Re: [ccp4bb] Finding partial occupancy monomer by MR ?
Hi Phil, It’s always a difficult balance between looking hard in a difficult case and possibly finding an answer with a weak signal, and stopping when the search is really unlikely to succeed. Phaser perhaps errs too much toward trying really hard. There’s one really blunt tool in Phaser, the KILL TIME command that tells it to stop after a certain number of minutes have passed. I don’t particularly like or recommend that option. A more nuanced approach is to use the PURGE commands to control the amount of branching that takes place in the searches — this is where the CPU time can really build up. What I like is to set something like PURGE RNP NUM 20, in which case only 20 possible partial solutions after refinement are preserved as starting points to look for the next copy. You can choose the number depending on your case and how long each rotation search and each translation search takes. There are PURGE commands to control things at the rotation and translation stages as well, but I usually find that the RNP one gives enough control. Best wishes, Randy > On 10 Dec 2020, at 16:00, Phil Jeffrey wrote: > > Thanks for the suggestions. > > The idea that it's related to a trigonal space group and twinning or pseudo > space group is an interesting one, but this is C2221 and the intensity stats > don't show twinning. Twinned P21 -> C2221 doesn't solve the non-unit > occupancy in this case. Since the other monomers are full-occupancy it can't > be 3 overlapping dimers so the phenomenon is rather unusual in my finite > experience. (Also only one set of Se peaks for this 4th monomer). > > I used Herman's suggestion of finding 3 monomers first (with very large > RFZ/TFZ/LLG since the monomers had been refined against the data) since > that's very fast. And then Phaser took a long while to not find the 4th > monomer. Once I figure out how to make modern versions of phaser to "fail > quickly" like the older versions I'll scan a range of homology% and see if > that changes anything. > > Phil > > > On 12/10/20 9:46 AM, Schreuder, Herman /DE wrote: >> Dear Phil, >> 0.32 is awfully close to 1/3, which brings a nice mathematical puzzle to my >> mind to see if the 1/3 occupancy is somehow related to the 3 fully occupied >> monomers... It may also be related to a (trigonal??) space group... >> You probably have already tried it, but phaser has the option to give it >> already solved molecules and ask it to search for additional molecules. Here >> I would indeed lower the expected % homology significantly, to crudely >> compensate for the low occupancy. In contrast to the advice of Dale, I would >> play around with the % homology to find the value which works best. >> My 2 cents, >> Herman >> -Ursprüngliche Nachricht- >> Von: CCP4 bulletin board Im Auftrag von Phil Jeffrey >> Gesendet: Donnerstag, 10. Dezember 2020 14:49 >> An: CCP4BB@JISCMAIL.AC.UK >> Betreff: [ccp4bb] Finding partial occupancy monomer by MR ? >> Preamble: >> I have an interesting crystal form with 3 monomers (~400aa) at full >> occupancy and apparently one at much reduced occupancy. It was built >> recently from Se-SAD and was in moderately good condition: Rfree=32% for >> trimer, 2.6 Å. In recent refinement cycles it became obvious that there was >> a 4th monomer in a region of weaker/choppy 2Fo-Fc and Fo-Fc density that >> corresponded to a "confusing" set of low-occupancy SeMet sites found by >> SHELXD and Phaser-EP. The experimental map was bad in that region and was >> probably flattened during density modification anyway, in retrospect. >> Question: >> Phaser failed to find the 4th monomer after trivially finding the other >> 3 with a recent version of the monomer. I'm wondering if there's a way to >> indicate "this one is partial occupancy" to Phaser, or if there's a way to >> improve the odds of success beyond just lowering the expected % homology. >> Or if anyone has had success with other programs. This is perhaps a rare >> edge case but I naively expected Phaser to work. >> In the end I used the weak SeMet sites to locate the monomer and the >> occupancy appears to be around 0.32 in refinement. >> Cheers, >> Phil Jeffrey >> Princeton >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1data=04%7C01%7CHerman.Schreuder%40SANOFI.COM%7C5ee2ee2e87f04d853f0408d89d126615%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C1%7C63743205007789%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=G28AUNQrAgQYblmaYBVnXESTXiekmWzfTLPMMX%2B%2BOgw%3Dreserved=0 >> This message was issued to members of >>
Re: [ccp4bb] Finding partial occupancy monomer by MR ?
