Re: [ccp4bb] PDB passes 100,000 structure milestone
So, a bit like Fold-it but with actual data? :-D Dr David C Briggs PhD http://about.me/david_briggs On 16 May 2014 06:19, Pavel Afonine pafon...@gmail.com wrote: What about structures that are obviously wrong based on inspection of the density, but no one has bothered to challenge yet? The TWILIGHT database helps some, if that counts, but it doesn't catch everything. How about this utopia.. Imagine PDB has two versions: one is the original data and model deposited as is, and never ever changed no matter what. Another version is a curated one obtained in a quest-like way: anyone can take an original entry, improve it and deposit (into the curated version) with his/her name tag on it. And of course anyone can take and update that improved entry and re-deposit it again with his/her name tag, etc. If desired one could keep track of all the revisions, like in svn or so. Sounds like a sport with an element of public service that might yield crowd-perfected models -:) ! Pavel
[ccp4bb] AW: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Dear Matt, 95% solvent is highly unlikely, but not impossible. Did you have a look at the crystal packing? Are there continuous crystal contacts in all three dimensions, or are there layers of molecules that are not connected? Are you sure your space group is P212121 and not one of the other seven P2x2x2x space groups? You have to test all possible space groups. With presumably so many molecules in the asymmetric unit, you may have non-crystallographic symmetry mimicking crystallographic symmetry. If you are sure you have used the correct space group and you have a decent resolution (say better than 2.5Å) you could also try to just refine your solution and see if some density for the missing molecule(s) appears. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Matthew Bratkowski Gesendet: Freitag, 16. Mai 2014 00:51 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement Hello all, I am working on the structure of a small protein in space group P212121. The protein is monomeric in solution based on gel filtration analysis. The Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit results in ~50% solvent content, while 1 molecule per asymmetric unit results in ~95% solvent. I tried molecular replacement with a search model which is essentially identical in sequence to my protein, and searched for 9 or 10 molecules/asu. Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 1-1.5. However, when using Phaser, I get a solution with one molecules/asu. Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a contrast score of 3.12. This model still has some issues, but looks more correct compaired to models created with 9 or 10 molecules/asu. It seems highly unlikely that a crystal would contain 95% solvent, but is there any possiblility that this could be the case? Assuming that the Matthews coefficient is correct, does anyone have an idea why MR seems to work better for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% solvent content? Alternatively, is there any reason why the Matthews coefficient could be calculating incorrectly? Any suggestions would be helpful. Thanks, Matt
[ccp4bb] AW: [ccp4bb] PDB passes 100,000 structure milestone
I am also in favor of two versions of the pdb: one archive version with all models as originally deposited including retracted and corrected versions, which are useful for educational purposes, and a curated version with only models that meet a minimum of validation criteria, including credible ligand density. Whether crowd-perfected or original submissions that pass the validation criteria does not matter to me. Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Pavel Afonine Gesendet: Freitag, 16. Mai 2014 07:19 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] PDB passes 100,000 structure milestone What about structures that are obviously wrong based on inspection of the density, but no one has bothered to challenge yet? The TWILIGHT database helps some, if that counts, but it doesn't catch everything. How about this utopia.. Imagine PDB has two versions: one is the original data and model deposited as is, and never ever changed no matter what. Another version is a curated one obtained in a quest-like way: anyone can take an original entry, improve it and deposit (into the curated version) with his/her name tag on it. And of course anyone can take and update that improved entry and re-deposit it again with his/her name tag, etc. If desired one could keep track of all the revisions, like in svn or so. Sounds like a sport with an element of public service that might yield crowd-perfected models -:) ! Pavel
Re: [ccp4bb] report mosaicity
