Hi, I just replied to the copy of this email on the CCP4 BB without seeing this response! Kevin has spotted something I missed, which is the solvent content of 87%. There could easily be several copies of the protein related by multiples of the same tNCS vector, i.e. 0,0,0; 0.157,0,0.666; 0.314,0,0 (with a unit cell translation of one being equivalent to zero) and maybe even more. Are there corresponding peaks in the native Patterson for the multiples of this vector? As I said in my other response, Phaser doesn’t seem to have actually taken tNCS into account, placing one copy at a time, so I would need to see the log file to understand why that was. Best wishes, Ramndy

On 25 Feb 2021, at 21:12, Kevin Jude wrote:

Hi Bashir, it looks like tNCS is correctly accounted for by Phaser; your native Patterson map has a peak at {0.157 -0.000 -0.333}, which corresponds to the translation vector between your two molecules in solution 1.

Your problem here is probably related to this warning:

Warning: The composition you have entered gives a solvent content of 87%, which is extremely unlikely. You should increase the composition.

You don't have a complete crystal lattice in your model. Check near the top of the Phaser log file for the Matthews coefficient calculation, which will suggest the most probable multiples of your input molecular weight. You can also calculate these probabilities with the ccp4 program matthews_coeff or Bernard Rupps server at http://www.ruppweb.org/mattprob/default.html.

Best wishes Kevin -- Kevin Jude, PhD (he/him/his) Structural Biology Research Specialist, Garcia Lab Howard Hughes Medical Institute Stanford University School of Medicine Beckman B177, 279 Campus Drive, Stanford CA 94305 Phone: (650) 723-6431

On Thu, Feb 25, 2021 at 12:57 PM Muhammad Bashir Khan wrote: Dear All;

I collected data set which can be easily processed in a space grouop P21 (P1211) at approx 3.7A. I procedeed with MR using phaser in phenix which give a solution with two copies.

** There were 8 solutions

** Pdb and/or Mtz files have been written with results for 1 of these solutions

** Solution #1 written to PDB file: Khan_phaser.1.pdb ** Solution #1 written to MTZ file: Khan_phaser.1.mtz Solution #1 annotation (history): SOLU SET RFZ=3.4 TFZ=4.2 PAK=0 LLG=15 TFZ==5.5 RFZ=2.8 TFZ=27.6 PAK=5 LLG=1173 TFZ==36.5 LLG=1210 TFZ==36.8 PAK=6 LLG=1210 TFZ==36.8 SOLU SPAC P 1 2 1 SOLU 6DIM ENSE ense_1 EULER 7.6 86.4 3.3 FRAC 0.20 0.00 0.18 BFAC -1.90 #TFZ==5.5 SOLU 6DIM ENSE ense_1 EULER 7.6 86.4 3.2 FRAC 0.05 0.00 0.51 BFAC 1.60 #TFZ==36.8 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0444 #RMSD 1.29 #VRMS 1.64

Solution #2 annotation (history): SOLU SET RFZ=3.0 TFZ=5.5 PAK=0 LLG=10 RFZ=2.7 TFZ=25.6 PAK=0 LLG=1165 TFZ==34.8 LLG=1198 TFZ==34.0 PAK=4 LLG=1198 TFZ==34.0 SOLU SPAC P 1 2 1 SOLU 6DIM ENSE ense_1 EULER 10.7 83.5 177.9 FRAC 0.24 -0.00 0.14 BFAC -3.15 SOLU 6DIM ENSE ense_1 EULER 169.3 96.4 357.9 FRAC 0.91 -0.00 0.52 BFAC 1.82 #TFZ==34.0 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0705 #RMSD 1.29 #VRMS 1.65

Solution #3 annotation (history): SOLU SET RFZ=3.4 TFZ=4.1 PAK=1 LLG=-1 RFZ=2.8 TFZ=34.6 PAK=1 LLG=1146 TFZ==39.7 LLG=1185 TFZ==39.2 PAK=3 LLG=1186 TFZ==39.2 SOLU SPAC P 1 21 1 SOLU 6DIM ENSE ense_1 EULER 8.2 86.4 2.0 FRAC 0.01 -0.00 0.01 BFAC -1.69 SOLU 6DIM ENSE ense_1 EULER 8.3 86.4 2.0 FRAC -0.14 -0.00 0.34 BFAC 1.72 #TFZ==39.2 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0406 #RMSD 1.29 #VRMS 1.64

Solution #4 annotation (history): SOLU SET RFZ=3.5 TFZ=5.0 PAK=6 LLG=13 RFZ=2.9 TFZ=30.5 PAK=6 LLG=1156 TFZ==34.4 LLG=1183 TFZ==33.6 PAK=7 LLG=1183 TFZ==33.6 SOLU SPAC P 1 21 1 SOLU 6DIM ENSE ense_1 EULER 359.5 82.0 2.7 FRAC -0.18 -0.00 0.47 BFAC -2.89 SOLU 6DIM ENSE ense_1 EULER 359.5 82.1 2.8 FRAC -0.02 -0.00 0.14 BFAC 2.57 #TFZ==33.6 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0529 #RMSD 1.29 #VRMS 1.64

