Re: [ccp4bb] help regarding structure solution - high R values after MR
I wonder if this could have happened here? Some one in the lab has yet again been trapped by a feature?? of REFMAC. Say your MR solution is found to be in P21212 after you searched various orthorhombic SFs, but the input MTZ file has the space group still listed as P222 (i.e. the point group) then REFMAC will treat the solution as in P222, and NOT use the SG given on the PDB CRYST1 card.. You need to change the space group in the mtz header using one of the reflection utilities. CAD or SFTOOLS or REINDEX (without any reindexing..) all will rewrite the mtz file with the correct space group. It would be nice if REFMAC reported any discrepancy in SG assignment as a fatal error.. Eleanor On 22 Jun 2012, at 00:53, Valerie Pye wrote: Hi Sonali, You could try wide-search MR: https://portal.sbgrid.org/d/apps/wsmr/ Best of luck, val On 20 June 2012 19:13, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution - high R values after MR
Dear All, Thanks a lot for all the suggestions and help. Dear Eleanor, I will check the mtz file as u mentioned for the spacegroup and Dear Pye, Thanks for the link, i will run it and will let you knw if i find any luck. Thanks again, Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Fri, 22/6/12, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: From: Eleanor Dodson eleanor.dod...@york.ac.uk Subject: Re: [ccp4bb] help regarding structure solution - high R values after MR To: CCP4BB@JISCMAIL.AC.UK Date: Friday, 22 June, 2012, 3:30 PM I wonder if this could have happened here? Some one in the lab has yet again been trapped by a feature?? of REFMAC. Say your MR solution is found to be in P21212 after you searched various orthorhombic SFs, but the input MTZ file has the space group still listed as P222 (i.e. the point group) then REFMAC will treat the solution as in P222, and NOT use the SG given on the PDB CRYST1 card.. You need to change the space group in the mtz header using one of the reflection utilities.CAD or SFTOOLS or REINDEX (without any reindexing..) all will rewrite the mtz file with the correct space group. It would be nice if REFMAC reported any discrepancy in SG assignment as a fatal error.. Eleanor On 22 Jun 2012, at 00:53, Valerie Pye wrote: Hi Sonali, You could try wide-search MR: https://portal.sbgrid.org/d/apps/wsmr/ Best of luck, val On 20 June 2012 19:13, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Dear Raaij, We have not done mass-spec on the band from SDS-PAGE to confirm if it is our desired protein or any other contaminant. So, cant say for sure. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 21/6/12, Mark J van Raaij mjvanra...@cnb.csic.es wrote: From: Mark J van Raaij mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] help regarding structure solution To: sonali dhindwal sonali11dhind...@yahoo.co.in Date: Thursday, 21 June, 2012, 11:33 AM you didn't answer the most important question - are you 100% sure the protein in the crystal is not a contaminant? Unfortunately, these things happen... Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 21 Jun 2012, at 06:47, sonali dhindwal wrote: Dear All, Thanks a lot for your replies. Glad to found so much help. Clemens, cell parameters are, 38.0020 78.0240 56.3800 90. 102.2770 90., in P21 spacegroup. Raaij, Savvas, we have checked for the twining and no twining was detected. Nat, DEN is a good suggestion, i will definitely try it today. Roger, Molrep didn't fail but as i mentioned in the mail, it do give solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. But none of the solution fits in the electron density. And phaser didn't give any solution. We checked in pointless also, it was suggesting the same spacegroup. Matthew, Yes, we don't have the same crystal now. Garib, I will run the balbes server, and will let you know then. Robert, I tried using your server, and found few hits. Will run those templates for molecular replacement. Peter, We didnt had MBP tag in the protein. But your idea of doing limited proteolysis sounds good, and will definitely try that. Thanks again to all, for their kind and valuable help. I will write after trying all the things as suggested. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 21/6/12, Peter Hsu hsuu...@u.washington.edu wrote: From: Peter Hsu hsuu...@u.washington.edu Subject: Re: [ccp4bb] help regarding structure solution To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 21 June, 2012, 5:08 AM Hi Sonali, Did you use MBP as your purification tag? That's around 45-50kDa if I remember right. If not, I've had a decent amount of luck using in situ proteolysis to get crystals of degraded fragments. Try a limited proteolysis first overnight at 4C at varying concentrations of trypsin, see which one gives a nice stable band after overnight. Use that same concentration to add to your protein stock before setting up drops and then try another screen. I always use freshly prepared trypsin stock instead of frozen solutions to make sure that the freeze thaw doesn't reduce activity of the trypsin and that batch to batch is reproducible. Best of luck, Peter
Re: [ccp4bb] help regarding structure solution
Hi Sonali, You could try wide-search MR: https://portal.sbgrid.org/d/apps/wsmr/ Best of luck, val On 20 June 2012 19:13, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
[ccp4bb] help regarding structure solution
Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure.Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
