3221) or two (P31, P32) copies of the model. Runs in P32 or P3221 gave no solutions or solutions with TFZ around 4-5. When run in P31 or P3121, phaser output solutions wi
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Hi Qing - Just a couple of observations on your story, without a real solution: - Your space group is almost certainly P31 2 1. Your scaling statistics and Phaser results point to this - if your true space group was P31, then your statistics would be noticeably (even dramatically) worse in the higher symmetry space group. - Your twinning result does not mean that your crystal is twinned. In space group P31, you see a two-fold rotation axis along the ab unit cell edge; the twin fraction of 0.475 means that this is nearly perfect. Adding that two-fold to P31 gives you the P3121 space group. - I suspect that your molecular replacement solution is correct, although I've had Z scores that high before and not gotten a usable solution. I believe your data is simply too weak to permit any sort of refinement. I'm also a bit dubious about the 4.3 A limit; your useful data may be ending around 4.6 instead, despite the high I/sigma numbers. I'm guessing that you have very high redundancy in this dataset? Sadly, I have no advice for you other than to pursue larger crystals, with better X-rays. (I'm assuming the data you show us is from a synchrotron; if not, take these crystals there immediately!) How radiation sensitive are these crystals? Perhaps a microfocus synchrotron beamline would help; even if your crystals are large, you could shoot small parts of the crystal until they burned out, then move to a new region. You also have to consider whether a 3.5 A structure (probably the best you could hope for, barring a breakthrough in crystal quality) will answer the questions you have regarding this system, and be publishable. Best of luck, Matt On 9/6/12 7:48 PM, Qing Luan wrote: I have a ~4.3 angstrom data set of a trigonal crystal of a seven subunit protein complex which I can scale in P3, P31, P32, P321, P3121 and P3221 with similar statistics: P3 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 9.25 1296.889.223.5 1.233 0.064 0.077 9.25 7.35 356.318.5 9.7 1.512 0.065 0.066 7.35 6.4297.1 8.2 7.5 1.584 0.143 0.140 6.42 5.8355.2 8.3 8.1 1.503 0.247 0.241 5.83 5.4251.4 9.4 9.3 1.438 0.297 0.284 5.42 5.1047.010.510.5 1.469 0.374 0.345 5.10 4.8448.311.811.9 1.421 0.398 0.383 4.84 4.6343.612.913.1 1.474 0.488 0.449 4.63 4.4540.314.114.2 1.530 0.546 0.477 4.45 4.3030.814.715.0 1.601 0.732 0.631 All reflections203.819.612.3 1.477 0.125 0.085 P3121: Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 9.14 1242.951.818.3 1.200 0.057 0.068 9.14 7.26 314.011.2 6.5 1.454 0.070 0.069 7.26 6.3586.9 5.3 5.0 1.499 0.158 0.152 6.35 5.7751.9 5.5 5.3 1.248 0.264 0.252 5.77 5.3546.9 6.1 6.0 1.213 0.330 0.305 5.35 5.0444.3 6.9 6.7 1.137 0.393 0.363 5.04 4.7943.4 7.7 7.4 1.109 0.434 0.407 4.79 4.5839.2 8.5 8.1 1.128 0.533 0.478 4.58 4.4034.2 9.1 8.6 1.115 0.634 0.549 4.40 4.2524.9 9.9 9.3 1.064 0.872 0.766 All reflections199.012.4 8.1 1.216 0.127 0.080 Unit cell parameters: 129.653 129.653 358.28090.00090.000 120.000 The systematic absences are consistent with either P31, P32, P3121, or P3221. Analyzing the cell contents in P3121 suggests either 1 (Matthews coefficient of 3.86, 68.2% solvent) or 2 mol/ASU (Matthews coefficient of 1.93, 36.38% solvent) I built a molecular replacement model (a polyala model containing about 2/3 of the protein complex) and ran phaser in multiple space groups with one (for P3121 or P3221) or two (P31, P32) copies of the model. Runs in P32 or P3221 gave no solutions or solutions with TFZ around 4-5. When run in P31 or P3121, phaser output solutions with TFZ> 11.0 and what appeared to be good packing. Rigid body refinement on the P3121 solution failed to improve the Rfactor (it hovered around 55.3%). Adding the missing subunits (as polyala chains) based on the phaser solution and refining with rigid body refinement resulted in a model with an Rfree to 48.5. Refining with torsion angle dynamics and restrained group B-factor refinement made the Rfree worse – it jumped up to about 55.6%. The Rwork values were similar to the Rfree values for each attempt. I also tried DEN refinement with similar results. Rigid body refinement of the P31 phaser solution gave an Rfree of about 54.4%. Adding the missing subunits and running rigid body refinement again improve
