[ccp4bb] Modeling residues with very poor density
I am trying to build a model of a 6 Da protein from the diffraction data collected at 2.0 A resolution. There is a 10-residue stretch that has such bad electron density that even at 0.4 sigma level one can hardly see any well defined density for residues with long side chains. My question is: are such poorly defined regions left unmodeled in protein structures? Or is it conventional to model the whole chain no matter how poor the density? The region in question is in the middle of the chain and has several long side chain residues - both charged and uncharged. AM
[ccp4bb] Summary: Modeling residues with very poor density
Thank you Afonine, Hans, Kumar, Deliang, Mark, Jose, Tim, Eleanor and Ed for sharing your thoughts. Here is a summary of the responses I received- 1. One should try to model residues based on 2Fo-Fc maps contoured at 1.0 sigma level. Structures of mobile regions/loops can sometimes be modeled based on maps carved at 0.5-0.6 sigma, but in such cases extreme care must be taken to avoid modeling based on noise signals and not to omit genuine signals visible in the region. 2. 2Fo-Fc maps contoured below 0.5 sigma and Fo-Fc maps contoured below 1.5 sigma are full of noise signals and hence need to be carefully analyzed. It is advisable to avoid modeling anything based solely on signals seen below the above-mentioned sigma cut offs. Fo-Fc maps sometimes improve significantly after refining the initial model a couple of times. 3. There seems to be disagreement over modeling residues that one can not see in maps and leaving out residues that one can see in maps. Thanks a lot for your responses. AM
[ccp4bb] Refmac5 and dual conformation of a dual conformation
I am currently trying to refine a structure where a 5 residue stretch of a chain is in 2 conformations. Oddly enough, 1 of these 5 residues is in dual conformations in both the conformations! Is there a conventional nomenclature for defining such dual-dual conformations? Refmac5 does not accept the intuitive way of naming such an atom. For example: A normal dual conformer GLY would be named as AGLY and BGLY in PDB file and this is acceptable to Refmac5 When I name a "dual-dual" GLY as AAGLY and BAGLY, Refmac5 fails! Any idea, WHY?? Thank you! Here is the error log: Logical name: ATOMSF, Filename: /programs/ccp4-6.0.2/lib/data/atomsf.lib *** * Information from CCP4Interface script *** The program run with command: refmac5 XYZIN "/home/../myfile.pdb" XYZOUT "...tmp" HKLIN "mtz" HKLOUT "tmp" LIBOUT "..._56_lib.cif" has failed with error message fmt: read unexpected character apparent state: internal I/O last format: (I4) lately reading sequential formatted internal IO *** #CCP4I TERMINATION STATUS 0 fmt: read unexpected character apparent state: internal I/O last format: (I4) lately reading sequential formatted internal IO
[ccp4bb] Rigid body refinement as last refinement?
I am told by a mentor that rigid body refinement should never be the last refinement before submission to pdb. Any idea why? Structures in which every round of retrained refinement messes up a conformation, is there any alternative to not using rigid body refinement? Andy
Re: [ccp4bb] Rigid body refinement as last refinement?
Thank you for your responses. Restrained refinement does a good job at refining most of the structure except a small region with poor density. Every time I refine it, it puts this region completely out of the map. It doesn't have much effect on overall geometrical factors as its a very small part of a relatively large molecule. I am annoyed at the fact that Refmac doesn't seem to have much respect for the electron density map while refining this region, and all my attempts to keep the residues within the map go in vain. The refined residues are still ok according to Fo-Fc map, but they're completely out of the 2Fo-Fc map boundaries. Also, my model and the refined model both look bad on Ramachandran plot. But I think its a unique feature of the molecule, at least thats what the density suggests. Also, packing score and quality value plot suggest that there is nothing seriously wrong with my model. As you've suggested I should try altering the restraints and see how it goes. Thank you. Andy
[ccp4bb] Refinement: Basic questions
I have some very basic questions about model refinement process. 1. Why Refmac5 refinement from Coot GUI gives different results than Refmac5 refinement from CCP4i? One obvious difference is that COOT refinements are local whereas CCP4i refinements are global. But why should it make a major difference? 2. Is it acceptable to deposit a model to PDB which has been last refined using Refmac5 from within COOT GUI? 3. Which cases warrant use of TLS and NCS restraints? When is it most appropriate to use anisotropic refinement? 4. PHENIX permits defining different refinement strategies for different parts of the structure. Can a structure part of which has been rigid body refined in the last step, be deposited to PDB? 5. How to prevent Refmac from introducing close contacts during refinement? Setting up geometric parameters (Distance restraint overall wt 1.0) does not seem to be of any help. Thanks! AM
