Re: [ccp4bb] side chain density
before modelling a long side-chain in non-existing or dubious density, also make sure it is really there in the protein by sequencing your expression plasmid. Your arginine (for example) may in fact be a serine or glycine...databases are not 100% accurate and neither is PCR if it was used in the cloning. Quoting Ed Pozharski: OK, here we go again. This has been argued ad nauseam, see for example http://www.dl.ac.uk/list-archive-public/ccp4bb/msg19738.html or http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20268.html (hard to believe we have gone more than a year without another version of "what to do with disordered side chains" 250-post long discussion :) I do not have much to add to the above, however On 11/09/2012 05:22 PM, Matthew Franklin wrote: I think we can all agree that virtually every structure in the PDB will have a few residues where some of the atoms are not visible in the density. So the "trim the side chains" crowd is a well-represented minority at 30%, but 70% of depositors chose another option. This maybe the historical average, I suspect that currently the "trim the side chains crowd" may be at least at 50% (but what about Ohio? :). Majority, however, is not always right (don't get me started on I-over-sigma ratios). I personally like to leave all atoms on the side chains; they look wrong to me when beheaded. I just try to put the invisible atoms in a stereochemically plausible conformation, leave the occupancy set to 1, and let the refinement program deal with them. With all due respect, to model something where there is no density (aka experimental evidence) cannot be justified by aesthetics. On the contrary, there is some evidence suggesting that modelling disordered side chains in the way you describe adds small, but detectable error to the rest of the model. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] side chain density
OK, here we go again. This has been argued ad nauseam, see for example http://www.dl.ac.uk/list-archive-public/ccp4bb/msg19738.html or http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20268.html (hard to believe we have gone more than a year without another version of "what to do with disordered side chains" 250-post long discussion :) I do not have much to add to the above, however On 11/09/2012 05:22 PM, Matthew Franklin wrote: I think we can all agree that virtually every structure in the PDB will have a few residues where some of the atoms are not visible in the density. So the "trim the side chains" crowd is a well-represented minority at 30%, but 70% of depositors chose another option. This maybe the historical average, I suspect that currently the "trim the side chains crowd" may be at least at 50% (but what about Ohio? :). Majority, however, is not always right (don't get me started on I-over-sigma ratios). I personally like to leave all atoms on the side chains; they look wrong to me when beheaded. I just try to put the invisible atoms in a stereochemically plausible conformation, leave the occupancy set to 1, and let the refinement program deal with them. With all due respect, to model something where there is no density (aka experimental evidence) cannot be justified by aesthetics. On the contrary, there is some evidence suggesting that modelling disordered side chains in the way you describe adds small, but detectable error to the rest of the model. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] side chain density
Hi Guys, I'm dealing with the same issue with a couple of structures at the moment. Matt you said that you like to leave the side chains in a plausible conformation and let refinement deal with the problem. Generally when I try this their backbone geometry gets all bent out of shape in refinement. What's the best 'kind' of refinement to do in order to deal with this problem? Cheers, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew Franklin [mfrank...@nysbc.org] Sent: 09 November 2012 22:22 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] side chain density Dear Remy and Faisal - Just a geeky bioinformatics note: searching my local copy of the PDB (somewhat out of date), shows that 22,165 out of 72,000 structures contain a 'REMARK 470' line, which is how the PDB notes that atoms are missing from a residue. Some of these are no doubt mistakes by the depositors, but most probably reflect the decision to remove invisible side chain atoms from the coordinates (e.g. trimming back to Cbeta) while keeping the correct residue name. I think we can all agree that virtually every structure in the PDB will have a few residues where some of the atoms are not visible in the density. So the "trim the side chains" crowd is a well-represented minority at 30%, but 70% of depositors chose another option. I personally like to leave all atoms on the side chains; they look wrong to me when beheaded. I just try to put the invisible atoms in a stereochemically plausible conformation, leave the occupancy set to 1, and let the refinement program deal with them. - Matt On 11/9/12 3:47 PM, Remy Loris wrote: > Dear Faisal, > > You definitely do not mutate to alanine as that would imply for the > future "user" of your pdb file that it is a mutant. > Some people feel they have to keep the side chain but put the > occupancies at zero. I think this is a bad practice and strongly > oppose to it as for the future "user" of your deposited pdb file, who > often is not a crystallographer, you suggest a specific conformation > for your side chain that may be interpreted in terms of biology, while > in reality it addopts a huge, disordered ensemble of conformations. > Personally I am of the opinion that you should simply remove the side > chain atoms (but keep