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
And also - I presume p6 does not work? On 09/06/2012 07:48 PM, Qing Luan wrote: which I can scale in P3, P31, P32, P321, P3121 and P3221 with similar statistics:
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
On 09/06/2012 07:48 PM, Qing Luan wrote: I built a molecular replacement model What is the model based on (i.e. how much sequence identity you have)? Did you try something other than CNS (specifically for twinning detection)? Did you check the patterson map and/or self-rotation for off-origin peaks? 36% solvent is highly unlikely given the resolution. Did you try molecular replacement with individual domains? etc, etc, etc
Re: [ccp4bb] protein interactions
Search for homologous binding partners with close or remote homologies with your own sequences can also be addressed from the InterEvol database http://biodev.cea.fr/interevol/ It seems you can upload the sequences there : http://biodev.cea.fr/interevol/interevalign.aspx Hope this can help Marie-Ln Dr Marie-Hélène LeDu Laboratoire de Biologie Structurale et Radiobiologie CEA/DSV/IBiTec-S, CEA Saclay Bât 144, pièce 25 91191 Gif-sur-Yvette, France Tel : 33 (0)1 69 08 71 35 E-mail : marie-helene.l...@cea.fr Le 06/09/12 14:20, « moham...@strubi.ox.ac.uk » a écrit : >Hi Careina, > >In answer to your first question you could also try the iPATCH server: > >http://portal.stats.ox.ac.uk/userdata/proteins/i-Patch/home.pl > >This takes two reference structures for proteins that interact, and >combined with multiple sequence alignments of their homologs attempts to >predict the surface contact residues between them. > >As far as your second question is concerned, a quick google search using >the term "protein interaction prediction from sequence" gave some useful >links, one of which is Struct2Net: > >http://groups.csail.mit.edu/cb/struct2net/webserver/ > >This tool attempts to predict protein-protein interactions purely from >sequence data. However, it does use a structure-based threading approach, >so your sequences will be run against the pdb. If they are unique to >anything in the structural databases, it may not be useful. > > >Hope this helps, > >Mohammad > > > >Dr. Mohammad W. Bahar >Division of Structural Biology >Wellcome Trust Centre for Human Genetics >University of Oxford
Re: [ccp4bb] [COOT] CCP4 6.3.0 released
On 17 Jul 2012, at 22:34, William G. Scott wrote: > On Jul 17, 2012, at 6:25 AM, Felix Frolow wrote: > >> I will wait for fink version if it will be one… :-\ > > Does anyone use or want this anymore? Apologies for reanimating this long-dead discussion, but did you reach a conclusion about putting ccp4 6.3.0 in fink? I'd rather use the dmg installer (choosing alternative install locations seems a little wonky) but will bite the bullet if it's not going to be finkified. Thanks, once again, for all your hard work on fink over the years. Chris -- Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
[ccp4bb] Fwd: [ccp4bb] poorly diffracting and twinned trigonal crystal
