Hey, I played around with the RMSD value that helped at least for me. Try to make it higher. Best, Daniel On 15.03.2011, at 16:45, Zhang yu wrote:

Hi, Pozharski

I attached part of log file.

Fast Rotation Function Table: 1 ------------------------------- #SET Top (Z) Second (Z) Third (Z) 1 -1129.09 32.53 --- --- --- --- ---- ---------- ----- ---------- ----- ---------- -----

--------------- FINAL SELECTION ---------------

Mean used for final selection = -5302.48 Cutoff used for final selection = -2172.44 Number of sets stored before final selection = 1 Number of solutions stored before final selection = 1 Number of sets stored (deleted) after final selection = 1 (0) Number of solutions stored (deleted) after final selection = 1 (0)

Select by Percentage of Top value: 75 Top TF = -5085.32 Mean TF used for final selection = -7813.43 LLG Cutoff used for final selection = -5767.35 Number of solutions stored before final selection = 1 Number of solutions stored (deleted) after final selection = 1 (0) Top TFZ = 21.3024

----------------- TABLES OF RESULTS -----------------

Fast Translation Function Table: Space Group P 1 2 1 ---------------------------------------------------- #SET #TRIAL Top (Z) Second (Z) Third (Z) Ensemble 1 1 -5085.32 (21.30) - - - - 1 ---- ------

Solutions: ===================== Solution #1: Likelihood Gain -4625.06 ENSE 1 - EULER 326.300, 61.849, 83.068 - FRAC 0.529, -0.001, 0.102

Unit cell: (186.376, 103.164, 295.884, 90, 98.789, 90) Space group: P 1 2 1 (No. 3)

SPACE GROUP OF SOLUTION: 'P 1 2 1'

By the way, the Xtrige in Phenix tells that translational pseudo-symmetry is very likely present. I also attached the log file.

Twinning and intensity statistics summary (acentric data):

Statistics independent of twin laws /<I>^2 : 2.148 <F>^2/ : 0.803 <|E^2-1|> : 0.729 <|L|>, : 0.391, 0.215 Multivariate Z score L-test: 9.366

The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it.

No (pseudo)merohedral twin laws were found.

Patterson analyses - Largest peak height : 56.002 (corresponding p value : 2.915e-05)

The analyses of the Patterson function reveals a significant off-origin peak that is 56.00 % of the origin peak, indicating pseudo translational symmetry. The chance of finding a peak of this or larger height by random in a structure without pseudo translational symmetry is equal to the 2.9153e-05. The detected tranlational NCS is most likely also responsible for the elevated intensity ratio. See the relevant section of the logfile for more details. The results of the L-test indicate that the intensity statistics are significantly different than is expected from good to reasonable, untwinned data. As there are no twin laws possible given the crystal symmetry, there could be a number of reasons for the departure of the intensity statistics from normality. Overmerging pseudo-symmetric or twinned data, intensity to amplitude conversion problems as well as bad data quality might be possible reasons. It could be worthwhile considering reprocessing the data.

Yu Zhang

2011/3/15 Ed Pozharski On Mon, 2011-03-14 at 18:32 -0400, Zhang yu wrote:

...

I am working on a DNA-protein complex, the protein apo structure is already known. Recently I got a dataset of the DNA-protein complex, I tried to find a solution by autoMR in Phenix with the known protein coordinate as template. I got only one solution with very negative LLG (around -4000), and after rigid body refinement, the both Rwork and Rfree is around 0.55. Was there anyone in the same situation as me? What does that mean if there is just one solution while with very high negative LLG?

Could mean several things, but one thing is for sure - R~55% suggests that molecular replacement did not work. One possibility is that your protein undergoes conformational change when it binds to DNA. If it has distinct domains, you may be able to get a solution if running them as separate models.

Negative LLG could mean that you did not correctly guess the unit cell content. Or maybe the space group is wrong. You may want to post the phaser log-file, since it's not obvious to me what you mean by only one solution. What were the Z-scores at rotation/translation steps?