Thanks for the suggestions. The idea that it's related to a trigonal space group and twinning or pseudo space group is an interesting one, but this is C2221 and the intensity stats don't show twinning. Twinned P21 -> C2221 doesn't solve the non-unit occupancy in this case. Since the other monomers are full-occupancy it can't be 3 overlapping dimers so the phenomenon is rather unusual in my finite experience. (Also only one set of Se peaks for this 4th monomer). I used Herman's suggestion of finding 3 monomers first (with very large RFZ/TFZ/LLG since the monomers had been refined against the data) since that's very fast. And then Phaser took a long while to not find the 4th monomer. Once I figure out how to make modern versions of phaser to "fail quickly" like the older versions I'll scan a range of homology% and see if that changes anything. Phil On 12/10/20 9:46 AM, Schreuder, Herman /DE wrote: Dear Phil, 0.32 is awfully close to 1/3, which brings a nice mathematical puzzle to my mind to see if the 1/3 occupancy is somehow related to the 3 fully occupied monomers... It may also be related to a (trigonal??) space group... You probably have already tried it, but phaser has the option to give it already solved molecules and ask it to search for additional molecules. Here I would indeed lower the expected % homology significantly, to crudely compensate for the low occupancy. In contrast to the advice of Dale, I would play around with the % homology to find the value which works best. My 2 cents, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board Im Auftrag von Phil Jeffrey Gesendet: Donnerstag, 10. Dezember 2020 14:49 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Finding partial occupancy monomer by MR ? Preamble: I have an interesting crystal form with 3 monomers (~400aa) at full occupancy and apparently one at much reduced occupancy. It was built recently from Se-SAD and was in moderately good condition: Rfree=32% for trimer, 2.6 Å. In recent refinement cycles it became obvious that there was a 4th monomer in a region of weaker/choppy 2Fo-Fc and Fo-Fc density that corresponded to a "confusing" set of low-occupancy SeMet sites found by SHELXD and Phaser-EP. The experimental map was bad in that region and was probably flattened during density modification anyway, in retrospect. Question: Phaser failed to find the 4th monomer after trivially finding the other 3 with a recent version of the monomer. I'm wondering if there's a way to indicate "this one is partial occupancy" to Phaser, or if there's a way to improve the odds of success beyond just lowering the expected % homology. Or if anyone has had success with other programs. This is perhaps a rare edge case but I naively expected Phaser to work. In the end I used the weak SeMet sites to locate the monomer and the occupancy appears to be around 0.32 in refinement. Cheers, Phil Jeffrey Princeton To unsubscribe from the CCP4BB list, click the following link: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1data=04%7C01%7CHerman.Schreuder%40SANOFI.COM%7C5ee2ee2e87f04d853f0408d89d126615%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C1%7C63743205007789%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=G28AUNQrAgQYblmaYBVnXESTXiekmWzfTLPMMX%2B%2BOgw%3Dreserved=0 This message was issued to members of https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.jiscmail.ac.uk%2FCCP4BBdata=04%7C01%7CHerman.Schreuder%40SANOFI.COM%7C5ee2ee2e87f04d853f0408d89d126615%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C1%7C63743205007789%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=WitzjV%2F3hzx1SzMmmDzKVX56uVBD1fXluDDAcyY8Y1g%3Dreserved=0, a mailing list hosted by https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.jiscmail.ac.uk%2Fdata=04%7C01%7CHerman.Schreuder%40SANOFI.COM%7C5ee2ee2e87f04d853f0408d89d126615%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C1%7C63743205007789%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=wv3OjpY8AGsTj0RfiiEMjWPLDD5QOMvfV1TwcaRXczs%3Dreserved=0, terms & conditions are available at https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fpolicyandsecurity%2Fdata=04%7C01%7CHerman.Schreuder%40SANOFI.COM%7C5ee2ee2e87f04d853f0408d89d126615%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C1%7C63743205007789%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=mEy2Z2rqn8IBmLklgmDe0%2BZOXq4gcLHqSqze6fW%2Fhx4%3Dreserved=0 To unsubscribe from the CCP4BB list, click the following link:
Re: [ccp4bb] Finding partial occupancy monomer by MR ?
Dear Phil, What is the space group? Could it be that the 4th monomer makes also a trimer by symmetry, in an alternative SG? A. > On 10 Dec 2020, at 14:49, Phil Jeffrey wrote: > > Preamble: > I have an interesting crystal form with 3 monomers (~400aa) at full occupancy > and apparently one at much reduced occupancy. It was built recently from > Se-SAD and was in moderately good condition: Rfree=32% for trimer, 2.6 Å. In > recent refinement cycles it became obvious that there was a 4th monomer in a > region of weaker/choppy 2Fo-Fc and Fo-Fc density that corresponded to a > "confusing" set of low-occupancy SeMet sites found by SHELXD and Phaser-EP. > The experimental map was bad in that region and was probably flattened during > density modification anyway, in retrospect. > > Question: > Phaser failed to find the 4th monomer after trivially finding the other 3 > with a recent version of the monomer. I'm wondering if there's a way to > indicate "this one is partial occupancy" to Phaser, or if there's a way to > improve the odds of success beyond just lowering the expected % homology. Or > if anyone has had success with other programs. This is perhaps a rare edge > case but I naively expected Phaser to work. > > In the end I used the weak SeMet sites to locate the monomer and the > occupancy appears to be around 0.32 in refinement. > > Cheers, > Phil Jeffrey > Princeton > > > > 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/ 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/