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Hongshi, if the numbers do not vary too much, e.g. between 0.2 and 0.6, I would deposit the calculated mean. I would say that the reported mosaicity is not too important a figure for a publication. It is interesting when it comes to data collection and planning your strategy, and may be something to consider if it is very, very large, but for a normal structural study it won't matter too much. Best, Tim On 05/16/2014 01:26 AM, hongshi WANG wrote: Hello everyone, I am gonna report the mosaicity of my data set as required by the journal. I processed the data using HKL2000. So I checked the denzo log file. I found many different mosaicity values. The first one is default input (0.3), the rest are corresponding to specific images. I think the mosaicity value required should be an overall value or averaged value. Could you please let me know how I can get it. Or some other software can determine it. I really appreciate your help and response! Hongshi - -- - -- 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/ iD8DBQFTdcyKUxlJ7aRr7hoRAk+GAKD9+jnmvpV+TFw0t8jtSWXemlKsfgCaA7vn s8YBbU/1ghs+C5cYsJrur/A= =Ra/3 -END PGP SIGNATURE-
Re: [ccp4bb] report mosaicity
Hi I'm sure that a real HKL or Denzo/Scalepack expert will correct me, but my recollection is that you don't use any of the values from Denzo, but the value from Scalepack (see http://www.hkl-xray.com/sites/default/files/HKL2000manual/chapter3/step14-1.htm, for example). On 16 May 2014, at 00:26, hongshi WANG wrote: Hello everyone, I am gonna report the mosaicity of my data set as required by the journal. I processed the data using HKL2000. So I checked the denzo log file. I found many different mosaicity values. The first one is default input (0.3), the rest are corresponding to specific images. I think the mosaicity value required should be an overall value or averaged value. Could you please let me know how I can get it. Or some other software can determine it. I really appreciate your help and response! Hongshi 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] Two postdoctoral positions available at UCL/Magnus Life Science
Hi ccp4bb Two postdoctoral positions in X-ray crystallography/drug discovery are currently available at University College London/Magnus Life Science. Please see link for further details. http://www.magnuslifescience.co.uk/careers/ Best
Re: [ccp4bb] FW: [ccp4bb] PDB passes 100,000 structure milestone
2014-05-15 9:53 GMT-03:00 Colin Nave colin.n...@diamond.ac.uk: Of course exponential growth can’t go on forever – the hidden point behind my question. A nice example from another biological database is Swissprot. It had an exponential-like growth until 2009, and now it's somewhat linear: http://web.expasy.org/docs/relnotes/relstat.html I didn't looked much into that, but I guess it's because the annotators (Swissprot is human curated) simply can't keep up with everything coming from all genome projects. The other Uniprot database, automatically annotated Trembl, is still growing in a exponential-like fashion: http://www.ebi.ac.uk/uniprot/TrEMBLstats
Re: [ccp4bb] PDB passes 100,000 structure milestone
16-May-2014 Dear Patrick, Proteopedia [http://proteopedia.org] uses exactly the same style for referencing published material. Proteopedia allows for the easy insertion of Pubmed and DOI references by only requesting from the user to enter the Pubmed or DOI ids. We have extended the same software used in Wikipedia for the internal Proteopedia engine to, based on this reference ID, retrieve, format and insert the correctly formatted reference at the bottom of the page. For example, type refPMID 18673581/ref or refdoi 10.1093/nar/gku213/ref in the wikitext box and save the page. If you type the reference in this manner, the properly formatted reference will be created automatically at the bottom of the page (or wherever you place the necessary wikitext references/). See http://proteopedia.org/w/Help:Editing#Citing_Literature_References and Proteopedia pages for actual examples. best regards, Jaime Joel On 15May, 2014, at 13:48, Patrick Shaw Stewart patr...@douglas.co.ukmailto:patr...@douglas.co.uk wrote: I may be missing something here, but I don't think you have to rebut anything. You simply report that someone else has rebutted it. Along the lines of Many scientists regard this published structure as unreliable since a misconduct investigation by the University of Alabama at Birmingham has concluded that it was, more likely than not, faked [1] [1] http://www.nature.com/news/2009/091222/full/462970a.html On 15 May 2014 18:00, Nat Echols nathaniel.ech...@gmail.commailto:nathaniel.ech...@gmail.com wrote: On Thu, May 15, 2014 at 9:53 AM, Patrick Shaw Stewart patr...@douglas.co.ukmailto:patr...