[ccp4bb] Molecular replacement of a multidomain protein
Dear All, I am working with a Bifunctional protein of molecular weight ~60 kDa. I have a 3.3 angstrom native dataset. The matthews number show there are 6 molecules in the asymmetric unit. The structures of the individual domains are already known from prokaryotes. The sequence identity with the known structures are about 30%. I have tried molecular replacement using the two parts as models respectively with CNS, MOLREP, PHASER etc. However I always get the solution for one domain. I have also tried to fix that domain and find the other one. But none of the programs can find a solution. I am trying to model build the correct sequence of one domain using a density modified (using CNS), NCS averaged (using RAVE) map but the map does not look very good. The side chains are not clear. That might be due to the fact that I am only having a partial model. Any suggestion will be appreciated. Thanks. Ms. Anjali Mehta
Re: [ccp4bb] Molecular replacement
The answer to your question is called Phaser. Using Phaser, you can input multiple search models in one search routine to look for solutions. You should read the online manual and tutorials, if you are not already familiar with Phaser. You can also 'modify' or appropriately 'trim' your search manual. All in the manual. Good luck. Raji -Included Message-- Dear All, I wanted to solve one of my new structures by molecular replacement. I got three short search motifs from three different structures available in the PDB. My question is, how do I use all the search models to solve the structure. With by best regards, Sekar (\_/) (='.'=) ()_() -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -End of Included Message--
[ccp4bb] Molecular replacement
Dear All, I wanted to solve one of my new structures by molecular replacement. I got three short search motifs from three different structures available in the PDB. My question is, how do I use all the search models to solve the structure. With by best regards, Sekar (\_/) (='.'=) ()_() -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Molecular Replacement of a Known Anomalous Substructure
Dear All, The authors of the following paper used molecular replacement of the substructure to locate the heavy atoms. The known 11-fold symmetry meant that the heavy atoms had to be in a circle - molecular replacement with circles of different radii did the trick. Antson et al. Nature, Vol. 374 (1995), pp. 693-700. You simply need to use the DANO field of the mtz instead of the intensities if using anomalous data. I have found the known Se substructure by molecular replacement this way as a proof-of-principle (after finding the substructure with shelxd). Whether molecular replacement of the substructure is in general a useful thing to do is another question, modern software is extremely good at locating heavy atom sites. James Dr. James Murray Biochemistry Building Department of Biological Sciences Imperial College London London, SW7 2AZ Tel: +44 (0)20 7594 5276 Dr. James Murray Biochemistry Building Department of Biological Sciences Imperial College London London, SW7 2AZ Tel: +44 (0)20 7594 5276
Re: [ccp4bb] Molecular Replacement issues with a WD-40 7-bladed beta propeller
Hello Scott, hmmm - it is quite difficult to do a good analysis of your problem, remotely. You've tried the enantiomorphic space group I4(3), just to be sure? In principle, the molecular replacement solution given by Phaser sounds good, but this is no proof of whether it's correct. What sounds good is, that you have high Z-scores, the packing looks good to you and the starting R-factor is quite low, not high! For a first model, the working-R and Free-R are usually very close (the initial unrefined model explains the whole set and a random subset equally well), and it is normal, that the Free-R rises in the first xyzB refinement cycles (the free set decouples from the working set, so to say). You don't say how much it rises, but make sure that you use _very_ tight geometry restraints at this resolution to reduce overfitting. I suggest a starting weight of 0.01 or even 0.005 for the X-ray term, resulting in final RMSD values for the bonds of ~0.010-0.012 (there is no exact rule). Your could also use tight NCS restraints for the two molecules to even further reduce any potential overfitting. In contrast, you can leave the B-factor restraints as they are, or even loosen their restraints (I use always values of 2.0, 3.0, 4.5, 6.0 - you can find them in REFMAC under the Geometrical Parameters tab)! On one hand, this gives the model more freedom which could potentially result in more overfitting, on the other hand, however, this additional freedom, at least in my experience, effectively _reduces_ model bias by allowing wrongly placed atoms to vanish. In general, for molecular replacement, never believe any solution until you see the resulting electron density maps. For a true solution, the electron densities should tell you, which parts are wrong and which are missing. At 2.9 A, resolution, this might be difficult. I would suggest that you look at the NCS-averaged electron density map in Coot. However, there could be a lot of other problems ... I would suggest that you could find a local crystallographer at Berkeley that helps you on site. I hope that helps a little bit. Good luck, Dirk. Scott Coyle wrote: Hello, I'm an undergraduate and recently crystallized and obtained 2.9A diffraction data for a protein which is predicted to fold into a WD40 7-bladed beta-propeller structure (which has been crudely verified by cryo-EM by another lab). The space group appears to be I4(1) with unit cell parameters 118.936 118.93685.45690.00090.000 90.000. Using a number of different search models (which I trimmed and aligned to my protein's sequence using Chainsaw) I have obtained a number of MR solutions placing 2 molecules in the AU with Phaser with high Z-scores (ranging from Z=9 to 12) that seem to pack together nicely, so I was hoping to use this technique to solve my structure. However, the initial Rfree for my best solution is relatively high (0.49) and all attempts to refine the structure result in the Rfree blowing up almost immediately. This makes me worry that the maps I'm generating may be too model-biased to use to generate a solution. I've tried using Prime and Switch to remove model bias but the resulting map looks worse than the starting map. As the predicted structure possesses so much radial symmetry (7-fold), I'm worried that my MR solutions will never be oriented correctly enough for me to be able to build a model. If anyone has any suggestions for tackling this kind of molecular replacement woe, I would greatly appreciate it. Otherwise I guess I'll just plan to collect experimental phasing information sometime in the near future. I'm not sure if this is the right place to be asking this question, perhaps you guys could direct me elsewhere. Thanks! -Scott -- Dirk Kostrewa Paul Scherrer Institut Biomolecular Research, OFLC/110 CH-5232 Villigen PSI, Switzerland Phone: +41-56-310-4722 Fax:+41-56-310-5288 E-mail: [EMAIL PROTECTED] http://sb.web.psi.ch
[ccp4bb] Molecular Replacement issues with a WD-40 7-bladed beta propeller
Hello, I'm an undergraduate and recently crystallized and obtained 2.9A diffraction data for a protein which is predicted to fold into a WD40 7-bladed beta-propeller structure (which has been crudely verified by cryo-EM by another lab). The space group appears to be I4(1) with unit cell parameters 118.936 118.93685.45690.000 90.00090.000. Using a number of different search models (which I trimmed and aligned to my protein's sequence using Chainsaw) I have obtained a number of MR solutions placing 2 molecules in the AU with Phaser with high Z-scores (ranging from Z=9 to 12) that seem to pack together nicely, so I was hoping to use this technique to solve my structure. However, the initial Rfree for my best solution is relatively high (0.49) and all attempts to refine the structure result in the Rfree blowing up almost immediately. This makes me worry that the maps I'm generating may be too model-biased to use to generate a solution. I've tried using Prime and Switch to remove model bias but the resulting map looks worse than the starting map. As the predicted structure possesses so much radial symmetry (7- fold), I'm worried that my MR solutions will never be oriented correctly enough for me to be able to build a model. If anyone has any suggestions for tackling this kind of molecular replacement woe, I would greatly appreciate it. Otherwise I guess I'll just plan to collect experimental phasing information sometime in the near future. I'm not sure if this is the right place to be asking this question, perhaps you guys could direct me elsewhere. Thanks! -Scott
[ccp4bb] Molecular replacement and refinement trouble
Hi, Dear all, Firstly ,I'd like to thank all the people for their kind help, especially Miguel, Bernie ,Mark, Moody, Eleanor, Eaton , Peter and lots of others Without your help, I wouldn't make it this far. Secondly, I'd like to share some of the experiences. We had a hard time trying to do MR for this protein. The data was indexed very well in R32, and we can only find two molecules,we just couldn't find the other.We stocked for a long time, not until recently, we figured it's actually a C2 space group. But when we tried to search with one monomer, we couldn't find the solution either because the signal is really low.So we search by those two molecules ( from R32 ), and bingo, we found 6 molecules.So on and so forth, now we are able to find 9 molecules and we believe that's all.Three molecules are bundled together and form three trimers and these three trimers are 3-fold symmetry related.We were excited and went for the next step refining...But after rigid body refinement, the Rfree is still very high , 44%.We calculated the map and looks like density around one loop region is incomplete.I was told that I should give this up, because there is no way to build the model because the density is missing. But I was just wondering if there are any better ways to do refinement, is there any way that could possibly determine the structure.The data is 2.1A,but my protein is really small, 10kD, looks like the packing is really not that great.We are now using CNS to do the refinement. Any suggestions would be highly appreciated. Thanks. Jenny