Seeing Herman's message reminds me that I forgot to send this reply to the BB as well. Herman makes many of the same points, and a very good additional point that you want to examine whether the molecular replacement solution in the lower symmetry space group actually has the higher symmetry. One way to do this manually is to check whether the Fcalcs from the MR solution have higher symmetry, but I think the Zanuda server is doing that kind of thing automatically. Best wishes, Randy Read Begin forwarded message: > From: Randy Read > Date: 7 September 2012 09:10:19 GMT+01:00 > To: Qing Luan > Subject: Re: [ccp4bb] poorly diffracting and twinned trigonal crystal > > Hi, > > There are a number of issues here. > > Refinement at 4.3A resolution is difficult at the best of times. I'm > assuming that, if you've chosen to use polyAla models for molecular > replacement, the sequence identity between the template and the target is > rather low, in which case you would expect very poor convergence for > refinement at this resolution. > > As an aside, there's very convincing evidence from a number of studies > (starting with a paper by Schwarzenbacher et al) that useful information is > lost by trimming back as far as a polyAla model. You keep a significantly > larger number of reasonably well-placed atoms by keeping all identical side > chains and keeping up to the gamma atom of non-conserved side chains (unless, > of course, the target amino acid is a glycine or an alanine). Various > programs, including the CCP4 chainsaw program and our sculptor program, can > carry out such model modification. > > It's not clear which twinning tests you are reporting. Twinning tests that > compare the intensities of potentially twin-related reflections to see if > they are more closely related than expected randomly (e.g. the H test or the > Britton plot) can't tell the difference between twinning and either higher > symmetry or pseudosymmetry. So you need to use a test based on intensity > distributions independent of twin laws (like the intensity moments or the L > test) to decide if your crystal is twinned, then if it is twinned you will > get a better idea of the twin fraction from tests comparing the twin-related > reflections. It's instructive to take a tetragonal lysozyme data set (true > space group P43212), merge it in P43, then run twinning tests to see what it > looks like when you assign too low symmetry. Based on the fact that you can > solve the structure in both P31 and P3121, either you have true P3121 > symmetry or P31 with pseudosymmetry close to P3121, so you have to be > cautious in your conclusions about twinning. Note that, as Garib has shown, > the R-factors are always lower when you assume the crystal is twinned! > > Best wishes, > > Randy Read > > On 7 Sep 2012, at 00:48, Qing Luan wrote: > >> I have a ~4.3 angstrom data set of a trigonal crystal of a seven subunit >> protein complex which I can scale in P3, P31, P32, P321, P3121 and P3221 >> with similar statistics: >> >> P3 >> Shell Lower Upper Average Average Norm. Linear Square >> limitAngstrom I error stat. Chi**2 R-fac R-fac >> 50.00 9.25 1296.889.223.5 1.233 0.064 0.077 >> 9.25 7.35 356.318.5 9.7 1.512 0.065 0.066 >> 7.35 6.4297.1 8.2 7.5 1.584 0.143 0.140 >> 6.42 5.8355.2 8.3 8.1 1.503 0.247 0.241 >> 5.83 5.4251.4 9.4 9.3 1.438 0.297 0.284 >> 5.42 5.1047.010.510.5 1.469 0.374 0.345 >> 5.10 4.8448.311.811.9 1.421 0.398 0.383 >> 4.84 4.6343.612.913.1 1.474 0.488 0.449 >> 4.63 4.4540.314.114.2 1.530 0.546 0.477 >> 4.45 4.3030.814.715.0 1.601 0.732 0.631 >> All reflections203.819.612.3 1.477 0.125 0.085 >> >> >> P3121: >> >> Shell Lower Upper Average Average Norm. Linear Square >> limitAngstrom I error stat. Chi**2 R-fac R-fac >> 50.00 9.14 1242.951.818.3 1.200 0.057 0.068 >> 9.14 7.26 314.011.2 6.5 1.454 0.070 0.069 >> 7.26 6.3586.9 5.3 5.0 1.499 0.158 0.152 >> 6.35 5.7751.9 5.5 5.3 1.248 0.264 0.252 >> 5.77 5.3546.9 6.1 6.0 1.213 0.330 0.305 >> 5.35 5.0444.3 6.9 6.7 1.137 0.393 0.363 >> 5.04 4.7943.4 7.7 7.4 1.109 0.434 0.407 >> 4.79 4.5839.2 8.5 8.1 1.128 0.533 0.478 >> 4.58 4.4034.2 9.1 8.6 1.115 0.634 0.549 >> 4.40 4.2524.9 9.9 9.3 1.064 0.872 0.766 >> All reflections199.012.4 8.1 1.216 0.127 0.080 >> >> Unit cell parameters: 129.653 129.653 358.28090.00090.000 >> 120.000 >> >> The systematic absences are consistent with either P31, P32, P3121, or >> P3221. Analyzing the
Re: [ccp4bb] poorly diffracting and twinned trigonal crystal
