Re: [ccp4bb] MolRep of coiled coils
MR on CCs is generally a pain. The problem is that sliding any CC along its own supertwist (say by one heptad) will give you a solution that lines up very well with the "right" model. There are a lot of solutions of this type, so you basically have a multitude of models that are all "okay" and the "right" one is not that much better than any of them. Hence, the Z-score is low. This problem gets even worse if your CC crystallizes end-to-end with another copy of itself (CCs like to do that). I'd be willing to bet that you actually already have the right solution. Take the "best" one from your favorite program and start refining it. Do rigid body first and keep re-applying rigid-body refinement until the model stops moving. This is important. Don't just stop when the Rcryst flattens out. Let the MODEL settle down. Then break the CC in half. Rigid-body each half until they stop moving. Keep sub-dividing like this. Eventually, you will be refining individual heptads from each chain. This is a poor-man's way to do "rubber body refinement" which should let you refine a bent CC. "rubber body refinement" in general lets you make much bigger leaps in coordinate space than all-atom refinement. Using this methodology, I was once able to solve an antiparallel trimer CC using a parallel trimer as the starting model. (the reversed chain was apparent in the refined map, but not in the starting map). It can also be useful to start with idealized coiled-coil models. The PDB does not always contain what you want. I wrote a little jiffy program for generating an idealized coiled-coil in PDB format here: http://bl831.als.lbl.gov/~jamesh/scripts/supertwist.awk The top of this file (text) contains instructions on how to use it. The idealized CC MR method proved useful in solving this structure: R. Howard et. al. Neuron 53(5), 663-75. and the methodology is described in that paper. Using idealized models allows you to "scan" over the various superhelical parameters and then plot the Z-score, CC, or whatever you like vs the parameter. Once you have optimized each parameter, you can do a multi-dimensional "MR meta-search" over a small range of all the parameters. -James Holton MAD Scientist Thomas Edwards wrote: Dear BB, I am attempting molecular replacement with a 2.8A data set from crystals of a coiled coil of about 150 residues. Probably p21212 but maybe p2221. So far, Phaser, MolRep, Amore, Mr Bump, have not provided a good solution as judged by Z-scores, CCs, Rfactors, and whether there is any density outside the model (there should be - I'm using a slightly shorter model to search with). One possible problem is that the coiled coil may not be straight. It may have a slight curve to it. I have used models from the PDB that are straight or slightly curved, so far with no success. I have tried a few different resolution cutoffs too. I have tried single helix chains or dimeric coiled coils. Is there anything special about MR with coiled coils? Can anybody provide any tips/hints on MR with coiled coils?? Thanks Ed
Re: [ccp4bb] MolRep of coiled coils
Hi Thomas, MR of coiled coils can be quite tricky including considerations of being curved, etc. If conventional MR is failing (assuming you have tried different kinds of parameter and search model tweaks, you can also play around with the search thresholds in phaser), you may try the following: If you have multiple copies in the asu, you may try to find the orientation of the NCS axis. If you think that the molecules may be aligned along a particular direction, you can then generate a list of rotation search angles manually in fine increments (couple of degrees) and then force a translation search around all these rotation angles and then follow it up with a packing function search. This is like a brute force rotation/translation search across all rotation angles. Therefore identification of the ncs axis/molecular orientation will provide some search time advantage. You can do this in PHASER. Check out the manual to run stand alone scripts for these steps. Although different from the above, the follg. reference may provide useful reading: Gonzalez L Jr, Brown RA, Richardson D, Alber T. Crystal structures of a single coiled-coil peptide in two oligomeric states reveal the basis for structural polymorphism. Nat Struct Biol. 1996 Dec;3(12):1002-9. Also check tropomysin structure MR attempts from Carolyn Cohen's group. Regards, Debanu. -- Debanu Das, Structure Determination Core, Joint Center for Structural Genomics. Stanford Synchrotron Radiation Laboratory, Menlo Park, CA. -Original Message- From: CCP4 bulletin board on behalf of Thomas Edwards Sent: Fri 3/28/2008 5:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] MolRep of coiled coils Dear BB, I am attempting molecular replacement with a 2.8A data set from crystals of a coiled coil of about 150 residues. Probably p21212 but maybe p2221. So far, Phaser, MolRep, Amore, Mr Bump, have not provided a good solution as judged by Z-scores, CCs, Rfactors, and whether there is any density outside the model (there should be - I'm using a slightly shorter model to search with). One possible problem is that the coiled coil may not be straight. It may have a slight curve to it. I have used models from the PDB that are straight or slightly curved, so far with no success. I have tried a few different resolution cutoffs too. I have tried single helix chains or dimeric coiled coils. Is there anything special about MR with coiled coils? Can anybody provide any tips/hints on MR with coiled coils?? Thanks Ed
Re: [ccp4bb] MolRep of coiled coils
My short experience with coiled-coil is that molecular replacement can be difficult for "classical software" (due to the very anysotropic shape of the protein). In our case (a short parallel dimeric coiled-coil), molecular replacement trials using AMoRe or MOLREP were unsuccessful. We solved the structure using EPMR (evolutionary search molecular replacement software). http://www.msg.ucsf.edu/local/programs/epmr/epmr.html Michel.
Re: [ccp4bb] MolRep of coiled coils
Sorry - I should have added that, yes, there are 2 identical peptide chains that should be parallel coiled-coil. Any advice gratefully received. Ed -Original Message- From: cockburn [mailto:[EMAIL PROTECTED] Sent: Fri 3/28/2008 1:35 PM To: Thomas Edwards Subject: Re: [ccp4bb] MolRep of coiled coils Hi Thomas, Does your coiled-coil consist of two polypeptide chains, and are their sequences identical? Do you expect it to be a parallel or anti-parallel coiled-coil? Yours Joe Thomas Edwards a écrit : > Dear BB, > > I am attempting molecular replacement with a 2.8A data set from crystals of a > coiled coil of about 150 residues. > Probably p21212 but maybe p2221. > > So far, Phaser, MolRep, Amore, Mr Bump, have not provided a good solution as > judged by Z-scores, CCs, Rfactors, and whether there is any density outside > the model (there should be - I'm using a slightly shorter model to search > with). > > One possible problem is that the coiled coil may not be straight. It may have > a slight curve to it. > I have used models from the PDB that are straight or slightly curved, so far > with no success. > I have tried a few different resolution cutoffs too. > I have tried single helix chains or dimeric coiled coils. > > Is there anything special about MR with coiled coils? > Can anybody provide any tips/hints on MR with coiled coils?? > > Thanks > Ed >
