Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Hello all, Thank you for you suggestions. I took a look at the crystal packing for the solution with one molecule per asu, and the next closest molecule is 50 angstroms away, suggesting that this is not likely the correct solution. I have also tried MR with a number of different molecules per asu. In some cases I get better packing, but I have not yet gotten a solution that looks to refine well. In addition to my previous information, I would like to add the following: 1. The resolution of my data is not particular great. I get ~4 A resolution at best and spots are rather weak even with almost no beam attenuation. 2. The search model that gives the best solution (in terms of contrast score in MolRep) is an NMR structure. I have heard that MR with NMR structures can possibly give false solutions. An alternate crystal structure that I tried using gave much poorer contrast socres overall, regardless of the number of molecules in the asu to search for. If anyone has any additonal suggestion, I would appreciate them. Thanks, Matt On Fri, May 16, 2014 at 8:46 AM, R. M. Garavito rmgarav...@gmail.comwrote: 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 %28517%29%20355-9724 Lab: (517) 353-9125 %28517%29%20353-9125* *FAX: (517) 353-9334 %28517%29%20353-9334 Email: rmgarav...@gmail.com garav...@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
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Did you look at the maps for extra density/molecules? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew Bratkowski Sent: Monday, May 19, 2014 4:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement Hello all, Thank you for you suggestions. I took a look at the crystal packing for the solution with one molecule per asu, and the next closest molecule is 50 angstroms away, suggesting that this is not likely the correct solution. I have also tried MR with a number of different molecules per asu. In some cases I get better packing, but I have not yet gotten a solution that looks to refine well. In addition to my previous information, I would like to add the following: 1. The resolution of my data is not particular great. I get ~4 A resolution at best and spots are rather weak even with almost no beam attenuation. 2. The search model that gives the best solution (in terms of contrast score in MolRep) is an NMR structure. I have heard that MR with NMR structures can possibly give false solutions. An alternate crystal structure that I tried using gave much poorer contrast socres overall, regardless of the number of molecules in the asu to search for. If anyone has any additonal suggestion, I would appreciate them. Thanks, Matt On Fri, May 16, 2014 at 8:46 AM, R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.com wrote: 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) 355-9724tel:%28517%29%20355-9724 Lab: (517) 353-9125tel:%28517%29%20353-9125 FAX: (517) 353-9334tel:%28517%29%20353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com On May 15, 2014, at 6:50 PM, Matthew Bratkowski mab...@cornell.edumailto: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
[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
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] 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
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Have you tried fixing the molecule that looks correct and searching for others? You might have greater than one but less than 9 molecules per ASU. When you do this, try imposing severe restraints on the packing function. This worked for me in Phaser with a difficult case. My anecdotal experience is that, when you have lots of molecules per asu, the correct solution gets swamped by poorly-packed solutions if the default packing penalties are used. Good luck. Sent from my iPhone 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
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
For P212121, I would put money on something divisible by 2 for the total molecules per asu. Anything from 6-12 might be likely. One of the early structures I worked on had 6 molecules per asu, which was darn near impossible to find using momomers (at the time). The way it was eventually solved was by finding a reasonable tetramer solution, then looking at the packing of that model to see what was missing and determine how much more would fit into the asu. Turned out to be another (obvious) dimer, which did the trick. The Matthews coefficient predicted something like 9 molecules per asu, which was not close to the actual answer. When n=large number, the Matthews coefficient does not easily identify a unique, most likely solution, but a range of reasonable solutions. One possible strategy would be to look for some partial solutions to give you a better clue of the actual packing in the asu. If you find there is a reasonable looking dimer pair, try searching with multiple dimers, etc. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/15/2014 8:29 PM, Toth, Eric wrote: Have you tried fixing the molecule that looks correct and searching for others? You might have greater than one but less than 9 molecules per ASU. When you do this, try imposing severe restraints on the packing function. This worked for me in Phaser with a difficult case. My anecdotal experience is that, when you have lots of molecules per asu, the correct solution gets swamped by poorly-packed solutions if the default packing penalties are used. Good luck. Sent from my iPhone 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