@douglas.co.uk wrote: It seems to me that the Wikipedia mechanism works wonderfully well. One rule is that you can't make assertions yourself, only report pre-existing material that is attributable to a reliable published source. This rule would be a little problematic for annotating the PDB. It requires a significant amount of effort to publish a peer-reviewed article or even just a letter to the editor, and none of us are being paid to write rebuttals to dodgy structures. -Nat -- patr...@douglas.co.ukmailto:patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.ukhttp://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Matt, In addition to the suggestions of the others, have you done a simple self rotation function? It can tell you quite a bit about how things are packed and give you strict criteria for choosing one solution over another. As Roger said, choosing an even number of monomers in the ASU is a good strategy, particularly if the self rotation function shows NCS 2-folds. Also, a calculated Matthews coefficient is NEVER correct, it is a mere guideline; it only has validity for any particularly crystal form AFTER the fact. Let the number of monomers in the ASU vary from 6-10; I have had MR cases that have had as little as 40% solvent to 70% solvent, where the calculated Matthews coefficient was quite wrong (i.e., the most common value observed in OTHER crystals). Two things to watch out for are: (1) An odd number of monomers in the ASU. I have had 1 1/2 dimers in an ASU (the 1/2 dimer is paired with another in a neighboring ASU). It is sometimes confusing to people and occasionally difficult to solve with some MR programs due to clashes. (2) Translation symmetry, which still can confuse some programs (but they are are getting better at detecting it). Finally, as Herman pointed out, look at the packing of any solution you are considering. It is surprising how a correct solution looks correct: nice intermolecular contacts and a pleasing distribution of mass throughout the unit cell (meaning expand out to at least a unit cell volume, which is easy in Pymol). Any unexplained gaps (meaning not caused by a missing domain) should be viewed critically. Regards and Good Luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 15, 2014, at 6:50 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hello all, I am working on the structure of a small protein in space group P212121. The protein is monomeric in solution based on gel filtration analysis. The Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit results in ~50% solvent content, while 1 molecule per asymmetric unit results in ~95% solvent. I tried molecular replacement with a search model which is essentially identical in sequence to my protein, and searched for 9 or 10 molecules/asu. Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 1-1.5. However, when using Phaser, I get a solution with one molecules/asu. Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a contrast score of 3.12. This model still has some issues, but looks more correct compaired to models created with 9 or 10 molecules/asu. It seems highly unlikely that a crystal would contain 95% solvent, but is there any possiblility that this could be the case? Assuming that the Matthews coefficient is correct, does anyone have an idea why MR seems to work better for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% solvent content? Alternatively, is there any reason why the Matthews coefficient could be calculating incorrectly? Any suggestions would be helpful. Thanks, Matt
[ccp4bb] PDB Storage of Diffraction Images
Hello, for convenience I might ask the CCP4BB: does anyone know what the status of the PDB is with regard to the storage of diffraction images? I had the impression that this matter had been discussed as far back as ~15 years ago but what came out of it? Short of storing images, which is the ultimate preservation of primary information, I have always been puzzled by the fact that the PDB only stores unique reflections i.e. no Friedel pairs even when provided. Is this outdated perhaps ? I remember that my deposited SFs in the past where reduced to not contain Friedel pairs. If there had been a concern about increasing the storage space by actually less than twice the space for unique SFs, this may be invalid today and is still far less than the space required for images. However, it is possible that the information content in Friedel pairs is deemed insignificant compared to their extra costs. I for one would appreciate having access to Friedel pairs very much. Thanks, Lothar
Re: [ccp4bb] PDB Storage of Diffraction Images
