Meisam:
Probabilities are just that: many of us have had structures with large
solvent contents that are statistically unlikely.
Pedantic quibble: "It scales in P21 Space group with 7% linear
Rfactor." really means that it scales in primitive monoclinic with a
reasonable Rsymm, and I hope you also checked P2 as well as P21 when
doing molecular replacement. P2 is rare, but not unprecedented.
When you say "refine just the back bone" do you mean you're refining
just a poly-ALA model or a non-mutated one ? Because if so, absent the
side-chains and any waters, an R-factor of 31% quite good. If so, add
side-chains and then waters and continue refining and see how things
look. 3-fold averaging across the current monomers plus your decent
resolution should make the sequence interpretation straightforward.
But:
* if you can see interpretable density for the fourth molecule, build in
secondary structure elements, refine those, repeat until you can see a
substructure you recognize, then place the monomer manually.
* use Arp/wArp to autobuild your structure - this has the benefit that
often the map you'll get out will be a very good one even if you build
the remaining monomer manually. If you're lucky it'll build it all for you.
* Autobuild in Phenix can also do much the same
(I would do the first two, and perhaps all three, in parallel until one
emerges a clear winner)
* if the above still doesn't resolve things, consider the possibility
that the fourth molecule is not what you think it is, or may be
statistically disordered
Phil Jeffrey
Princeton
On 11/21/13 12:35 PM, Meisam wrote:
Dear CCP4ers
I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7%
linear Rfactor.
The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric
unit, 53% for 4 molecules, and 36% for 5 molecules.
Molecular replacement just finds 3 molecules in the asymmetric unit. Running
Phaser also gives a partial solution with 3 molecules.
When I refine just the back bone of the protein for the 3 molecules the
Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular
replacement on the refined structure again, and I fix it as a model to search
for another molecule, it still does not find it.
I have attached a photo to show the density for the fourth molecule in the
asymmetric unit.
What is the solution to this?
Thanks in advance for your help
Meisam
