At the risk of being somewhat cheeky - perhaps I could claim second author? I too have successfully solved the structure - and I totally concur with Phil. Placing a second molecule in the asymmetric unit, essentially resolves the perceived R-factor problem.
A good thorough manual inspection and rebuilding is *ALWAYS* good practice for newcomers to the field. Tony. On 27 Mar 2013, at 17:14, Petr Leiman <[email protected]> wrote: > 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 >>>