On Fri, May 16, 2014 at 7:12 AM, esse...@helix.nih.gov wrote: Short of storing images, which is the ultimate preservation of primary information, I have always been puzzled by the fact that the PDB only stores unique reflections i.e. no Friedel pairs even when provided. Is this outdated perhaps ? I remember that my deposited SFs in the past where reduced to not contain Friedel pairs. If there had been a concern about increasing the storage space by actually less than twice the space for unique SFs, this may be invalid today and is still far less than the space required for images. However, it is possible that the information content in Friedel pairs is deemed insignificant compared to their extra costs. I for one would appreciate having access to Friedel pairs very much. They definitely store Friedel pairs! Maybe you're confused by the layout of the mmCIF file, which (like MTZ) usually lists just the unique (non-anomalous) indices, but with separate values for F+/F- when they are available. I've been making extensive use of anomalous data depositions - unfortunately there aren't as many as we would like, either because many people do not realize that this is useful information even when the experiment was not specifically looking for anomalous signal, or because the complexity of PDB deposition discourages providing the most complete data. An even more useful improvement would be to make deposition of unmerged intensities straightforward - the JCSG does this somehow but it is non-trivial for the average user. Hopefully this will also change soon. -Nat
[ccp4bb] Difficult MR with MBP fusion protein
Dear All, Recently we collected some data of a MBP fusion protein, at around 4A resolution. The protein itself is about half of the MBP size. However when we tried to solve it with MR, it failed. We tried to use MBP alone, homology model of target protein alone, and MBP+model. It is very strange that MBP alone can not yield any reasonable solution at all, so does searching with MBP and model together. While searching with model alone could get some better results, but when fix it to search MBP, it failed. There are 1 molecule per ASU with solvent content 55%. The spacegroup should be right and we tried to search all possible alternatives in each run, we also tried to lower it down, but did not work either. When running Phenix.phaser, there is a warning at the beginning saying eLLG suggests placing of ensembles will be very difficult. I wonder if anybody has encountered similar situation before. Any suggestions will be greatly appreciated! Regards, Niu
Re: [ccp4bb] PDB Storage of Diffraction Images
Hi Nat, okay. Looks like I missed this. Perhaps the data sets that I wish had Fiedel pairs didn't and this solidified my incorrect assumption(s) about pdb policy. So Fiedel pairs are there when deposited. What about storage of images? Is it coming soon - it feels like that it is about time Lothar On Fri, May 16, 2014 at 7:12 AM, esse...@helix.nih.gov wrote: Short of storing images, which is the ultimate preservation of primary information, I have always been puzzled by the fact that the PDB only stores unique reflections i.e. no Friedel pairs even when provided. Is this outdated perhaps ? I remember that my deposited SFs in the past where reduced to not contain Friedel pairs. If there had been a concern about increasing the storage space by actually less than twice the space for unique SFs, this may be invalid today and is still far less than the space required for images. However, it is possible that the information content in Friedel pairs is deemed insignificant compared to their extra costs. I for one would appreciate having access to Friedel pairs very much. They definitely store Friedel pairs! Maybe you're confused by the layout of the mmCIF file, which (like MTZ) usually lists just the unique (non-anomalous) indices, but with separate values for F+/F- when they are available. I've been making extensive use of anomalous data depositions - unfortunately there aren't as many as we would like, either because many people do not realize that this is useful information even when the experiment was not specifically looking for anomalous signal, or because the complexity of PDB deposition discourages providing the most complete data. An even more useful improvement would be to make deposition of unmerged intensities straightforward - the JCSG does this somehow but it is non-trivial for the average user. Hopefully this will also change soon. -Nat
Re: [ccp4bb] PDB passes 100,000 structure milestone
