Hi Napo, Just in case nobody suggested this option I want to add it. Very recently I’ve solved structure of Fab with 8 molecules per au. It wasn’t easy to do because some of tNCS were dropped by programs. After contacting the developer of MolRep Alexey Vagin and asking him to help understand why is that happening he found a way to improve the program. After that improvement it worked fine. Give it a try. If I remember correctly you’ve said that sequence identity is around 50%. That is considered (from previous life in Structural Genomics center) extremely similar to target. Our cut-off was 30%. Anything above this value was considered similar and wasn’t within new fold designation. Let me know personally (off the bb) if you need help.
Regards, Vaheh Oganesyan, Ph.D. Scientist, Biologic Therapeutics ____________________________________________________________________ AstraZeneca R&D | Antibody Discovery and Protein Engineering One Medimmune Way, Gaithersburg, MD 20878 T: 301-398-4640 M: 240-398-0046 [email protected]<mailto:[email protected]> From: CCP4 bulletin board <[email protected]> On Behalf Of Napoleão Sent: Thursday, September 5, 2019 10:04 PM To: [email protected] Subject: Re: [ccp4bb] Another difficult MR case Dear all, Thank you for your kind expert suggestions, and sorry for the late reply, I'm still trying all the suggested approaches. Some comments: - I can't use buccaneer yet, since I'm still working to resolve the structure. - Robyn Stanfield's suggestion of using Shelxe to try to verify the validity of the MR solutions was very helpful. I'm trying it a lot, however without success. Thank you Robyn and all Shelx developers! - The maps phased with partial molecular replacement solutions look reasonable, but do not show any sign of the rest of the molecules or of the missing side chains. - ContaMiner gave me green lights, so it does not appear to be a contaminant (thank you Ivan Shabalin). - Similarly, SAUC indicates there is no other PDB with cell parameters similar to this data set (thank you Jonathan Cooper). - I am currently using EPMR to try to resolve the structure by MR (thank you Roger Rowlett). - I am not sure if there is translational NCS, as Phil Jeffrey suggested despite phenix.xtriage's green lights. I'm not experienced with this issue, so I'm sending the log file to Randy Read as he kindly offered to help. I think one of my problems is that the AU is big (at least to me), apparently containing from 9 to 12 copies of my protein, so the MR solutions contain less than 10% of the scatters in the AU. Maps look good when I refine one chain of the MR solutions in Phenix (blue maps over most of the protein, with reasonable main chain continuity and very few green or red blobs), but the R values are 0.50 and I can't see any feature suggesting the solution is good (like a well-defined new side chain). I will keep trying. Thank you all again! And please let me know if any ideas cross your wise minds. Regards, Napo Em 2019-08-30 06:29, Randy Read escreveu: Dear Napoleão, Yes, I agree with Phil that it looks like a case where there *is* translational NCS but it's not being used by Phaser, either because it wasn't automatically detected (native Patterson peak just under the default limit? more than one off-origin Patterson peak?) or because Phaser was told to ignore tNCS. You should look in detail at the relevant section of the logfile, and manually override Phaser's automated decision if you think there's good evidence for tNCS. I would say that, as Phil noted, the fact that two molecules are in basically the same orientation separated by a translation is pretty strong evidence. In particular, the fact that placing the second molecule in the same orientation gave such a large increase in both LLG and TFZ is strong evidence: this tells us that having two molecules in the same orientation (even if they're the wrong molecule or in the wrong orientation) explains some feature of the data, i.e. the modulation caused by tNCS. I'd be happy to look at the log file for you if you find it hard to interpret. Best wishes, Randy Read On 29 Aug 2019, at 17:24, Phil Jeffrey <[email protected]<mailto:[email protected]>> wrote: Are you *sure* there's no translational NCS ? For example your first molecular replacement solution out of Phenix shows EULER 293.6 27.7 288.7 FRAC -0.02 0.02 0.02 (that's "first