Re: [ccp4bb] MR problem in muti-domain structure
Dear Randy, Eleanor and Jon, Thank you for your kind rely. I apologize for about the tutorial files. I actually got it from my institution's sever and did not check the link. Thanks to Randy we now have the correct link. To John: I did put the correct domain 1 chain A and domain 2 chain B as fixed model but it seems domain 2 chain A is extra hard to be added in the third MR. To Randy: Your approach solved many questions in my mind on that misplaced domain. I tried single job with phaser before but it failed too; at the same time, I actually tried to put the domains 1 or domain 3 as the third domain in Molrep (also failed). Maybe I should inspect more in the map when putting domain 3. I also found the misplaced second domain one was in completely wrong orientation that the N-terminus and C-terminus shall be switched. Although I got quiet heave work load this week, I will definitely try your steps later. I cannot be more grateful for your help, especially there are limited learning materials online. Thanks again. best, Lande To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] MR problem in muti-domain structure
Brief followup: 1. Typo: Phaser struggled with the second copy of domain *2*, not domain 1. 2. I thought the single job searching for 1+1+2+3+3+2 would work, but it failed at the last step. The easiest way to solve this with Phaser might be to do one job with 1+1+2+3+3, and then do a second job adding the second copy of domain 2. The reason for this, I think, is that Phaser refines all of the placed models simultaneously, including their relative B factors and estimated RMS errors, at the end of the whole job, not after placing every model. After each intermediate placement, Phaser only refines the parameters of the last component placed and not the estimated RMS error. The improvement the full refinement gives in the 5-domain solution is just enough that the second copy of domain 2 can finally be found. 3. If you want to find this and some other tutorials, go to http://legacy.ccp4.ac.uk/tutorials/targets/standard/, expand the section on “Basic phasing tutorials” under “ccp4i tutorials”, and download phasing.tgz, which contains what you need to run 9 tutorials in this section (of which this is #7). Best wishes, Randy Read > On 11 May 2021, at 14:57, Randy John Read wrote: > > Hi, > > I had a hard time tracking down the data in the legacy CCP4 site but > eventually found it! Then I tried solving the MR tutorial case with Phaser. > > Instead of using the tutorial domain 1, 2 and 3 files, I prepared them from > the deposited PDB file using a sequence alignment followed by using sculptor > to trim off non-conserved ends of side chains and any missing loops. Then I > did a default run of Phaser, searching for 2 copies of each domain. Phaser > automatically set the search order to domain 1, then domain 2 then domain 3 > (they have similar sequence identities and that sorts them from biggest to > smallest). It was clear from the statistics (increase in log-likelihood gain > or LLG and TFZ score) that both copies of domain 1 were correct, the first > copy of domain 2, and both copies of domain 3, even though an incorrect > placement of domain 2 occurred before looking for domain 3. Unfortunately, > the second copy of domain 3 was rejected because of packing problems with the > badly-placed domain 2 copy. > > So I went back to the partial solution with 2 copies of domain 1 and one of > domain 2, then added both copies of domain 3 followed by the second copy of > domain 2. This all worked well, giving clear solutions. No intermediate > refinement necessary, though if it hadn’t worked it would have been possible > to refine the intermediate solution and grab the improved models for any > missing domains. Fifty cycles of jelly-body refinement of the final MR > solution in Refmac gave an R-free of 43.7%. > > Note that in these jobs Phaser struggled with the second copy of domain 1, > only pulling it out in the refinement step, probably because the B-factor had > to be increased substantially to agree with the data. Another search order > might have been more efficient (maybe 1, 2, 3, 1 2, 3), but this worked. > Note that you can over-ride the automatic choice of search order, but Phaser > will by default choose its own search order (which is usually but not always > better). > > Hope that helps on the Phaser front! > > Best wishes > > Randy Read > >> On 11 May 2021, at 02:53, Lande wrote: >> >> Hi everyone, I started my work on data processing in protein crystallography >> about half year ago and have done a fair number of “easy” structures. >> Recently, I processed a tutorial structure containing 3 different domains >> and two chains of them (in particular, it’s “ mth685, high resolution data” >> from ccp4 legacy tutorial), but it was failed during MR process. >> >> According to this tutorial, I did MR with MOLREP by adding each domain in >> order. The first and second added domains, which are domain 1 from chain A >> and domain 2 from chain B, are correct; however, when I tried to add the >> next domain, R work/R free did not drop and clearly MOLREP did not find the >> correct solution according to the density map. I