Hi Joel and Jaime - very nice to hear from you. I hope everything is going well in Rehovot. Proteopedia is the natural place to put comments etc. However it might look more natural if there was more info there in the first place - ie if people gave more explanation about the significance of their and other people's structures, then comments like the one I suggested could be added. I don't know how you get people to become more active at Proteopedia. Maybe your students could post occasional messages to eg the CCP4bb with comments that show how useful Proteopedia can be. Tricky though! Best wishes, Patrick On 16 May 2014 14:35, Joel Sussman joel.suss...@weizmann.ac.il wrote: 16-May-2014 Dear Patrick, *Proteopedia* [*http://proteopedia.org] http://proteopedia.org%5D* uses exactly the same style for referencing published material. *Proteopedia* allows for the easy insertion of Pubmed and DOI references by only requesting from the user to enter the Pubmed or DOI ids. We have extended the same software used in Wikipedia for the internal *Proteopedia* engine to, based on this reference ID, retrieve, format and insert the correctly formatted reference at the bottom of the page. For example, *type refPMID 18673581/ref or refdoi 10.1093/nar/gku213/ref* in the wikitext box and save the page. If you type the reference in this manner, the properly formatted reference will be created automatically at the bottom of the page (or wherever you place the necessary wikitext references/). See *http://proteopedia.org/w/Help:Editing#Citing_Literature_References http://proteopedia.org/w/Help:Editing#Citing_Literature_References* and Proteopedia pages for actual examples. best regards, Jaime Joel On 15May, 2014, at 13:48, Patrick Shaw Stewart patr...@douglas.co.uk wrote: I may be missing something here, but I don't think you have to rebut anything. You simply report that someone else has rebutted it. Along the lines of Many scientists regard this published structure as unreliable since a misconduct investigation by the University of Alabama at Birmingham has concluded that it was, more likely than not, faked [1] [1] http://www.nature.com/news/2009/091222/full/462970a.html On 15 May 2014 18:00, Nat Echols nathaniel.ech...@gmail.com wrote: On Thu, May 15, 2014 at 9:53 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: It seems to me that the Wikipedia mechanism works wonderfully well. One rule is that you can't make assertions yourself, only report pre-existing material that is attributable to a reliable published source. This rule would be a little problematic for annotating the PDB. It requires a significant amount of effort to publish a peer-reviewed article or even just a letter to the editor, and none of us are being paid to write rebuttals to dodgy structures. -Nat -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Difficult MR with MBP fusion protein
Hi Niu, Several things come to mind. First, it may not be trivial when the first component to be placed is ~20kDa and the second component (SU) is ~43kDa. The signal after placing the first component may be weak. Also, if the model for the smaller SU has low sequence identity with the target and you have 4 Angstrom data, it would not be surprising to encounters problems after searching with the smaller SU. But the question of why MR with MBP failed is worth investigating. Here are couple of quick questions: (1) When you say you got some better results with the smaller component, what are the TFZ and LLG values? (2) Assuming you have very good 4A data, the correct space group, and a successful rotation and translation search with the first component so that maps are not entirely crappy, do you actually see a big mass of unaccounted density that could potentially fit MBP when you look at the maps after placing the first component? If yes, how about physically placing MBP in there yourself and starting from there. (3) What is the sequence identity of model with the target 20kDa component? (4) Do you know for sure that you have MBP in the crystal? Unlikely concern but I ask nonetheless. Also, I'm curious whether you ran Phaser jobs with the default settings or whether you tried tweaking some of the parameters? I'm happy to speak further about this offline. Good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University On Fri, May 16, 2014 at 11:03 AM, Niu Tou niutou2...@gmail.com wrote: Dear All, Recently we collected some data of a MBP fusion protein, at around 4A resolution. The protein itself is about half of the MBP size. However when we tried to solve it with MR, it failed. We tried to use MBP alone, homology model of target protein alone, and MBP+model. It is very strange that MBP alone can not yield any reasonable solution at all, so does searching with MBP and model together. While searching with model alone could get some better results, but when fix it to search MBP, it failed. There are 1 molecule per ASU with solvent content 55%. The spacegroup should be right and we tried to search all possible alternatives in each run, we also tried to lower it down, but did not work either. When running Phenix.phaser, there is a warning at the beginning saying eLLG suggests placing of ensembles will be very difficult. I wonder if anybody has encountered similar situation before. Any suggestions will be greatly appreciated! Regards, Niu
Re: [ccp4bb] Difficult MR with MBP fusion protein