Dear Quing, My first suggesting is a no-brainer: try to get better data. At 4.3Å model building and refinement will remain painful, whatever you try. Getting better data includes improving your crystals (ligands, additives etc.) and the cryoconditions. You could also try to collect data on say 100 crystals to see if there is one crystal that diffracts better, or is less twinned. However, from your question this is probably not an option. Concerning your current data set, I would also try molecular replacement with the complete model (no poly-ala), at least with the complete core. E.g. if your protein has an Ile at a certain position and your model has a Leu, this Leu is closer to your protein than the Ala you are using now. I would run molecular replacement in the lower symmetry P3x space groups since in this case no asumptions are being made whether the 2-folds are crystallographic, non-crystallographic, or generated by twinning. Then I would analyze the packing to look if 2-folds are present and whether they could be crystallographic, or must be non-crystallographic and whether the packing makes sense. If there are no 2-folds present, the twofold symmetry of your data must be caused by twinning. If there are 2-folds present, there still could be twinning and I would try to generate the twin-related molecule as well and examine them together on the graphics to see what the implications are. At low resolution, you get severe model bias giving a large split between R and Rfree and having twinned data or data with pseudo-crystallographic symmetry will not improve the situation either. Probably Randy or Garib could tell you more precisely what that means for your R/Rfree's. My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Qing Luan Sent: Friday, September 07, 2012 1:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] poorly diffracting and twinned trigonal crystal I have a ~4.3 angstrom data set of a trigonal crystal of a seven subunit protein complex which I can scale in P3, P31, P32, P321, P3121 and P3221 with similar statistics: P3 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 9.25 1296.889.223.5 1.233 0.064 0.077 9.25 7.35 356.318.5 9.7 1.512 0.065 0.066 7.35 6.4297.1 8.2 7.5 1.584 0.143 0.140 6.42 5.8355.2 8.3 8.1 1.503 0.247 0.241 5.83 5.4251.4 9.4 9.3 1.438 0.297 0.284 5.42 5.1047.010.510.5 1.469 0.374 0.345 5.10 4.8448.311.811.9 1.421 0.398 0.383 4.84 4.6343.612.913.1 1.474 0.488 0.449 4.63 4.4540.314.114.2 1.530 0.546 0.477 4.45 4.3030.814.715.0 1.601 0.732 0.631 All reflections203.819.612.3 1.477 0.125 0.085 P3121: Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 9.14 1242.951.818.3 1.200 0.057 0.068 9.14 7.26 314.011.2 6.5 1.454 0.070 0.069 7.26 6.3586.9 5.3 5.0 1.499 0.158 0.152 6.35 5.7751.9 5.5 5.3 1.248 0.264 0.252 5.77 5.3546.9 6.1 6.0 1.213 0.330 0.305 5.35 5.0444.3 6.9 6.7 1.137 0.393 0.363 5.04 4.7943.4 7.7 7.4 1.109 0.434 0.407 4.79 4.5839.2 8.5 8.1 1.128 0.533 0.478 4.58 4.4034.2 9.1 8.6 1.115 0.634 0.549 4.40 4.2524.9 9.9 9.3 1.064 0.872 0.766 All reflections199.012.4 8.1 1.216 0.127 0.080 Unit cell parameters: 129.653 129.653 358.28090.00090.000 120.000 The systematic absences are consistent with either P31, P32, P3121, or P3221. Analyzing the cell contents in P3121 suggests either 1 (Matthews coefficient of 3.86, 68.2% solvent) or 2 mol/ASU (Matthews coefficient of 1.93, 36.38% solvent) I built a molecular replacement model (a polyala model containing about 2/3 of the protein complex) and ran phaser in multiple space groups with one (for P3121 or P3221) or two (P31, P32) copies of the model. Runs in P32 or P3221 gave no solutions or solutions with TFZ around 4-5. When run in P31 or P3121, phaser output solutions with TFZ> 11.0 and what appeared to be good packing. Rigid body refinement on the P3121 solution failed to improve the Rfactor (it hovered around 55.3%). Adding the missing subunits (as polyala chains) based on the phaser solution and refining with rigid body refinement resulted in a model with an Rfree to 48.5. Refining with torsion angle dynamics and restrained group B-factor refinement made the Rfree worse - it jumped up to about 55.6%. The Rwork values were similar to the Rfree
