It may be worth trying rigid body-refinement of the solution, using two rigid bodies, one per monomer, if you haven’t tried already. Then perform positional refinement.
When refining with the original sequence, first remember that R/Rfree are bound to be and stay high with at most 25% homology. Does the map look reasonable? Can you see density matching the true sequence, even if refining with the molecular replacement model, in places where the side-chains are ordered (not facing the solvent) but there is an unmistakable side-chain difference (i.e. from a small side chain such as Ala or Val etc. to Phe Tyr Trp, or vice-versa) If so you can either use an automated approach to fit the new sequence or do it “manually” in Coot, then refine again. R’s should drop quickly. I’ve had good success using the above approach, all refinements performed with autoBUSTER. Thierry From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Robert S Phillips Sent: Thursday, June 18, 2020 9:01 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Molecular replacement problem EXTERNAL EMAIL – Use caution with any links or file attachments. I've been pulling out my hair with this for a few months now. I have data sets to 2.6 A for a new enzyme in the aminotransferase superfamily. Unfortunately, the closest structure is only 25% identity. MR with PHASER using the monomer was a complete failure. Since the minimum structure of enzymes in the family is a dimer (the active site is formed at the monomer-monomer interface), I used dimers for MR with PHASER. Most of the results were marginal, but one looks good. However, it will not refine. Everything I have done with this solution has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not give any useable models, since they have poor statistics and low completeness. The output from PHASER is below. ** SINGLE solution ** Solution written to PDB file: DGL_phaser.1.pdb ** Solution written to MTZ file: DGL_phaser.1.mtz Solution annotation (history): SOLU SET RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 LLG=994 TFZ==20.1 SOLU SPAC P 2 2 21 SOLU 6DIM ENSE ense_1 EULER 360.0 0.0 0.0 FRAC -0.00 -0.00 -0.00 BFAC -0.12 MULT 2 #TFZ==20.1 SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD 2.08 #VRMS 0.89 With LLG = 994 and TFZ = 20.1, isn't this a real solution? 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: rsphill...@chem.uga.edu<mailto:rsphill...@chem.uga.edu> Web: http://tryptophan.net<https://pod51004.outlook.com/owa/redir.aspx?C=ccbf42ffea5f48b1bf8e9bb950454bab&URL=http%3a%2f%2ftryptophan.net> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (2000 Galloping Hill Road, Kenilworth, New Jersey, USA 07033), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