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It indicates that pseudo translational symmetry is present in the dataset. I didn't do anything when I first try MR. Is that possible I merged two lattice together during data process, or should I reprocess the data to P1? I attached the scale log file, Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 10.29 1030.9 76.3 20.0 1.095 0.105 0.118 10.29 8.18 791.3 52.2 18.1 1.108 0.093 0.101 8.18 7.15 350.8 23.5 11.3 1.160 0.108 0.106 7.15 6.49 195.8 17.1 11.4 1.090 0.138 0.131 6.49 6.03 143.7 15.7 12.1 1.029 0.167 0.154 6.03 5.67 130.8 15.6 13.2 1.101 0.187 0.173 5.67 5.39 129.9 16.8 14.6 1.074 0.198 0.184 5.39 5.16 136.0 17.9 15.6 1.078 0.202 0.186 5.16 4.96 138.3 19.2 16.8 1.044 0.207 0.192 4.96 4.79 150.9 20.7 18.0 1.082 0.209 0.191 4.79 4.64 151.3 21.1 18.8 1.140 0.225 0.210 4.64 4.50 143.1 22.4 20.3 1.070 0.236 0.226 4.50 4.39 127.5 22.5 21.1 1.027 0.262 0.251 4.39 4.28 117.7 22.8 22.3 1.051 0.291 0.279 4.28 4.18 105.8 24.2 23.7 0.988 0.322 0.317 4.18 4.09 93.0 25.0 24.9 0.947 0.365 0.371 4.09 4.01 84.9 25.3 25.2 0.884 0.409 0.403 4.01 3.94 76.6 27.4 27.3 0.843 0.457 0.490 3.94 3.87 66.5 27.5 27.5 0.778 0.505 0.559 3.87 3.80 59.3 28.9 28.8 0.766 0.575 0.696 All reflections 222.9 26.7 19.5 1.037 0.165 0.128 2011/3/15 Francis E Reyes

This is probably the most revealing to me.. What did you do about this when you did the MR?

On Mar 15, 2011, at 9:45 AM, Zhang yu wrote:

The analyses of the Patterson function reveals a significant off-origin

...

peak that is 56.00 % of the origin peak, indicating pseudo translational symmetry.

--------------------------------------------- Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder

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*Xtrige log file of P21 * The full list of Patterson peaks is: x y z height p-value(height) ( 0.500, 0.500, 0.000 ) : 56.002 (2.915e-05) ( 0.483, 0.500, 0.034 ) : 6.152 (6.778e-01) If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible: space group operator unit cell of reference setting C 1 2 1 (a-1/4,b-1/4,c) x+1/2, y+1/2, z (186.38, 103.16, 295.88, 90.00, 98.79, 90.00) * Xtrige log file of P2* The full list of Patterson peaks is: The Xtrige log is as follows, x y z height p-value(height) ( 0.500, 0.500, 0.000 ) : 56.000 (2.916e-05) ( 0.483, 0.500, 0.034 ) : 6.151 (6.781e-01) If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible: space group operator unit cell of reference setting C 1 2 1 x+1/2, y+1/2, z (186.38, 103.16, 295.88, 90.00, 98.79, 90.00) *Results from rotation functions for P2 (Search two monomers in ASU)* ****************************** ******************************************************* *** Phaser Module: MOLECULAR REPLACEMENT FAST ROTATION FUNCTION 2.2.4 *** ************************************************************************************* --------------------- ANISOTROPY CORRECTION --------------------- No refinement of parameters ------------------------------- DATA FOR FAST ROTATION FUNCTION ------------------------------- Outliers with a probability less than 1e-06 will be rejected There were 2 (0.0000%) reflections rejected H K L reso F probability 5 5 62 4.48 42.404 4.049e-07 18 2 -26 8.12 112.092 7.520e-09 Space-Group Name (Number): P 1 2 1 (3) Resolution of All Data (Number): 3.82 49.18 (87989) Resolution of Selected Data (Number): 3.82 49.18 (87985) -------------------------- ENSEMBLE FOR DECOMPOSITION -------------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 394.48 295.80 293.03 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------- WILSON DISTRIBUTION ------------------- Parameters set for Wilson log-likelihood calculation E = 0 and variance 1 for each reflection Without correction for SigF to the variances, Wilson log(likelihood) = - number of acentrics (83981) - half number of centrics (4004/2) = -85983 With correction for SigF, Wilson log(likelihood) = -89650.5 Configuring ensembles --------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 914.73 520.03 508.93 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------------------ FAST ROTATION FUNCTION #1 OF 1 ------------------------------ Search Ensemble: 1 Known MR solutions (empty solution set - no components) Fraction of asymmetric unit modelled: 1.0047 of which the "moving" fraction is 1.0047 Fraction of asymmetric unit modelled (after correction): 0.99 of which the "moving" fraction is 0.99 Spherical Harmonics ------------------- Elmn for Data Elmn Calculation for Data 0% 100% |=========================================================================| DONE Elmn for Search Ensemble Elmn Calculation for Search Ensemble 0% 100% |===========================================================================| DONE Scanning the Range of Beta Angles --------------------------------- Performing a 1.3513 degree search. Clmn Calculation 0% 100% |====================================================================| DONE Top 59 rotations before clustering will be rescored Calculating Likelihood for RF #1 of 1 0% 100% |============================================================| DONE Scoring 500 randomly sampled rotations Generating Statistics for RF #1 of 1 0% 100% |========================================================================| DONE Mean Score (Sigma): -7144.27 (152.138) Highest Score (Z-score): -2269.9 (32.0392) Top Peaks With Clustering ------------------------- # Rank of the peak after rescoring search points (#) Rank of the peak before rescoring search points LLG Log-Likelihood Gain Z-Score Number of standard deviations of LLG above the mean FSS Fast Search Score You requested peaks over 75% of top (i.e. 0.75*(top-mean)+mean) There was 1 site over 75% of top The sites over 75% are: # (#) Euler1 Euler2 Euler3 LLG Z-score Split #Group raw/top 1 1 326.1 62.4 83.0 -2269.90 32.04 0.0 23 100.0/100.0 213.9 117.6 263.0 Fast Rotation Function Table: 1 ------------------------------- #SET Top (Z) Second (Z) Third (Z) 1 -2269.90 32.04 --- --- --- --- ---- ---------- ----- ---------- ----- ---------- ----- --------------- FINAL SELECTION --------------- Mean used for final selection = -7144.27 Cutoff used for final selection = -3488.49 Number of sets stored before final selection = 1 Number of solutions stored before final selection = 1 Number of sets stored (deleted) after final selection = 1 (0) Number of solutions stored (deleted) after final selection = 1 (0) ------------ *Results from rotation functions for P2 (Search one monomer in ASU)* ************************************************************************************* *** Phaser Module: MOLECULAR REPLACEMENT FAST ROTATION FUNCTION 2.2.4 *** ************************************************************************************* --------------------- ANISOTROPY CORRECTION --------------------- No refinement of parameters ------------------------------- DATA FOR FAST ROTATION FUNCTION ------------------------------- Outliers with a probability less than 1e-06 will be rejected There were 2 (0.0000%) reflections rejected H K L reso F probability 5 5 62 