[ccp4bb] NCS restraints of domains
I am refining a low (3A) resolution structure of a 3 domain protein. There are 4 copies in the ASU. I have been applying tight NCS restraints by domain in refmac and have pulled the weak MR solution down to Rfree below 30 (just). However my question is that in 2 of the 4 copies one of the domains is very poorly resolved. I can lower Rfree by around 0.5% by omitting the domains from the PDB entirely or not applying the NCS restraints to these copies of the domain. Clearly they are there and should resemble the moderately well resolved copies by coordinates but the way Bfactor restraints are applied between NCS copies seems to be the issue. If tight restraints are included the B factors are much lower (30-40) rather than 60-80 for the poor domains. I was wondering if there is a theoretically correct way to treat this? Would applying TLS scaling to each domain lead to the residual B factors being more balanced? Can a B factor offset be applied to the NCS restraints or could I only apply a coordinate restraint not a B factor restraint between certain copies? Comments welcomed especially from Garib. Happy New Year Nick -- Dr Nicholas H. Keep Dean of Faculty of Science Reader in Structural Biology School of Crystallography, Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6868 (Rosalind Franklin Laboratory) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
Re: [ccp4bb] NCS restraints of domains
It is worth doing sme rounds of non-NCS restrainded refinement then sending it to the Ethan Merrit server to get TLS groups suggested.. Eleanor Frank von Delft wrote: Two points: 1. B-factors tend to differ lots between NCS copies, so you want to set those restraints rather low (at least, I always do, by default) 2. NCS groups tend to need a far more fine-grained description than just plonking in the whole domain. For structures in my lab, we often see that tight NCS does not work -- until the group definitions are selected more carefully. Cheers phx Nicholas Keep wrote: I am refining a low (3A) resolution structure of a 3 domain protein. There are 4 copies in the ASU. I have been applying tight NCS restraints by domain in refmac and have pulled the weak MR solution down to Rfree below 30 (just). However my question is that in 2 of the 4 copies one of the domains is very poorly resolved. I can lower Rfree by around 0.5% by omitting the domains from the PDB entirely or not applying the NCS restraints to these copies of the domain. Clearly they are there and should resemble the moderately well resolved copies by coordinates but the way Bfactor restraints are applied between NCS copies seems to be the issue. If tight restraints are included the B factors are much lower (30-40) rather than 60-80 for the poor domains. I was wondering if there is a theoretically correct way to treat this? Would applying TLS scaling to each domain lead to the residual B factors being more balanced? Can a B factor offset be applied to the NCS restraints or could I only apply a coordinate restraint not a B factor restraint between certain copies? Comments welcomed especially from Garib. Happy New Year Nick
Re: [ccp4bb] NCS restraints of domains
Two points: 1. B-factors tend to differ lots between NCS copies, so you want to set those restraints rather low (at least, I always do, by default) 2. NCS groups tend to need a far more fine-grained description than just plonking in the whole domain. For structures in my lab, we often see that tight NCS does not work -- until the group definitions are selected more carefully. Cheers phx Nicholas Keep wrote: I am refining a low (3A) resolution structure of a 3 domain protein. There are 4 copies in the ASU. I have been applying tight NCS restraints by domain in refmac and have pulled the weak MR solution down to Rfree below 30 (just). However my question is that in 2 of the 4 copies one of the domains is very poorly resolved. I can lower Rfree by around 0.5% by omitting the domains from the PDB entirely or not applying the NCS restraints to these copies of the domain. Clearly they are there and should resemble the moderately well resolved copies by coordinates but the way Bfactor restraints are applied between NCS copies seems to be the issue. If tight restraints are included the B factors are much lower (30-40) rather than 60-80 for the poor domains. I was wondering if there is a theoretically correct way to treat this? Would applying TLS scaling to each domain lead to the residual B factors being more balanced? Can a B factor offset be applied to the NCS restraints or could I only apply a coordinate restraint not a B factor restraint between certain copies? Comments welcomed especially from Garib. Happy New Year Nick
Re: [ccp4bb] NCS restraints of domains
Sounds like a good candidate for (domain-level) TLS to me. Cheers Martyn -Original Message- From: CCP4 bulletin board on behalf of Nicholas Keep Sent: Thu 1/8/2009 10:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] NCS restraints of domains I am refining a low (3A) resolution structure of a 3 domain protein. There are 4 copies in the ASU. I have been applying tight NCS restraints by domain in refmac and have pulled the weak MR solution down to Rfree below 30 (just). However my question is that in 2 of the 4 copies one of the domains is very poorly resolved. I can lower Rfree by around 0.5% by omitting the domains from the PDB entirely or not applying the NCS restraints to these copies of the domain. Clearly they are there and should resemble the moderately well resolved copies by coordinates but the way Bfactor restraints are applied between NCS copies seems to be the issue. If tight restraints are included the B factors are much lower (30-40) rather than 60-80 for the poor domains. I was wondering if there is a theoretically correct way to treat this? Would applying TLS scaling to each domain lead to the residual B factors being more balanced? Can a B factor offset be applied to the NCS restraints or could I only apply a coordinate restraint not a B factor restraint between certain copies? Comments welcomed especially from Garib. Happy New Year Nick -- Dr Nicholas H. Keep Dean of Faculty of Science Reader in Structural Biology School of Crystallography, Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6868 (Rosalind Franklin Laboratory) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
Re: [ccp4bb] NCS restraints of domains
Dear Nick Using TLS sometimes improves behaviour of NCS restraints (it makes sense since remaining B values should be similar). However in other cases it does not improve. Perhaps removal of B value restraints for these domains may improve NCS restrained refinement. I have not done tests without B value restraints so I cannot say what would be behaviour. I would do several tests before making decision: 1) TLS (as Martyn suggests - domain level) with NCS restraints 2) TLS with NCS restraints without B value NCS 3) NCS without B value restraints regards Garib On 8 Jan 2009, at 10:54, Nicholas Keep wrote: I am refining a low (3A) resolution structure of a 3 domain protein. There are 4 copies in the ASU. I have been applying tight NCS restraints by domain in refmac and have pulled the weak MR solution down to Rfree below 30 (just). However my question is that in 2 of the 4 copies one of the domains is very poorly resolved. I can lower Rfree by around 0.5% by omitting the domains from the PDB entirely or not applying the NCS restraints to these copies of the domain. Clearly they are there and should resemble the moderately well resolved copies by coordinates but the way Bfactor restraints are applied between NCS copies seems to be the issue. If tight restraints are included the B factors are much lower (30-40) rather than 60-80 for the poor domains. I was wondering if there is a theoretically correct way to treat this? Would applying TLS scaling to each domain lead to the residual B factors being more balanced? Can a B factor offset be applied to the NCS restraints or could I only apply a coordinate restraint not a B factor restraint between certain copies? Comments welcomed especially from Garib. Happy New Year Nick -- Dr Nicholas H. Keep Dean of Faculty of Science Reader in Structural Biology School of Crystallography, Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6868 (Rosalind Franklin Laboratory) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
