Hi, I had a hard time tracking down the data in the legacy CCP4 site but eventually found it! Then I tried solving the MR tutorial case with Phaser.
Instead of using the tutorial domain 1, 2 and 3 files, I prepared them from the deposited PDB file using a sequence alignment followed by using sculptor to trim off non-conserved ends of side chains and any missing loops. Then I did a default run of Phaser, searching for 2 copies of each domain. Phaser automatically set the search order to domain 1, then domain 2 then domain 3 (they have similar sequence identities and that sorts them from biggest to smallest). It was clear from the statistics (increase in log-likelihood gain or LLG and TFZ score) that both copies of domain 1 were correct, the first copy of domain 2, and both copies of domain 3, even though an incorrect placement of domain 2 occurred before looking for domain 3. Unfortunately, the second copy of domain 3 was rejected because of packing problems with the badly-placed domain 2 copy. So I went back to the partial solution with 2 copies of domain 1 and one of domain 2, then added both copies of domain 3 followed by the second copy of domain 2. This all worked well, giving clear solutions. No intermediate refinement necessary, though if it hadn’t worked it would have been possible to refine the intermediate solution and grab the improved models for any missing domains. Fifty cycles of jelly-body refinement of the final MR solution in Refmac gave an R-free of 43.7%. Note that in these jobs Phaser struggled with the second copy of domain 1, only pulling it out in the refinement step, probably because the B-factor had to be increased substantially to agree with the data. Another search order might have been more efficient (maybe 1, 2, 3, 1 2, 3), but this worked. Note that you can over-ride the automatic choice of search order, but Phaser will by default choose its own search order (which is usually but not always better). Hope that helps on the Phaser front! Best wishes Randy Read > On 11 May 2021, at 02:53, Lande <fu...@northeastern.edu> wrote: > > Hi everyone, I started my work on data processing in protein crystallography > about half year ago and have done a fair number of “easy” structures. > Recently, I processed a tutorial structure containing 3 different domains and > two chains of them (in particular, it’s “ mth685, high resolution data” from > ccp4 legacy tutorial), but it was failed during MR process. > > According to this tutorial, I did MR with MOLREP by adding each domain in > order. The first and second added domains, which are domain 1 from chain A > and domain 2 from chain B, are correct; however, when I tried to add the next > domain, R work/R free did not drop and clearly MOLREP did not find the > correct solution according to the density map. I also tried Phaser but the > result was the same. > > screen shot for domain 2 in chain A: https://ibb.co/YB2JnRR > > <a href="https://ibb.co/YB2JnRR";><img > src="https://i.ibb.co/0yq14DD/screenshot-on-wrong-domain.png"; > alt="screenshot-on-wrong-domain" border="0"></a><br /><a target='_blank' > href='https://imgbb.com/'>pio pio 3 menu</a><br /> > > In addition, I found an article on that data > (https://journals.iucr.org/d/issues/2008/01/00/ba5117/index.html) but I > failed to get the same result expect the first two domains. > > I wonder if anyone has done this tutorial before or has faced similar > problems in his own work. I have no idea on how to deal with it. Any > suggestions or comments will be helpful. Thanks in ahead. > > Link for that tutorial: > ftp://ftp.ccp4.ac.uk/tutorials/ccp4_phasing/7_domains/mth685.pdf > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk ######################################################################## 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/
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