Hi Marco, To follow up: if your protein is something like WRN or BLM helicase - you mention recA like fold after all - (I worked on these in the past hehe) then the split into domains is almost mandatory - these proteins are very plastic and doing MR on them is equivalent to doing antibody MR where you absolutely have to treat domains as separately mobile rigid bodies.
I would love to help more but it would require more sharing that you might be justifiably unwilling or unable to do :) Best of luck, Artem On Mon, Feb 19, 2024 at 6:51 PM Artem Evdokimov <artem.evdoki...@gmail.com> wrote: > Hello Marco, > > First: compare your unit cell parameters with RCSB (there is an option for > that in the search, very helpful). Give it say 5% error margin. > > Worst case scenario - cry a little, having found that your protein is > something else... > > Next, check in depth for space group oddities, twinning, or general data > misbehavior. > > Next, split your helicase into two lobes (if this is the right kind of > helicase) and do MR with both halves, like it was a complex of two proteins > (requires a bit of skill with MR - but nothing terrifyingly hard tbh). If > there are more domains - split into more (RQC, HRDC, etc.) > > Hope this helps. > > Artem > > > > > On Mon, Feb 19, 2024, 5:54 PM Marco Bravo <mbrav...@ucr.edu> wrote: > >> Hello all, >> I recently collected data on some plate crystals for a previously >> uncharacterized protein at the ALS light source. The XDS auto-data >> processing log output indicates that my resolution is 2.8 angstroms. The >> protein is a helicase with homologs already in the protein data bank making >> it a suitable target for molecular replacement which I thought initially. >> However after trying molecular replacements with all known homologs in the >> protein data bank the R values remain high after MR >0.5. After an initial >> round of Rigid body or restrained refinement. The R values still remain >> very high at around >.5. I have tried MR with Rosetta and alphaphold models >> but the problem of high R values persists. The best solution I get is from >> the CCP4 cloud automatic molecular replacement and model building pipeline >> which gives me a free R value of 0.46.. However the solution is only for >> residues ~100-326 out of a 543 amino acid long protein. And even then the >> model still has a lot of missing residues and truncated sidechains and >> overall fits the map quite poorly. Does anyone have any suggestions about >> how I can solve my structure if at all possible at this point? I ran the >> crystals and pre-crystalized samples on a gel and it appears that the >> protein remains stable during crystallization as the molecular weight did >> not change or any degradation does not appear. >> >> ######################################################################## >> >> 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/ >> > ######################################################################## 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/