also tried Phaser but the >> result was the same. >> >> screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR >> >> https://ibb.co/YB2JnRR;>> src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png; >> alt="screenshot-on-wrong-domain" border="0">> href='https://imgbb.com/'>pio pio 3 menu >> >> In addition, I found an article on that data >> (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I >> failed to get the same result expect the first two domains. >> >> I wonder if anyone has done this tutorial before or has faced similar >> problems in his own work. I have no idea on how to deal with it. Any >> suggestions or comments will be helpful. Thanks in ahead. >> >> Link for that tutorial: >> ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf >> >> >> To unsubscribe from
Re: [ccp4bb] MR problem in muti-domain structure
Hi, I had a hard time tracking down the data in the legacy CCP4 site but eventually found it! Then I tried solving the MR tutorial case with Phaser. Instead of using the tutorial domain 1, 2 and 3 files, I prepared them from the deposited PDB file using a sequence alignment followed by using sculptor to trim off non-conserved ends of side chains and any missing loops. Then I did a default run of Phaser, searching for 2 copies of each domain. Phaser automatically set the search order to domain 1, then domain 2 then domain 3 (they have similar sequence identities and that sorts them from biggest to smallest). It was clear from the statistics (increase in log-likelihood gain or LLG and TFZ score) that both copies of domain 1 were correct, the first copy of domain 2, and both copies of domain 3, even though an incorrect placement of domain 2 occurred before looking for domain 3. Unfortunately, the second copy of domain 3 was rejected because of packing problems with the badly-placed domain 2 copy. So I went back to the partial solution with 2 copies of domain 1 and one of domain 2, then added both copies of domain 3 followed by the second copy of domain 2. This all worked well, giving clear solutions. No intermediate refinement necessary, though if it hadn’t worked it would have been possible to refine the intermediate solution and grab the improved models for any missing domains. Fifty cycles of jelly-body refinement of the final MR solution in Refmac gave an R-free of 43.7%. Note that in these jobs Phaser struggled with the second copy of domain 1, only pulling it out in the refinement step, probably because the B-factor had to be increased substantially to agree with the data. Another search order might have been more efficient (maybe 1, 2, 3, 1 2, 3), but this worked. Note that you can over-ride the automatic choice of search order, but Phaser will by default choose its own search order (which is usually but not always better). Hope that helps on the Phaser front! Best wishes Randy Read > On 11 May 2021, at 02:53, Lande wrote: > > Hi everyone, I started my work on data processing in protein crystallography > about half year ago and have done a fair number of “easy” structures. > Recently, I processed a tutorial structure containing 3 different domains and > two chains of them (in particular, it’s “ mth685, high resolution data” from > ccp4 legacy tutorial), but it was failed during MR process. > > According to this tutorial, I did MR with MOLREP by adding each domain in > order. The first and second added domains, which are domain 1 from chain A > and domain 2 from chain B, are correct; however, when I tried to add the next > domain, R work/R free did not drop and clearly MOLREP did not find the > correct solution according to the density map. I also tried Phaser but the > result was the same. > > screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR > > https://ibb.co/YB2JnRR;> src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png; > alt="screenshot-on-wrong-domain" border="0"> href='https://imgbb.com/'>pio pio 3 menu > > In addition, I found an article on that data > (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I > failed to get the same result expect the first two domains. > > I wonder if anyone has done this tutorial before or has faced similar > problems in his own work. I have no idea on how to deal with it. Any > suggestions or comments will be helpful. Thanks in ahead. > > Link for that tutorial: > ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] MR problem in muti-domain structure
Where did you find the tutorial files?? Eleanor On Tue, 11 May 2021 at 10:24, Jon Cooper < 488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > Hello, just to double check, I assume that you input the correctly > oriented and positioned structure of each domain from successive runs as a > 'fixed' model? Cheers, Jon.C. > > Sent from ProtonMail mobile > > > > Original Message > On 11 May 2021, 02:53, Lande < fu...