4.48 42.404 4.049e-07 18 2 -26 8.12 112.092 7.520e-09 Space-Group Name (Number): P 1 2 1 (3) Resolution of All Data (Number): 3.82 49.18 (87989) Resolution of Selected Data (Number): 3.82 49.18 (87985) -------------------------- ENSEMBLE FOR DECOMPOSITION -------------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 323.84 274.82 261.37 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------- WILSON DISTRIBUTION ------------------- Parameters set for Wilson log-likelihood calculation E = 0 and variance 1 for each reflection Without correction for SigF to the variances, Wilson log(likelihood) = - number of acentrics (83981) - half number of centrics (4004/2) = -85983 With correction for SigF, Wilson log(likelihood) = -89650.5 Configuring ensembles --------------------- - 显示引用文字 - This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 714.35 518.29 464.48 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------------------ FAST ROTATION FUNCTION #1 OF 1 ------------------------------ Search Ensemble: 1 Known MR solutions (empty solution set - no components) Fraction of asymmetric unit modelled: 0.831924 of which the "moving" fraction is 0.831924 Spherical Harmonics ------------------- Elmn for Data Elmn Calculation for Data 0% 100% |=========================================================================| DONE Elmn for Search Ensemble Elmn Calculation for Search Ensemble 0% 100% |===========================================================================| DONE Scanning the Range of Beta Angles --------------------------------- Performing a 1.5476 degree search. Clmn Calculation 0% 100% |===========================================================| DONE Top 41 rotations before clustering will be rescored Calculating Likelihood for RF #1 of 1 0% 100% |==========================================| DONE Scoring 500 randomly sampled rotations Generating Statistics for RF #1 of 1 0% 100% |========================================================================| DONE Mean Score (Sigma): -4412.88 (151.158) Highest Score (Z-score): -893.783 (23.2809) Top Peaks With Clustering ------------------------- # Rank of the peak after rescoring search points (#) Rank of the peak before rescoring search points LLG Log-Likelihood Gain Z-Score Number of standard deviations of LLG above the mean FSS Fast Search Score You requested peaks over 75% of top (i.e. 0.75*(top-mean)+mean) There was 1 site over 75% of top The sites over 75% are: # (#) Euler1 Euler2 Euler3 LLG Z-score Split #Group raw/top 1 1 70.6 39.1 10.6 -893.78 23.28 0.0 13 100.0/100.0 109.4 140.9 190.6 Fast Rotation Function Table: 1 ------------------------------- #SET Top (Z) Second (Z) Third (Z) 1 -893.78 23.28 --- --- --- --- ---- ---------- ----- ---------- ----- ---------- ----- --------------- FINAL SELECTION --------------- Mean used for final selection = -4412.88 Cutoff used for final selection = -1773.56 Number of sets stored before final selection = 1 Number of solutions stored before final selection = 1 Number of sets stored (deleted) after final selection = 1 (0) Number of solutions stored (deleted) after final selection = 1 (0) ------------ Results from rotation functions for P21 (Search two monomers in ASU) ************************************************************************************* *** Phaser Module: MOLECULAR REPLACEMENT FAST ROTATION FUNCTION 2.2.4 *** ************************************************************************************* --------------------- ANISOTROPY CORRECTION --------------------- No refinement of parameters ------------------------------- DATA FOR FAST ROTATION FUNCTION ------------------------------- Outliers with a probability less than 1e-06 will be rejected There were 2 (0.0000%) reflections rejected H K L reso F probability 5 5 62 4.48 42.404 4.046e-07 18 2 -26 8.12 112.092 7.516e-09 Space-Group Name (Number): P 1 21 1 (4) Resolution of All Data (Number): 3.82 49.18 (87983) Resolution of Selected Data (Number): 3.82 49.18 (87981) -------------------------- ENSEMBLE FOR DECOMPOSITION -------------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 323.83 274.82 261.37 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------- WILSON DISTRIBUTION ------------------- Parameters set for Wilson log-likelihood calculation E = 0 and variance 1 for each reflection Without correction for SigF to the variances, Wilson log(likelihood) = - number of acentrics (83976) - half number of centrics (4005/2) = -85978 With correction for SigF, Wilson log(likelihood) = -89646.3 Configuring ensembles --------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 714.35 518.29 464.48 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------------------ FAST ROTATION FUNCTION #1 OF 1 ------------------------------ Search Ensemble: 1 Known MR solutions (empty solution set - no components) Fraction of asymmetric unit modelled: 0.737308 of which the "moving" fraction is 0.737308 Spherical Harmonics ------------------- Elmn for Data Elmn Calculation for Data 0% 100% |=========================================================================| DONE Elmn for Search Ensemble Elmn Calculation for Search Ensemble 0% 100% |===========================================================================| DONE Scanning the Range of Beta Angles --------------------------------- Performing a 1.54708 degree