On Wed, Jun 20, 2012 at 11:13 AM, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. Have you tried using an automated building program on the best solutions you have so far? Refinement programs will often get stuck quickly if the MR solution is poor, but rebuilding from scratch can sometimes do a much better job. Other things to try in cases like this are DEN refinement or MR-Rosetta - both require significant computational resources but also have a wider radius of convergence. The other question to ask yourself in situations like this is did I really crystallize the protein I'm interested in, or something else? It's surprisingly easy to crystallize minor contaminants; at last count, I've met at least four different people who've done this. (One of them actually ended up with a decent paper describing a structure he'd never intended to solve.) I suspect there are dozens if not hundreds of datasets lying abandoned because they couldn't be phased or reproduced because they weren't what the researcher thought they were. If there is any way to obtain the mass spec of the crystallized protein, this will be the most useful confirmation either way. The next thing to try is searching for similar unit cells in the PDB, although this doesn't take into account changes in space group that result in a different unit cell without actually changing the lattice. (There are probably multiple tools that can account for this; I can point to one if you're interested.) As a last resort, I would recommend a brute-force approach: https://portal.sbgrid.org/d/apps/wsmr/ -Nat
Re: [ccp4bb] help regarding structure solution
Sonali, How did your MR search(es) fail? 1. Too many clashes? (allow more clashes or remove likely floppy bits of the protein, e.g. N- and/or C-termini) 2. Could not place all molecules in the asymmetric unit? (Consider searching for fewer molecules in asymmetric unit. A partial solution may give you a better idea of the number of molecules in the ASU by examining symmetry packing in Coot or Pymol. The Matthews Calculator does not always give a definitive solution, especially for 2 molecules per unit cell. Consider examining a partial solution as suggested above.) 3. Failure to converge? Phaser will almost always come up with something, even if it is not right. Examine symmetry packing in Coot or Pymol to see if it makes sense. Maybe you still have the wrong space group. 4. Twinning? I've forgotten to do this on occasion, and twinning can lead you on a merry chase that won't refine properly. With 46% identity, the likelihood of finding a solution with Phaser should normally be very high. An Rfree of 50% is normally indicative of a random fit (e.g., non-solution). If you can get an Rfree of 50% with the out-of-the-box MR solution, you may have a valid solution. I solved one structure where the good MR fit had an Rfree of 46-48%, and the random solutions were 52-55%. You may have considered these issues already, but if not, hope this helps, Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/20/2012 2:13 PM, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Dear Sonali - It seems very likely that your original protein (which did not crystallize) was proteolytically degraded over the one year storage, and you now have a fragment of the original protein which is capable of crystallizing. You should analyze all of the homologous structures in the PDB to see if there are break points, like domain boundaries, which could produce a fragment of the appropriate size upon cleavage. Then use those fragments as your search models. Don't forget to consider more distantly related proteins, or structures that match only a subdomain of your protein. However, it sounds as if you've already done that. Furthermore, if I read your email correctly, the original crystal is now gone, so there is no chance to collect additional data. (If this is not the case, you might attempt to get enough data for sulfur SAD phasing.) So what I suggest is that you try to reproduce the crystallization, not by waiting a year, but by limited proteolysis of the full-length protein. You should be able to get a stable fragment corresponding to the thing that's in your tray, and analyze it by mass spec and/or N-terminal sequencing. (You might be able to N-terminal sequence from your existing crystallization drop as well.) Once you know what the fragment is, you should try cloning and expressing just that fragment, then setting up more trays, perhaps with selenomethionine labeling. Good luck! - Matt On 6/20/12 2:13 PM, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali -- 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] help regarding structure solution
Dear Sonali have you run any diagnostics on your dataset e.g. via xtriage in PHENIX, or the ccp4 programs to detect issues such as twinning and pseudotranslation. You also did not provide any information regarding the data quality. Processing your dataset via Xia2 from the ccp4 suite could also provide an important (additional) dataset variant to work with. Best regards Savvas On 20 Jun 2012, at 20:13, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Dear Sonali, I think that first item on your possible to-do list is to verify that you have indeed crystallized the protein you purified. We, too, got great crystals once with protein X (100 kD) and noticed that 1) the lattice constants, space group symmetry, and Matthew's coefficient were within expected values (100 kD monomer in the ASU with moderate solvent content) and 2) the protein seemed to be cleaved into 50 kD fragments in the drop and in the crystal (as expected). However, it was a totally different protein that crystallized, which was why MR didn't work at all. After solving the structure by MIR, we found that a 50 kD minor contaminant ( 1%) crystallized, while our target protein did not. While there is still a good chance that the crystals you looked at contains at least a fragment of your target protein, make sure first, if you can. As Matt recommended, analyze your protein by mass spec and/or N-terminal sequencing to verify that it is what you think it is. Then, as he recommended, try cloning and expressing a truncated variant. Careful limited proteolysis of the full-length protein would also be worthwhile in getting crystals faster. 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 Jun 20, 2012, at 2:13 PM, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Dear Sonali I would do the following things 1) Check your space group. Although rare it could be p2 or even p1 2) Run balbes server and check all space groups (in your case only p21 and p2) If you want you can send data to me to see what might be going on Regards Garib On 20 Jun 2012, at 19:13, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] help regarding structure solution