@northeastern.edu> wrote: > > > Hi everyone, I started my work on data processing in protein > crystallography about half year ago and have done a fair number of “easy” > structures. Recently, I processed a tutorial structure containing 3 > different domains and two chains of them (in particular, it’s “ mth685, > high resolution data” from ccp4 legacy tutorial), but it was failed during > MR process. > > According to this tutorial, I did MR with MOLREP by adding each domain in > order. The first and second added domains, which are domain 1 from chain A > and domain 2 from chain B, are correct; however, when I tried to add the > next domain, R work/R free did not drop and clearly MOLREP did not find the > correct solution according to the density map. I also tried Phaser but the > result was the same. > > screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR > > https://ibb.co/YB2JnRR;>https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png; > alt="screenshot-on-wrong-domain" border="0"> href='https://imgbb.com/'>pio pio 3 menu > > In addition, I found an article on that data ( > https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I > failed to get the same result expect the first two domains. > > I wonder if anyone has done this tutorial before or has faced similar > problems in his own work. I have no idea on how to deal with it. Any > suggestions or comments will be helpful. Thanks in ahead. > > Link for that tutorial: > ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] MR problem in muti-domain structure
Hello, just to double check, I assume that you input the correctly oriented and positioned structure of each domain from successive runs as a 'fixed' model? Cheers, Jon.C. Sent from ProtonMail mobile Original Message On 11 May 2021, 02:53, Lande wrote: > Hi everyone, I started my work on data processing in protein crystallography > about half year ago and have done a fair number of “easy” structures. > Recently, I processed a tutorial structure containing 3 different domains and > two chains of them (in particular, it’s “ mth685, high resolution data” from > ccp4 legacy tutorial), but it was failed during MR process. > > According to this tutorial, I did MR with MOLREP by adding each domain in > order. The first and second added domains, which are domain 1 from chain A > and domain 2 from chain B, are correct; however, when I tried to add the next > domain, R work/R free did not drop and clearly MOLREP did not find the > correct solution according to the density map. I also tried Phaser but the > result was the same. > > screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR > > https://ibb.co/YB2JnRR;> src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png; > alt="screenshot-on-wrong-domain" border="0"> href='https://imgbb.com/'>pio pio 3 menu > > In addition, I found an article on that data > (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I > failed to get the same result expect the first two domains. > > I wonder if anyone has done this tutorial before or has faced similar > problems in his own work. I have no idea on how to deal with it. Any > suggestions or comments will be helpful. Thanks in ahead. > > Link for that tutorial: > ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf > > --- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] MR problem in muti-domain structure
Hi everyone, I started my work on data processing in protein crystallography about half year ago and have done a fair number of “easy” structures. Recently, I processed a tutorial structure containing 3 different domains and two chains of them (in particular, it’s “ mth685, high resolution data” from ccp4 legacy tutorial), but it was failed during MR process. According to this tutorial, I did MR with MOLREP by adding each domain in order. The first and second added domains, which are domain 1 from chain A and domain 2 from chain B, are correct; however, when I tried to add the next domain, R work/R free did not drop and clearly MOLREP did not find the correct solution according to the density map. I also tried Phaser but the result was the same. screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR pio pio 3 menu In addition, I found an article on that data (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I failed to get the same result expect the first two domains. I wonder if anyone has done this tutorial before or has faced similar problems in his own work. I have no idea on how to deal with it. Any suggestions or comments will be helpful. Thanks in ahead. Link for that tutorial: ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
Re: [ccp4bb] MR problem
Dear Ping, First thing to ask: Do you know with 100% certainty if your crystals contain a complex? If your crystals are large enough (and you have more than one) you can check this on SDS-PAGE or with mass-spec as long as you are sure to remove all surrounding mother liquid that may contaminate your result. Both proteins should be observed very clearly (80%-20% would be a contamination rather than a co-crystal!). Experience tells us that even with high affinity (nanomolar or better) and preformed complexes, still often only one of the two partners will crystallize. Second: does your second protein contain more than one domain? If so, there may be domain movements that obscure the desnity or even lead to a completely wrong MR solution. Try rigid body refinement with individual domains first. If this does not work, try MR with the individual domains. The MR solution of your second protein may also be wrong for any other reason. Check if you can trust the solution based on the statistics provided in the log file. If the LLG is only marginally better after adding the second protein, it will probably be wrong. Check also if there is no real solution hanging around but rejected by the program because (for example) it has just one clash too many. Remy Loris Vrije Universiteit Brussel and VIB On 04/12/11 05:18, Ping Wang wrote: Dear all, Recently I have a dataset with a protein complex including two proteins. Each structure of the single protein is available. When I use phaser to solve the phase, one protein got good density while the other was not so ideal. In order to get a better phase, I try to cut the flexible region of the bad protein. But the result shows no improvement. Does anyone have some suggestion for me? Thanks! Ping
