Hello Marc- My first thought is do you have the correct space group? Are you searching for other space groups in the point group? Second, helicases are multimers, but the orientation of the monomers that make up the multimers may deviate significantly. If you didn't use a monomer, it's worth doing so. Likewise, using the hexamer might be better. Is searching with a complete multimer sensible, ie would crystallographic symmetry cause overlaps due to coincidence of a non-crystallographic axis with a crystallographic axis? If you are searching with a multimer, even if you get a solution, you may very likely need to rigid body refine all "domains/subdomains" of the solution to proceed toward better r-factors. Lastly, trim as much as you can from the search model to get to the core, give that a shot.
Can you calculate the self-rotation function? What does it tell you about potential symmetry and orientation of symmetry vs. your best solution? What program are you using, phaser? what does the LLG score look like? I'm happy to discuss further if you like. Best- Todd ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of Marco Bravo <[email protected]> Sent: Monday, February 19, 2024 4:44 PM To: [email protected] <[email protected]> Subject: [ccp4bb] Difficult Molecular replacement 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://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=05%7C02%7Ctgreen%40UAB.EDU%7Ce2b2eaceb1c84962085e08dc319dcbbe%7Cd8999fe476af40b3b4351d8977abc08c%7C1%7C1%7C638439800998244676%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=EN%2FqVDVHvgyY9Ur8%2FGsV2eInIhU1N6N9fBdK5sNpeuE%3D&reserved=0<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> This message was issued to members of https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.jiscmail.ac.uk%2FCCP4BB&data=05%7C02%7Ctgreen%40UAB.EDU%7Ce2b2eaceb1c84962085e08dc319dcbbe%7Cd8999fe476af40b3b4351d8977abc08c%7C1%7C1%7C638439800998251769%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=y05Xe8HjIjkvCGxz5ZCg%2FrkjhXTX4Rpiq5of2bZCukM%3D&reserved=0<http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by https://nam12.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.jiscmail.ac.uk%2F&data=05%7C02%7Ctgreen%40UAB.EDU%7Ce2b2eaceb1c84962085e08dc319dcbbe%7Cd8999fe476af40b3b4351d8977abc08c%7C1%7C1%7C638439800998259001%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=auvuhLZsoyHa%2FQ64Grf%2Fe1Pw2w%2B0jQBRj7SuLl9Hp%2FU%3D&reserved=0<http://www.jiscmail.ac.uk/>, terms & conditions are available at https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fpolicyandsecurity%2F&data=05%7C02%7Ctgreen%40UAB.EDU%7Ce2b2eaceb1c84962085e08dc319dcbbe%7Cd8999fe476af40b3b4351d8977abc08c%7C1%7C1%7C638439800998263941%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=o3ZLf%2BCt%2BVy33BmVaN5WvHelbRyDMUMBGjor9isAK88%3D&reserved=0<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/
