Sorry it has been accepted already, see attachment, it will soon be online see the date for that.

Jürgen

......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:      +1-410-614-4894
Fax:      +1-410-955-2926
http://lupo.jhsph.edu

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On Mar 27, 2013, at 1:30 PM, Steiner, Roberto wrote:

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 <[log in to unmask]>
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 <[log in to unmask]>
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
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