Re: [ccp4bb] MR problem
Dear Ping, First thing to ask: Do you know with 100% certainty if your crystals contain a complex? If your crystals are large enough (and you have more than one) you can check this on SDS-PAGE or with mass-spec as long as you are sure to remove all surrounding mother liquid that may contaminate your result. Both proteins should be observed very clearly (80%-20% would be a contamination rather than a co-crystal!). Experience tells us that even with high affinity (nanomolar or better) and preformed complexes, still often only one of the two partners will crystallize. Second: does your second protein contain more than one domain? If so, there may be domain movements that obscure the desnity or even lead to a completely wrong MR solution. Try rigid body refinement with individual domains first. If this does not work, try MR with the individual domains. The MR solution of your second protein may also be wrong for any other reason. Check if you can trust the solution based on the statistics provided in the log file. If the LLG is only marginally better after adding the second protein, it will probably be wrong. Check also if there is no real solution hanging around but rejected by the program because (for example) it has just one clash too many. Remy Loris Vrije Universiteit Brussel and VIB On 04/12/11 05:18, Ping Wang wrote: Dear all, Recently I have a dataset with a protein complex including two proteins. Each structure of the single protein is available. When I use phaser to solve the phase, one protein got good density while the other was not so ideal. In order to get a better phase, I try to cut the flexible region of the bad protein. But the result shows no improvement. Does anyone have some suggestion for me? Thanks! Ping
[ccp4bb] MR problem
Dear all, Recently I have a dataset with a protein complex including two proteins. Each structure of the single protein is available. When I use phaser to solve the phase, one protein got good density while the other was not so ideal. In order to get a better phase, I try to cut the flexible region of the bad protein. But the result shows no improvement. Does anyone have some suggestion for me? Thanks! Ping
[ccp4bb] MR problem
Hi there I have a data of heterotrimer protein complex at 2.9A resolution. One protein consists of two domains. I tried phaser as well as molrep which gives a solution with only one domain. Rotation function and translation function were found to be fine for these solutions. I tried to find missing domain of the protein after fixing one of the domain or the other partner proteins using whole part or various truncations of missing domain. I also tried to find and build missing domain using Rosetta with the solutions of Molrep or Phaser as template. But, there were no solutions, or the solutions are clashed with other domain or proteins. Furthermore, R and Rfree is 30 and 40, respectively, and I could not reduce them further. There was almost no electron density map in the empty space so that I could not model manually. Plz guide me ,how can i look for the missing domain in the protein. regards -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] MR problem
Dear Intekhab, Before putting a lot of effort in trying to find the missing domain, you first should make sure it is there. It may be that only two proteins of the heterotrimer were packed in the crystal, or that one domain got clipped off during crystallization. The way to do it, is to run an sds page gel of a crystal, or a number of crystals if they are too small. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of intekhab alam Sent: Monday, August 22, 2011 9:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] MR problem Hi there I have a data of heterotrimer protein complex at 2.9A resolution. One protein consists of two domains. I tried phaser as well as molrep which gives a solution with only one domain. Rotation function and translation function were found to be fine for these solutions. I tried to find missing domain of the protein after fixing one of the domain or the other partner proteins using whole part or various truncations of missing domain. I also tried to find and build missing domain using Rosetta with the solutions of Molrep or Phaser as template. But, there were no solutions, or the solutions are clashed with other domain or proteins. Furthermore, R and Rfree is 30 and 40, respectively, and I could not reduce them further. There was almost no electron density map in the empty space so that I could not model manually. Plz guide me ,how can i look for the missing domain in the protein. regards -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] MR problem
