I am interested to know the resolution on the basis that the better it diffracts, the lower the solvent content, generally. Have you tried Bernard Rupp's server which gives you probabilities for the No mols per AU, partly based on resolution? The LLGs look challenging - I've seen higher ones refine with only random R-free values.
On 13 Nov 2019 17:42, "Tanner, John J." <tanne...@missouri.edu> wrote:
Dear Rob,
Based on our experience with difficult MR cases, I recommend performing refinement on the coordinates from MR (I prefer PHENIX simulated annealing for this step). Then send the refined map - WITHOUT the model - to automated building with density modification. The auto-built model may give you an indication of how many chains are in the asymmetric unit. You could do this procedure for each of the MR solutions (N=4,5,6) and compare the results. Also, if you enable automatic detection of NCS during DM, you may find that 6 NCS operators are found even when N=4 or 5; this would give you confidence that N=6 is possible.
Also look at the cross rotation function to see how many strong peaks are present. I’m sure the peaks are listed in Phaser, but it is easier for me to find them in the MOLREP output. MOLREP also provides a matrix that tells you which cross RF peaks are related by symmetry and which are unique, which is helpful.
And David Schuller mentioned inspecting the self-RF, which is a good idea.
Finally, do you know anything about the oligomeric state of the protein? If it is a pentamer, N=6 seems unlikely. If it is a dimer, N=5 in P212121 seems unlikely.
Jack
John J. Tanner
Professor of Biochemistry and ChemistryAssociate Chair of BiochemistryDepartment of BiochemistryUniversity of Missouri
117 Schweitzer Hall503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280Lab: Schlundt Annex rooms 3,6,9, 203B, 203COffice: Schlundt Annex 203A
On Nov 13, 2019, at 8:33 AM, Robert S Phillips <plp@UGA.EDU> wrote:
I have been working on a protein structure which has been hard to solve by molecular replacement.
Unit cell: (60.6, 172.34, 196.42, 90, 90, 90)
Space group: P 21 21 21
The problem is that the homologues have only ~20% identity, and there are multiple chains in the asymmetric unit. The question is how many. It could be 4, 5, or 6 chains.
N solvent P
4 0.602 3.09 0.225
5 0.502 2.47 0.3886 0.403 2.06 0.229
I have run PHASER with 4, 5 and 6 chains. I allowed it to search all possible space groups, and P212121 was the best solution. These are the results.
N LLG TFZ
4 104.9 7.55 137.5 7.76 166.2 8.3
Am I correct to conclude that there are 6 chains in the asymmetric unit?
Rob
Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphillips@chem.uga.edu
Web: http://tryptophan.net
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