the residue name). And that is the same as what > you do with a whole loop that is disordered. I think it is lso the > most common practice in deposited structures. > > Remy Loris > Vrije Universiteit Brussel > > On 09/11/12 20:22, Faisal Tarique wrote: >> Dear all >> >> i have solved a structure ( at 2A resolution) whose Rwork and Rfree >> is 22 and 25 respectively..the Ramachandran plot shows 90% of the >> residues in the most favorable region and with 6 residues in >> generously allowed and no residues in disallowed region. But in some >> areas i can see density missing for side chains ( in loop regions >> )..i have question do i need to mutate them to alanine or leave them >> as such..The density fit analysis in COOT ( traffic light) showing >> those regions with side chain as red.. >> >> thanx in advance >> >> Regards >> >> Faisal >> School of Life Sciences >> JNU >> > > -- 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] side chain density
Dear Remy and Faisal - Just a geeky bioinformatics note: searching my local copy of the PDB (somewhat out of date), shows that 22,165 out of 72,000 structures contain a 'REMARK 470' line, which is how the PDB notes that atoms are missing from a residue. Some of these are no doubt mistakes by the depositors, but most probably reflect the decision to remove invisible side chain atoms from the coordinates (e.g. trimming back to Cbeta) while keeping the correct residue name. I think we can all agree that virtually every structure in the PDB will have a few residues where some of the atoms are not visible in the density. So the "trim the side chains" crowd is a well-represented minority at 30%, but 70% of depositors chose another option. I personally like to leave all atoms on the side chains; they look wrong to me when beheaded. I just try to put the invisible atoms in a stereochemically plausible conformation, leave the occupancy set to 1, and let the refinement program deal with them. - Matt On 11/9/12 3:47 PM, Remy Loris wrote: Dear Faisal, You definitely do not mutate to alanine as that would imply for the future "user" of your pdb file that it is a mutant. Some people feel they have to keep the side chain but put the occupancies at zero. I think this is a bad practice and strongly oppose to it as for the future "user" of your deposited pdb file, who often is not a crystallographer, you suggest a specific conformation for your side chain that may be interpreted in terms of biology, while in reality it addopts a huge, disordered ensemble of conformations. Personally I am of the opinion that you should simply remove the side chain atoms (but keep the residue name). And that is the same as what you do with a whole loop that is disordered. I think it is lso the most common practice in deposited structures. Remy Loris Vrije Universiteit Brussel On 09/11/12 20:22, Faisal Tarique wrote: Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU -- 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] side chain density
Dear Faisal, You definitely do not mutate to alanine as that would imply for the future "user" of your pdb file that it is a mutant. Some people feel they have to keep the side chain but put the occupancies at zero. I think this is a bad practice and strongly oppose to it as for the future "user" of your deposited pdb file, who often is not a crystallographer, you suggest a specific conformation for your side chain that may be interpreted in terms of biology, while in reality it addopts a huge, disordered ensemble of conformations. Personally I am of the opinion that you should simply remove the side chain atoms (but keep the residue name). And that is the same as what you do with a whole loop that is disordered. I think it is lso the most common practice in deposited structures. Remy Loris Vrije Universiteit Brussel On 09/11/12 20:22, Faisal Tarique wrote: Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] side chain density
On Friday, 09 November 2012, Faisal Tarique wrote: > Dear all > > i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 > and 25 respectively..the Ramachandran plot shows 90% of the residues in the > most favorable region and with 6 residues in generously allowed and no > residues in disallowed region. But in some areas i can see density missing > for side chains ( in loop regions )..i have question do i need to mutate > them to alanine or leave them as such. Mutating to alanine is not an option. They are not alanine. If nothing else, when you get to the point of depositing your structure in the PDB it will fail validation checks because the sequence is not correct at those points. But if you mean should you delete sidechain atoms beyond CB, that is another question. That is a legitimate option. I suggest trying that and then looking in difference density maps to see if any density shows up to guide placement of the sidechain. Ethan > .The density fit analysis in COOT ( > traffic light) showing those regions with side chain as red.. > > thanx in advance > > Regards > > Faisal > School of Life Sciences > JNU >
Re: [ccp4bb] side chain density
Hi, There has been quite a lot of discussion about this and I think different opinions exist. I would try to keep the side chains, if there is some evidence where they are. Otherwise I would just delete atoms and I would not mutate them to alanine. http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20437.html Best regards, Lari __ Lari Lehtiö, PhD, Adjunct Professor Biocenter Oulu Department of Biochemistry P.O.Box 3000 FIN-90014 University of Oulu Finland http://cc.oulu.fi/~llehtio/ _ Quoting Faisal Tarique : Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
[ccp4bb] side chain density
Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