As already noted, it is possible that the other domain is not present, or not ordered. Try refining a bit without it, and see what your stats look like. It is also possible that it is present, but minor clashes are causing good solutions to be rejected. Go into the output file and examine the TF scores before overlap rejection. The R and Rfree sound pretty good for an MR solution. About the failure of these numbers to come down, I'm not sure what you tried. Refinement with REFMAC5 should bring them down. You should see indications in the 2fo-fc and fo-fc maps that they are accurate - i.e. that there are real features showing up that are not in your model. You can test that also by deleting some prominent side chains. On 08/22/11 03:12, intekhab alam wrote: Hi there I have a data of heterotrimer protein complex at 2.9A resolution. One protein consists of two domains. I tried phaser as well as molrep which gives a solution with only one domain. Rotation function and translation function were found to be fine for these solutions. I tried to find missing domain of the protein after fixing one of the domain or the other partner proteins using whole part or various truncations of missing domain. I also tried to find and build missing domain using Rosetta with the solutions of Molrep or Phaser as template. But, there were no solutions, or the solutions are clashed with other domain or proteins. Furthermore, R and Rfree is 30 and 40, respectively, and I could not reduce them further. There was almost no electron density map in the empty space so that I could not model manually. Plz guide me ,how can i look for the missing domain in the protein. regards -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
[ccp4bb] MR problem in determining the number of identical molecules in ASU
Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
Well P4 isnt a subgroup of P43212 - you would need P43 MR programs will often let you test several spacegroups. See Phaser MR or MOLREP - I would try that and choose the best Eleanor On 02/16/2011 02:48 PM, Ting-Wei Jiang wrote: Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
Ting-Wei Jiang wrote: Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei For molecular replacement: it does not matter if you carry out the rotation search in P422 or P43212. To understand this, ask youself the following question: what is the symmetry of the Patterson compared to that of the crystal? The symmetry of the Patterson is obtained from the symmetry of the crystal by converting all translation operators to the corresponding non-translation operators and adding a centre of symmetry. Hence for the rotation function, the symmetry of the Patterson will be P4/mmm for space group P422 and P4/mmm for P43212. Hence no difference for the rotation function. Different for P4 though: the symmetry of the Patterson there is P4/m. Hence the rotation function can be computed once for all space groups that have Patterson symmetry P4/mmm. No need to repeat the rotation function calculations several times. The situation is different for the translation function however. This is the stage at which you distinguish between all these space groups (including the pair of enantiomorphs P41212 and p43212). Failure to assign the proper space group cannot give you a satisfactory model. HTH, Fred.
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
I think the question was not concerning right vs wrong space group, but right vs a lower symmetry superset space group - P4322 vs P43 for example. I would also be interested in the answer. At first glance it appears harder in the lower symmetry because with each monomer you would be searching for a smaller fraction of the contents of the AU. But if the rotation function doesn't consider space group symmetry (?) does that matter? eab Vellieux Frederic wrote: Ting-Wei Jiang wrote: Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei For molecular replacement: it does not matter if you carry out the rotation search in P422 or P43212. To understand this, ask youself the following question: what is the symmetry of the Patterson compared to that of the crystal? The symmetry of the Patterson is obtained from the symmetry of the crystal by converting all translation operators to the corresponding non-translation operators and adding a centre of symmetry. Hence for the rotation function, the symmetry of the Patterson will be P4/mmm for space group P422 and P4/mmm for P43212. Hence no difference for the rotation function. Different for P4 though: the symmetry of the Patterson there is P4/m. Hence the rotation function can be computed once for all space groups that have Patterson symmetry P4/mmm. No need to repeat the rotation function calculations several times. The situation is different for the translation function however. This is the stage at which you distinguish between all these space groups (including the pair of enantiomorphs P41212 and p43212). Failure to assign the proper space group cannot give you a satisfactory model. HTH, Fred.