Dear Niu, When it works, crystallising a fusion protein can be great, with the big advantage that placing a model for the (known) carrier protein gives free phase information. Certainly there are examples of this working, but in the early days of this (20 years or so ago?), I remember hearing of more than one case where only one component was visible in the electron density, because the other component was unconstrained by crystal packing or by the flexible linker. So you might want to consider whether the MBP component might be disordered. It would be interesting to hear whether anyone has data on how frequently fusion proteins crystallise and how often both components are well-ordered, because I’m just working from a few anecdotes. The warning from Phaser reflects the fact that signal in the MR search will be limited by the low resolution, the incompleteness of the model, and possibly the quality of the model for the target protein. Best wishes, Randy Read On 16 May 2014, at 16:03, Niu Tou niutou2...@gmail.com wrote: Dear All, Recently we collected some data of a MBP fusion protein, at around 4A resolution. The protein itself is about half of the MBP size. However when we tried to solve it with MR, it failed. We tried to use MBP alone, homology model of target protein alone, and MBP+model. It is very strange that MBP alone can not yield any reasonable solution at all, so does searching with MBP and model together. While searching with model alone could get some better results, but when fix it to search MBP, it failed. There are 1 molecule per ASU with solvent content 55%. The spacegroup should be right and we tried to search all possible alternatives in each run, we also tried to lower it down, but did not work either. When running Phenix.phaser, there is a warning at the beginning saying eLLG suggests placing of ensembles will be very difficult. I wonder if anybody has encountered similar situation before. Any suggestions will be greatly appreciated! Regards, Niu -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] report mosaicity
Dear all, Thanks for all your reply and useful comment on this. Harry, You are right. I think there are individual mosaicity value corresponding to each image from scalepack. Clearly, we can get either average or range for report. best, Hongshi On Fri, May 16, 2014 at 2:26 AM, Harry Powell ha...@mrc-lmb.cam.ac.ukwrote: Hi I'm sure that a real HKL or Denzo/Scalepack expert will correct me, but my recollection is that you don't use any of the values from Denzo, but the value from Scalepack (see http://www.hkl-xray.com/sites/default/files/HKL2000manual/chapter3/step14-1.htm, for example). On 16 May 2014, at 00:26, hongshi WANG wrote: Hello everyone, I am gonna report the mosaicity of my data set as required by the journal. I processed the data using HKL2000. So I checked the denzo log file. I found many different mosaicity values. The first one is default input (0.3), the rest are corresponding to specific images. I think the mosaicity value required should be an overall value or averaged value. Could you please let me know how I can get it. Or some other software can determine it. I really appreciate your help and response! Hongshi 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)
Re: [ccp4bb] report mosaicity
If you refine crystal mosaicity (I assume there is a way to do that in the gui) then scalepack prints out a single value for the crystal (search the logfile for mosaicity) eab On 05/16/2014 12:07 PM, hongshi WANG wrote: Dear all, Thanks for all your reply and useful comment on this. Harry, You are right. I think there are individual mosaicity value corresponding to each image from scalepack. Clearly, we can get either average or range for report. best, Hongshi On Fri, May 16, 2014 at 2:26 AM, Harry Powell ha...@mrc-lmb.cam.ac.uk mailto:ha...@mrc-lmb.cam.ac.uk wrote: Hi I'm sure that a real HKL or Denzo/Scalepack expert will correct me, but my recollection is that you don't use any of the values from Denzo, but the value from Scalepack (see http://www.hkl-xray.com/sites/default/files/HKL2000manual/chapter3/step14-1.htm, for example). On 16 May 2014, at 00:26, hongshi WANG wrote: Hello everyone, I am gonna report the mosaicity of my data set as required by the journal. I processed the data using HKL2000. So I checked the denzo log file. I found many different mosaicity values. The first one is default input (0.3), the rest are corresponding to specific images. I think the mosaicity value required should be an overall value or averaged value. Could you please let me know how I can get it. Or some other software can determine it. I really appreciate your help and response! Hongshi 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)
Re: [ccp4bb] report mosaicity