Dear Sonali, Following Michael's suggestion that you just might have crystallized a contaminant (and all the signs point to that I'm afraid - a small crystal growing after a long time that diffracts surprisingly well), you might expect this crystal form already to have been characterised and deposited in the PDB. In which case can I suggest you use a tool developed by my student, Varun Ramraj, to check the unit cell against the PDB. Its quick and easy and the URL is http://www.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi It just might save a lot of wasted effort. Regards, Robert -- Dr. Robert Esnouf, University Research Lecturer and Head of Research Computing, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK Emails: rob...@strubi.ox.ac.uk Tel: (+44) - 1865 - 287783 and rob...@esnouf.comFax: (+44) - 1865 - 287547 Original message Date: Wed, 20 Jun 2012 16:00:06 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] help regarding structure solution To: CCP4BB@JISCMAIL.AC.UK Dear Sonali, I think that first item on your possible to-do list is to verify that you have indeed crystallized the protein you purified. We, too, got great crystals once with protein X (100 kD) and noticed that 1) the lattice constants, space group symmetry, and Matthew's coefficient were within expected values (100 kD monomer in the ASU with moderate solvent content) and 2) the protein seemed to be cleaved into 50 kD fragments in the drop and in the crystal (as expected). However, it was a totally different protein that crystallized, which was why MR didn't work at all. After solving the structure by MIR, we found that a 50 kD minor contaminant ( 1%) crystallized, while our target protein did not. While there is still a good chance that the crystals you looked at contains at least a fragment of your target protein, make sure first, if you can. As Matt recommended, analyze your protein by mass spec and/or N-terminal sequencing to verify that it is what you think it is. Then, as he recommended, try cloning and expressing a truncated variant. Careful limited proteolysis of the full-length protein would also be worthwhile in getting crystals faster. 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) 353-9334 Email: rmgarav...@gmail.com ** ** On Jun 20, 2012, at 2:13 PM, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Hi Sonali, Did you use MBP as your purification tag? That's around 45-50kDa if I remember right. If not, I've had a decent amount of luck using in situ proteolysis to get crystals of degraded fragments. Try a limited proteolysis first overnight at 4C at varying concentrations of trypsin, see which one gives a nice stable band after overnight. Use that same concentration to add to your protein stock before setting up drops and then try another screen. I always use freshly prepared trypsin stock instead of frozen solutions to make sure that the freeze thaw doesn't reduce activity of the trypsin and that batch to batch is reproducible. Best of luck, Peter
Re: [ccp4bb] help regarding structure solution
Dear All, Thanks a lot for your replies. Glad to found so much help. Clemens, cell parameters are, 38.0020 78.0240 56.3800 90. 102.2770 90., in P21 spacegroup. Raaij, Savvas, we have checked for the twining and no twining was detected. Nat, DEN is a good suggestion, i will definitely try it today. Roger, Molrep didn't fail but as i mentioned in the mail, it do give solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. But none of the solution fits in the electron density. And phaser didn't give any solution. We checked in pointless also, it was suggesting the same spacegroup. Matthew, Yes, we don't have the same crystal now. Garib, I will run the balbes server, and will let you know then. Robert, I tried using your server, and found few hits. Will run those templates for molecular replacement. Peter, We didnt had MBP tag in the protein. But your idea of doing limited proteolysis sounds good, and will definitely try that. Thanks again to all, for their kind and valuable help. I will write after trying all the things as suggested. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 21/6/12, Peter Hsu hsuu...@u.washington.edu wrote: From: Peter Hsu hsuu...@u.washington.edu Subject: Re: [ccp4bb] help regarding structure solution To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 21 June, 2012, 5:08 AM Hi Sonali, Did you use MBP as your purification tag? That's around 45-50kDa if I remember right. If not, I've had a decent amount of luck using in situ proteolysis to get crystals of degraded fragments. Try a limited proteolysis first overnight at 4C at varying concentrations of trypsin, see which one gives a nice stable band after overnight. Use that same concentration to add to your protein stock before setting up drops and then try another screen. I always use freshly prepared trypsin stock instead of frozen solutions to make sure that the freeze thaw doesn't reduce activity of the trypsin and that batch to batch is reproducible. Best of luck, Peter