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
In my experience, the success of molecular replacement depends primarily on the quality of the model. Thus if your model is good, even P1 will work. Two extreme cases that I encountered were searching with a monomer for what turned out to be 4 tetramers (thus first search only accounted for 1/16 of the asu) and getting a solid solution from 40% complete dataset at 3A-ish resolution with the search model being 25% of the asu. On the other end of the spectrum, I've seen molecular replacement fail to find the second copy using the first copy from the final refined structure. Bottomline: the limit to which MR can be pushed is inversely proportional to the quality of your model. Your question is that kind which is easier to answer if you specify what is that you are trying to accomplish. -- Hurry up, before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
The space group in which you do the RF makes no difference (assuming the model in each case is identical of course) because you're superposing the same amount of scattering matter: it has nothing to do with the fraction of the contents of the AU. In the high symmetry space group with say 1 instance of a differently oriented model per AU you are still only superposing 1 AU at a time, and each AU gives a separate peak in the RF (some of which may be related by the symmetry of the RF and some not: e.g. the 3-fold related peaks in cubic space groups). In the low symmetry space group you still get 1 peak per differently oriented instance of the model. This assumes that the high symmetry space group is the correct one; if it turns out that the subgroup is correct then of course that will give the better solution. -- Ian On Wed, Feb 16, 2011 at 5:57 PM, Edward A. Berry ber...@upstate.edu wrote: I think the question was not concerning right vs wrong space group, but right vs a lower symmetry superset space group - P4322 vs P43 for example. I would also be interested in the answer. At first glance it appears harder in the lower symmetry because with each monomer you would be searching for a smaller fraction of the contents of the AU. But if the rotation function doesn't consider space group symmetry (?) does that matter? eab Vellieux Frederic wrote: Ting-Wei Jiang wrote: Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei For molecular replacement: it does not matter if you carry out the rotation search in P422 or P43212. To understand this, ask youself the following question: what is the symmetry of the Patterson compared to that of the crystal? The symmetry of the Patterson is obtained from the symmetry of the crystal by converting all translation operators to the corresponding non-translation operators and adding a centre of symmetry. Hence for the rotation function, the symmetry of the Patterson will be P4/mmm for space group P422 and P4/mmm for P43212. Hence no difference for the rotation function. Different for P4 though: the symmetry of the Patterson there is P4/m. Hence the rotation function can be computed once for all space groups that have Patterson symmetry P4/mmm. No need to repeat the rotation function calculations several times. The situation is different for the translation function however. This is the stage at which you distinguish between all these space groups (including the pair of enantiomorphs P41212 and p43212). Failure to assign the proper space group cannot give you a satisfactory model. HTH, Fred.
Re: [ccp4bb] MR problem in determining the number of identical molecules in ASU
Another cause of difficulty - nothing really to do with the spacegroup selection - is when one copy of the model has much higher B factors than others. Most MR searches assume that the copies contribute more or less equally to scattering. If you assign too high a symmetry this will make MR less likely to work. This usually happens when the data is twinned. Eleanor On 02/16/2011 05:57 PM, Edward A. Berry wrote: I think the question was not concerning right vs wrong space group, but right vs a lower symmetry superset space group - P4322 vs P43 for example. I would also be interested in the answer. At first glance it appears harder in the lower symmetry because with each monomer you would be searching for a smaller fraction of the contents of the AU. But if the rotation function doesn't consider space group symmetry (?) does that matter? eab Vellieux Frederic wrote: Ting-Wei Jiang wrote: Dear experts, Sorry for a simple question but confusing me so much! Does it make bad effects on determining the number of identical molecules in ASU by choosing low symmetry space group. For example,If I choose lowest symmetry(p4) instead of higher one(p43212). Does it cause any trouble in determining structure while we try to find the solution by molrep. If molrep compare the patterson of observe and model in P1 space group, it doesn't matter in choosing a lower s.g.? Any suggestion will be appreciated. Thanks in advance. Ting wei For molecular replacement: it does not matter if you carry out the rotation search in P422 or P43212. To understand this, ask youself the following question: what is the symmetry of the Patterson compared to that of the crystal? The symmetry of the Patterson is obtained from the symmetry of the crystal by converting all translation operators to the corresponding non-translation operators and adding a centre of symmetry. Hence for the rotation function, the symmetry of the Patterson will be P4/mmm for space group P422 and P4/mmm for P43212. Hence no difference for the rotation function. Different for P4 though: the symmetry of the Patterson there is P4/m. Hence the rotation function can be computed once for all space groups that have Patterson symmetry P4/mmm. No need to repeat the rotation function calculations several times. The situation is different for the translation function however. This is the stage at which you distinguish between all these space groups (including the pair of enantiomorphs P41212 and p43212). Failure to assign the proper space group cannot give you a satisfactory model. HTH, Fred.
Re: [ccp4bb] MR- Problem-76 % seqeunce identity-no solution
Meetmr Ss wrote: Dear all, I struck with MR problem. MY target has 76% sequence identity with the model. I tried Phaser, AMoRe and Molrep. None of them gave me satisfactory solution. If you have any suggestions and or New programs I would like to try. Thanks somu Is your space group correct? Eleanor
Re: [ccp4bb] MR- Problem-76 % seqeunce identity-no solution
Dear all and specially Eleanor, Thank each and everyone who responded to my query. All your suggestions made me to think in all possible dimensions. I have good news to share with all of you. Balbes gave me marginal solution with model which had 29% sequence identity. At present I am doing model building and refinement with R-free 47 and R-factor 45. My special thanks to Garib…. (I don’t think I should mention once again for what ..) and his web server. Wish you a happy new year to all the members. With regards somu From: Eleanor Dodson c...@ysbl.york.ac.uk To: Meetmr Ss meetm...@yahoo.com Cc: CCP4BB@jiscmail.ac.uk Sent: Tuesday, December 30, 2008 2:04:24 PM Subject: Re: [ccp4bb] MR- Problem-76 % seqeunce identity-no solution Meetmr Ss wrote: Dear all, I struck with MR problem. MY target has 76% sequence identity with the model. I tried Phaser, AMoRe and Molrep. None of them gave me satisfactory solution. If you have any suggestions and or New programs I would like to try. Thanks somu Is your space group correct? Eleanor
Re: [ccp4bb] MR- Problem-76 % sequence identity-no solution
where to start? 1st what is the resolution and Rfree of your model? Have you cut the model down to 1 molecule in the ASU, and what does Matthew's predict for your crystals as far as number in ASU? If all of those answers are reasonable, floppy regions can often cause failure in MR. I would cut 15-20 residues of the N and C terminus and try in phaser and Molrep again. Kevin P Madauss V-161a US Structural Biology GlaxoSmithKline Office: 919.483.3759 5 Moore Drive Cell: 919.924.6436 RTP, NC 27709 Meetmr Ss meetm...@yahoo.com Sent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK 24-Dec-2008 02:07 Please respond to Meetmr Ss meetm...@yahoo.com To CCP4BB@JISCMAIL.AC.UK cc Subject [ccp4bb] MR- Problem-76 % seqeunce identity-no solution Dear all, I struck with MR problem. MY target has 76% sequence identity with the model. I tried Phaser, AMoRe and Molrep. None of them gave me satisfactory solution. If you have any suggestions and or New programs I would like to try. Thanks somu
Re: [ccp4bb] MR- Problem-76 % seqeunce identity-no solution
I had the same problem with an even higher %identity once (80%), eventually resorted to SIRAS phasing with a mercury compound, and I found a case of domain swapping creating a novel dimer interface. Flip
[ccp4bb] MR- Problem-76 % seqeunce identity-no solution
Dear all, I struck with MR problem. MY target has 76% sequence identity with the model. I tried Phaser, AMoRe and Molrep. None of them gave me satisfactory solution. If you have any suggestions and or New programs I would like to try. Thanks somu
Re: [ccp4bb] MR problem --molrep
Hi there, I think 55% completeness is insufficient - you really need to collect more data. Take a look at this movie from James Holton's website: http://ucxray.berkeley.edu/~jamesh/movies/osc.mpeg Stop the movie at 55% and check out how lousy the maps are compared to 95-100% - and I think these maps are generated with perfect phases, at 1.5A resolution. Molecular replacement programs and refinement programs are going to struggle with just over half a dataset - this is also why your R-factors are sky high. In addition, I know of cases where reviewers have cast doubt over structures (of a point mutant) with data of 80% completeness. I don't think a 55% complete dataset is going to get out there. I really think more data is the only way our of your predicament. I hope you have a few crystals ready to go! Good Luck, David. 2008/7/27 Carl Soja [EMAIL PROTECTED]: HI all, Thank you very much for your kindly answer. I also tried to use different program like phaser, amore, and some MR servers. actualy, I don`t know how many molecule in AU clearly. 2-6 molecules in 76%- 35% of solvent content. 4mer is 50% of solvent content. the homolgous structure has high solvent content over 70%. when i used phaser program to do MR, I can get several solutions but not good structure conformation. I don`t know how to use phaser do automatic more than 2 molecules search like molrep program. As spacegroup, I checked the C222 and C2221. the phaser program can give both solutions when I changed the search model or maximum solution limit. my question is how i can search more than 2 molecules by phaser program like molrp only input over 2. by the way, my diffraction data have low completeness(55%) and low redandancy(1.8). Thanks in advance!! carl roja -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] MR problem --molrep