HKL-2000 has a menu button at the top Report. If you click on that a report is generated with mosaicity range explicitly listed. It is probably not suitable to describe a crystal as having a single mosaicity value because mosaicity may be anisotropic. It should be obvious that a unit cell like 58 x 58 x 150 may have a bigger mosaicity along the 58 directions and a smaller mosaicity along the 150 direction and still have the Bragg reflections spatially separated. Thus, the mosaicity reported/used by diffraction image processing is probably just the best estimate for the mosaicity model used by the algorithm AND the orientation of the crystal in the experiment. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry [ber...@upstate.edu] Sent: Friday, May 16, 2014 11:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] report mosaicity If you refine crystal mosaicity (I assume there is a way to do that in the gui) then scalepack prints out a single value for the crystal (search the logfile for mosaicity) eab On 05/16/2014 12:07 PM, hongshi WANG wrote: Dear all, Thanks for all your reply and useful comment on this. Harry, You are right. I think there are individual mosaicity value corresponding to each image from scalepack. Clearly, we can get either average or range for report. best, Hongshi On Fri, May 16, 2014 at 2:26 AM, Harry Powell ha...@mrc-lmb.cam.ac.uk mailto:ha...@mrc-lmb.cam.ac.uk wrote: Hi I'm sure that a real HKL or Denzo/Scalepack expert will correct me, but my recollection is that you don't use any of the values from Denzo, but the value from Scalepack (see http://www.hkl-xray.com/sites/default/files/HKL2000manual/chapter3/step14-1.htm, for example). On 16 May 2014, at 00:26, hongshi WANG wrote: Hello everyone, I am gonna report the mosaicity of my data set as required by the journal. I processed the data using HKL2000. So I checked the denzo log file. I found many different mosaicity values. The first one is default input (0.3), the rest are corresponding to specific images. I think the mosaicity value required should be an overall value or averaged value. Could you please let me know how I can get it. Or some other software can determine it. I really appreciate your help and response! Hongshi 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)
Re: [ccp4bb] Difficult MR with MBP fusion protein
Dear Randy, Over the last ~6 years, we have tried to use the MBP tag to enhance crystallization on 10 proteins. Only one that couldn't crystallize by itself in the end crystallized with the fusion tag. However, unfortunately, the target protein (~10 kDa) turned out to be in the big cavity of the MBP lattice and was completely disordered. Therefore, I am personally not very optimistic about the method. Best wishes, Gang DONG On Fri, May 16, 2014 17:57, Randy Read wrote: Dear Niu, When it works, crystallising a fusion protein can be great, with the big advantage that placing a model for the (known) carrier protein gives free phase information. Certainly there are examples of this working, but in the early days of this (20 years or so ago?), I remember hearing of more than one case where only one component was visible in the electron density, because the other component was unconstrained by crystal packing or by the flexible linker. So you might want to consider whether the MBP component might be disordered. It would be interesting to hear whether anyone has data on how frequently fusion proteins crystallise and how often both components are well-ordered, because I’m just working from a few anecdotes. The warning from Phaser reflects the fact that signal in the MR search will be limited by the low resolution, the incompleteness of the model, and possibly the quality of the model for the target protein. Best wishes, Randy Read On 16 May 2014, at 16:03, Niu Tou niutou2...@gmail.com wrote: Dear All, Recently we collected some data of a MBP fusion protein, at around 4A resolution. The protein itself is about half of the MBP size. However when we tried to solve it with MR, it failed. We tried to use MBP alone, homology model of target protein alone, and MBP+model. It is very strange that MBP alone can not yield any reasonable solution at all, so does searching with MBP and model together. While searching with model alone could get some better results, but when fix it to search MBP, it failed. There are 1 molecule per ASU with solvent content 55%. The spacegroup should be right and we tried to search all possible alternatives in each run, we also tried to lower it down, but did not work either. When running Phenix.phaser, there is a warning at the beginning saying eLLG suggests placing of ensembles will be very difficult. I wonder if anybody has encountered similar situation before. Any suggestions will be greatly appreciated! Regards, Niu -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