molecule at origin in P1") and EULER 294.0 27.9 288.8 FRAC -0.37 0.02 0.02 which is essentially the same orientation, and a translation down one crystallographic axis (a*) And this suggests to me that either Xtriage or Phaser is missing something here. Does Phaser find translational NCS in its initial data analysis ? Unmodeled translational NCS could cause significant problems with the molecular replacement search. Phil Jeffrey Princeton On 8/29/19 11:28 AM, Napoleão wrote: Deal all, Sorry for the long post. I have a data set obtained from a crystal produced after incubating a protease with a protein which is mostly composed by an antiparallel beta sheet. I have tried numerous approaches to solve it, and failed. Molecular replacement using Phaser, and the protease or the protein as a template yields no solution. However, molecular replacement using only part of the beta sheet yields LLG=320 TFZ==28.0 (see below). The apparently good data extends to 1.9 A, as processed by XDS, and the space group is P1 (pointless agree). XDS info below: SPACE_GROUP_NUMBER= 1 UNIT_CELL_CONSTANTS= 44.43 72.29 77.30 97.802 89.939 101.576 a b ISa 9.647E-01 3.176E-03 18.07 RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 1.90 24890 19149 23814 80.4% 58.1% 63.7% 11482 0.77 82.2% 63.8* 3 0.694 492 total 163756 125884 146938 85.7% 10.6% 10.8% 75744 3.78 15.0% 99.0* -3 0.761 5834 Xtriage in Phenix 1.16-3549 gives me all green lights (print below), suggesting the data presents no twinning, no translational NCS, no ice rings and is not anisotropic. http://fullonline.org/science/phenix_xtriage_green.png<https://clicktime.symantec.com/3W2rCjVa7Hg6p9AZJtcrJHg6H2?u=http%3A%2F%2Ffullonline.org%2Fscience%2Fphenix_xtriage_green.png> Molecular replacement in Phaser yields single solutions like: Solution annotation (history): SOLU SET RFZ=3.0 TFZ=* PAK=0 LLG=29 RFZ=2.8 TFZ=8.8 PAK=1 LLG=310 TFZ==27.6 LLG=320 TFZ==28.0 SOLU SPAC P 1 SOLU 6DIM ENSE ensemble1 EULER 293.6 27.7 288.7 FRAC -0.02 0.02 0.02 BFAC -6.03 SOLU 6DIM ENSE ensemble1 EULER 294.0 27.9 288.8 FRAC -0.37 0.02 0.02 BFAC -6.52 SOLU ENSEMBLE ensemble1 VRMS DELTA -0.1983 RMSD 0.49 #VRMS 0.21 or partial solutions like: Partial Solution #1 annotation (history): SOLU SET RFZ=3.7 TFZ=* PAK=0 LLG=32 RFZ=2.8 TFZ=13.0 PAK=0 LLG=317 TFZ==30.2 LLG=331 TFZ==30.5 RFZ=2.4 TFZ=7.2 PAK=0 LLG=464 TFZ==18.5 RFZ=2.7 TFZ=5.7 PAK=1 LLG=501 TFZ==6.8 LLG=509 TFZ==6.6 SOLU SPAC P 1 SOLU 6DIM ENSE ensemble1 EULER 85.4 153.0 138.5 FRAC -0.01 -0.00 -0.00 BFAC -12.30 SOLU 6DIM ENSE ensemble1 EULER 86.2 153.2 139.5 FRAC -0.36 -0.01 -0.01 BFAC -9.16 SOLU 6DIM ENSE ensemble1 EULER 83.8 152.3 135.9 FRAC -0.00 0.00 -0.25 BFAC 1.52 SOLU 6DIM ENSE ensemble1 EULER 191.2 109.1 39.3 FRAC -0.27 -0.01 0.22 BFAC 10.18 SOLU ENSEMBLE ensemble1 VRMS DELTA -0.0447 RMSD 0.49 #VRMS 0.44 However, after 1 refinement round in Phenix_Refine (Final: r_work = 0.4881 r_free = 0.5009) I got densities that are part good and part bad, and if I delete the bad parts and refine again, the good parts become bad. Please check the prints: https://clicktime.symantec.com/37C7fcu5uSaN4Zcsf9kRaV86H2?u=http%3A%2F%2Ffullonline.org%2Fscience%2Fgood_part_of_density.png https://clicktime.symantec.com/34vHvp9AWiG7HRXfS4rqgc96H2?u=http%3A%2F%2Ffullonline.org%2Fscience%2Fbad_part_of_density.png What is the explanation for these molecular replacement results? What else should I try? Arcimboldo takes 2 days+ to run and yields no good solution. Thank you! Regards, Napo ------------------------------------------------------------------------ To unsubscribe from the CCP4BB list, click the following link: https://clicktime.symantec.com/37816eSGb8MY3NA1WbeDia26H2?u=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://clicktime.symantec.com/37816eSGb8MY3NA1WbeDia26H2?u=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1> ------ 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: [email protected]<mailto:[email protected]> Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk<https://clicktime.symantec.com/3KYZY19nnp4a65zBKnbLHmp6H2?u=http%3A%2F%2Fwww-structmed.cimr.cam.ac.uk> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://clicktime.symantec.com/37816eSGb8MY3NA1WbeDia26H2?u=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://clicktime.symantec.com/37816eSGb8MY3NA1WbeDia26H2?u=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
