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
