Dear Tom, I think that only way for you is to make this structure yourself because it is only way to learn something (at first you must ask in your lab). Anyway, as student I can make some brief suggestions and guides for your refinement 1) Find as much info about your protein as possible, especially look at previous structure (read 2VAD related structure as first step) 2) Before refinement you must find conservative domains inside your protein or in other words red fluorescent protein motifs - these residues will be your starting point 3) As this motif is conserved and probably MR will place them in correct orientation you must move forth and appropriately mutate incorrect residues, if their CA fits good. If your density becomes bad you will remove incorrect residues from this point, later by FOFC map you will place the rest of them in correct orientation. Now move back from the conserved motif in the same manner. 4) After this run 8-10 rounds of refinement, generate map. Repeat step 3 Hope this will help you somehow
Important note for you: do not show your data before publication of pdb. 2013/3/27 Tom Van den Bergh <[email protected]> > Dear members of ccp4bb, > > I need some help with the refinement of my structure of a variant of mRFP > (monomer red fluorescent protein, sequence in attachment). I have done > molecular replacement with phaser with model 2VAD of protein database. Then > i have done some model building phenix.autobuild. (2 pdb's (overall...), > freeR flags and log file attached) When i refine with phenix.refine my > structure i get a R-value of 0,42 which is still way too high. > (redfluorescent protein.pdb, .mtz and logfile attached) When i look at the > structure in coot i find many unmodelled blobs and many outliers in density > analysis and rotamer analysis. The problem is that there are so many > problems with my structure, that i dont know where to begin. Could you try > some refinement for me, because this is first structure that i need to > solve as a student and i dont have too many experience with it. > > Greetings, > > Tom > > -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: [email protected]