Solution #5 annotation (history): SOLU SET RFZ=3.5 TFZ=5.0 PAK=6 LLG=13 RFZ=3.0 TFZ=29.0 PAK=6 LLG=1115 TFZ==34.1 LLG=1178 TFZ==33.6 PAK=7 LLG=1178 TFZ==33.6 SOLU SPAC P 1 21 1 SOLU 6DIM ENSE ense_1 EULER 359.9 80.8 0.7 FRAC -0.18 -0.00 0.47 BFAC -1.57 SOLU 6DIM ENSE ense_1 EULER 180.1 99.2 180.7 FRAC 0.34 0.50 0.20 BFAC 2.47 #TFZ==33.6 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0405 #RMSD 1.29 #VRMS 1.64

Solution #6 annotation (history): SOLU SET RFZ=3.5 TFZ=4.6 PAK=1 LLG=-0 RFZ=2.8 TFZ=36.2 PAK=1 LLG=1136 TFZ==35.0 LLG=1169 TFZ==34.6 PAK=1 LLG=1169 TFZ==34.6 SOLU SPAC P 1 21 1 SOLU 6DIM ENSE ense_1 EULER 359.2 83.0 1.5 FRAC 0.06 0.01 0.03 BFAC -2.58 SOLU 6DIM ENSE ense_1 EULER 359.2 82.9 1.4 FRAC -0.09 0.01 0.36 BFAC 2.08 #TFZ==34.6 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0869 #RMSD 1.29 #VRMS 1.66

Solution #7 annotation (history): SOLU SET RFZ=3.5 TFZ=4.6 PAK=1 LLG=-0 RFZ=2.8 TFZ=35.3 PAK=1 LLG=1122 TFZ==35.4 LLG=1149 TFZ==34.5 PAK=1 LLG=1148 TFZ==34.5 SOLU SPAC P 1 21 1 SOLU 6DIM ENSE ense_1 EULER 359.2 82.5 2.4 FRAC 0.06 0.01 0.03 BFAC -1.93 SOLU 6DIM ENSE ense_1 EULER 180.8 97.5 182.4 FRAC 0.78 -0.49 0.31 BFAC 2.44 #TFZ==34.5 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0809 #RMSD 1.29 #VRMS 1.65

Solution #8 annotation (history): SOLU SET RFZ=3.4 TFZ=4.1 PAK=1 LLG=-1 RFZ=2.8 TFZ=33.9 PAK=1 LLG=1083 TFZ==38.5 LLG=1146 TFZ==38.5 PAK=4 LLG=1146 TFZ==38.5 SOLU SPAC P 1 21 1 SOLU 6DIM ENSE ense_1 EULER 8.1 86.1 2.6 FRAC 0.01 -0.00 0.00 BFAC -1.77 SOLU 6DIM ENSE ense_1 EULER 8.1 86.2 2.6 FRAC 0.17 -0.00 -0.33 BFAC 1.66 #TFZ==38.5 SOLU ENSEMBLE ense_1 VRMS DELTA +1.0762 #RMSD 1.29 #VRMS 1.65

CPU Time: 0 days 6 hrs 35 mins 53.63 secs ( 23753.63 secs) Finished: Tue Feb 16 23:27:36 2021 Got the following warnings:

WARNINGS --------

------------------------------------------------------------------------------------------ Warning: Intensity moments suggest possibility of twinning. Tests based on possible twin laws will be more definitive. ------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------ Warning: The composition you have entered gives a solvent content of 87%, which is extremely unlikely. You should increase the composition.

After running xtraige for the .mtz, I get this warning about NCS Frac. coord. : 0.157 -0.000 -0.333 Distance to origin : 119.294 Height relative to origin : 33.056 % p_value(height) : 8.504e-04.

Translational pseudo-symmetry is very likely present in these data. Be aware that this will change the intensity statistics and may impact subsequent analyses, and in practice may lead to higher R-factors in refinement.

Upon an attempt at initial refinement with different stratageis, the R-fators are stuck at

Start R-work = 0.5066, R-free = 0.5233 Final R-work = 0.4831, R-free = 0.4965

The question is that there is something that is not being recognized for example merohedral twinning or that the NCS/pseudosymmetry is not being addressed correctly. Any suggestions would be highly appreciated. Regards;

Bashir -- ------------------------------------------------------ Muhammad Bashir Khan, Ph.D. Research Associate Department of Biochemistry Medical Science Bldg. Lab 3-27 University of Alberta Edmonton AB, T6G 2H7

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----- 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] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk