we should all chip in a water molecule or two and the second author becomes "the CCP4 community"….
On 27 Mar 2013, at 17:22, Antony Oliver <[email protected]> wrote: > 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 >>>> > Roberto A. Steiner Group Leader Randall Division of Cell and Molecular Biophysics King's College London [email protected] Room 3.10A New Hunt's House Guy's Campus SE1 1UL London Phone 0044 20 78488216 Fax 0044 20 78486435