HI all, Thank you very much for your kindly answer. I also tried to use different program like phaser, amore, and some MR servers. actualy, I don`t know how many molecule in AU clearly. 2-6 molecules in 76%- 35% of solvent content. 4mer is 50% of solvent content. the homolgous structure has high solvent content over 70%. when i used phaser program to do MR, I can get several solutions but not good structure conformation. I don`t know how to use phaser do automatic more than 2 molecules search like molrep program. As spacegroup, I checked the C222 and C2221. the phaser program can give both solutions when I changed the search model or maximum solution limit. my question is how i can search more than 2 molecules by phaser program like molrp only input over 2. by the way, my diffraction data have low completeness(55%) and low redandancy(1.8). Thanks in advance!! carl roja
[ccp4bb] MR problem --molrep
Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja
Re: [ccp4bb] MR problem --molrep
Hi, I dare to say about the possible way to do molrep from my recent experience. You can choose rotation and translation function job at first and do the self rotation fuction (for multimer) after that.Each of these run will generate two different outputs *_rf.molrep_rf ans *_srf.molrep_rf. Make the third run inputting both of these peak values. Give NMON as 4. You can also use the protein sequence hare. Then refine it with Refmac, that again should decrease your R factor. I will be highly benefitted if some one point out my mistake. regards Debajyoti On Fri, 25 Jul 2008 Carl Soja wrote : Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja
Re: [ccp4bb] MR problem --molrep
Dear Debajyoti Thank you very much for your help. I input the each peak values as 10 and carried out the rotation and translation function again. I didn't get a improved solution by molrep. can i edit the self-rotaion function and translation function solution file as input ? best regards, carl soja Hi, I dare to say about the possible way to do molrep from my recent experience. You can choose rotation and translation function job at first and do the self rotation fuction (for multimer) after that.Each of these run will generate two different outputs *_rf.molrep_rf ans *_srf.molrep_rf. Make the third run inputting both of these peak values. Give NMON as 4. You can also use the protein sequence hare. Then refine it with Refmac, that again should decrease your R factor. I will be highly benefitted if some one point out my mistake. regards Debajyoti On Fri, 25 Jul 2008 Carl Soja wrote : Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja
Re: [ccp4bb] MR problem --molrep
Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation-translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R-factor under 50%. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] MR problem --molrep
Perhaps obvious - are you sure the space group is C222 not C2221? Phil On 25 Jul 2008, at 14:19, Roger Rowlett wrote: Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation- translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R- factor under 50%. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] MR problem --molrep
In addition to Bert's statistics argument (I've never had the pleasure of working w/ a C222 crystal), do check your self-Patterson map. I recently had a difficult MR case; the crystal masqueraded as P21212 or P2221, but it was actually P212121 with the two molecules in the AU related by a non-crystallographic translation. Dave Borhani -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Roger Rowlett Sent: Friday, July 25, 2008 10:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] MR problem --molrep Good point. One should always check all the screw axis combos. Not fun for P422, but Phaser makes this very easy, as it will do all the possible screw axis combinations in one job. The correct space group and MR solution is usually very obvious if the model is acceptable and the MR parameters have been set reasonably. I think Open-EPMR will also examine screw axis combinations now, but I've never used it to do this. Cheers, Roger Rowlett Phil Evans wrote: Perhaps obvious - are you sure the space group is C222 not C2221? Phil On 25 Jul 2008, at 14:19, Roger Rowlett wrote: Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation- translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R- factor under 50%. Cheers, -- -- -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
