Since this is now public domain knowledge and if this gets ever published, Phil has my vote to be the first author!
Petr On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote: > That's quite brave - shipping your entire structure to people that could be > actual competitors. But it was fun to play at 1.4 Angstrom over lunch. > > Practical points: > > * not everyone loves 12Mb of attachments in one email in their inbox, so if > you do this again please put the files on a webserver and point us there > > Structural points: > > * the map looks pretty good, but I think the sequence is misassigned in some > regions (e.g. A118-A122 etc). Automation is a good tool but a poor master, > and extreme caution is required before taking the results too literally. > Usually you'd expect a 1.4 Angstrom to be easy to autobuild but I recently > had a sequence misassignment at just that resolution. That map was trivial to > interpret with the correct sequence however - one of the joys of working with > Arp/wArp at 1.4 Angstrom. > > * the large number of positive difference density blobs and water molecules > clustered in what otherwise would be the solvent void strongly suggest that > there's a second molecule present. > > > If I take redfluorescentprotein_refine_10.pdb (waters removed) and > exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, > it finds them quite successfully. (for the record an LLG of 15111 using > nominal sequence identity of 90%). I will send this to you off-list. Please > note that Phaser is using a different origin for this molecular replacement > solution so the coordinates and your previous map do not overlap. > > This rather nicely explains why your structure had an R-factor in the 40's > despite being a half-way decent model. The new MR solution has an R-free in > the 30's in the phenix.refine job I'm running right now. > > > Going forward I suggest you utilize the Arp/wArp program to autobuild your > structure for you, starting from the molecular replacement solution (or, > perhaps with it stripped to ALA). While you could use Autobuild, this is the > CCP4 list and so you should use CCP4 programs. > > Phil Jeffrey > Princeton > > > On 3/27/13 12:22 PM, Tom Van den Bergh wrote: >> 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 >>