search. Clmn Calculation 0% 100% |===========================================================| DONE Top 47 rotations before clustering will be rescored Calculating Likelihood for RF #1 of 1 0% 100% |================================================| DONE Scoring 500 randomly sampled rotations Generating Statistics for RF #1 of 1 0% 100% |========================================================================| DONE Mean Score (Sigma): -3258.27 (128.907) Highest Score (Z-score): -242.259 (23.3968) Top Peaks With Clustering ------------------------- # Rank of the peak after rescoring search points (#) Rank of the peak before rescoring search points LLG Log-Likelihood Gain Z-Score Number of standard deviations of LLG above the mean FSS Fast Search Score You requested peaks over 75% of top (i.e. 0.75*(top-mean)+mean) There was 1 site over 75% of top The sites over 75% are: # (#) Euler1 Euler2 Euler3 LLG Z-score Split #Group raw/top 1 1 70.7 39.1 10.6 -242.26 23.40 0.0 18 100.0/100.0 109.3 140.9 190.6 Fast Rotation Function Table: 1 ------------------------------- #SET Top (Z) Second (Z) Third (Z) 1 -242.26 23.40 --- --- --- --- ---- ---------- ----- ---------- ----- ---------- ----- --------------- FINAL SELECTION --------------- Mean used for final selection = -3258.27 Cutoff used for final selection = -996.261 Number of sets stored before final selection = 1 Number of solutions stored before final selection = 1 Number of sets stored (deleted) after final selection = 1 (0) Number of solutions stored (deleted) after final selection = 1 (0) ------------ Results from rotation functions for P21 (Search One monomers in ASU) ************************************************************************************* *** Phaser Module: MOLECULAR REPLACEMENT FAST ROTATION FUNCTION 2.2.4 *** ************************************************************************************* --------------------- ANISOTROPY CORRECTION --------------------- No refinement of parameters ------------------------------- DATA FOR FAST ROTATION FUNCTION ------------------------------- Outliers with a probability less than 1e-06 will be rejected There were 2 (0.0000%) reflections rejected H K L reso F probability 5 5 62 4.48 42.404 4.046e-07 18 2 -26 8.12 112.092 7.516e-09 Space-Group Name (Number): P 1 21 1 (4) Resolution of All Data (Number): 3.82 49.18 (87983) Resolution of Selected Data (Number): 3.82 49.18 (87981) -------------------------- ENSEMBLE FOR DECOMPOSITION -------------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 323.83 274.82 261.37 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------- WILSON DISTRIBUTION ------------------- Parameters set for Wilson log-likelihood calculation E = 0 and variance 1 for each reflection Without correction for SigF to the variances, Wilson log(likelihood) = - number of acentrics (83976) - half number of centrics (4005/2) = -85978 With correction for SigF, Wilson log(likelihood) = -89646.3 Configuring ensembles --------------------- This pdb file contains 1 models The (input) RmsD of this model with respect to the real structure is 0.8 Unit Cell: 714.35 518.29 464.48 90.00 90.00 90.00 Electron Density Calculation 0% 100% |=======| DONE ------------------------------ FAST ROTATION FUNCTION #1 OF 1 ------------------------------ Search Ensemble: 1 Known MR solutions (empty solution set - no components) Fraction of asymmetric unit modelled: 0.737308 of which the "moving" fraction is 0.737308 Spherical Harmonics ------------------- Elmn for Data Elmn Calculation for Data 0% 100% |=========================================================================| DONE Elmn for Search Ensemble Elmn Calculation for Search Ensemble 0% 100% |===========================================================================| DONE Scanning the Range of Beta Angles --------------------------------- Performing a 1.54708 degree search. Clmn Calculation 0% 100% |===========================================================| DONE Top 47 rotations before clustering will be rescored Calculating Likelihood for RF #1 of 1 0% 100% |================================================| DONE Scoring 500 randomly sampled rotations Generating Statistics for RF #1 of 1 0% 100% |========================================================================| DONE Mean Score (Sigma): -3258.27 (128.907) Highest Score (Z-score): -242.259 (23.3968) Top Peaks With Clustering ------------------------- # Rank of the peak after rescoring search points (#) Rank of the peak before rescoring search points LLG Log-Likelihood Gain Z-Score Number of standard deviations of LLG above the mean FSS Fast Search Score You requested peaks over 75% of top (i.e. 0.75*(top-mean)+mean) There was 1 site over 75% of top The sites over 75% are: # (#) Euler1 Euler2 Euler3 LLG Z-score Split #Group raw/top 1 1 70.7 39.1 10.6 -242.26 23.40 0.0 18 100.0/100.0 109.3 140.9 190.6 Fast Rotation Function Table: 1 ------------------------------- #SET Top (Z) Second (Z) Third (Z) 1 -242.26 23.40 --- --- --- --- ---- ---------- ----- ---------- ----- ---------- ----- --------------- FINAL SELECTION --------------- Mean used for final selection = -3258.27 Cutoff used for final selection = -996.261 Number of sets stored before final selection = 1 Number of solutions stored before final selection = 1 Number of sets stored (deleted) after final selection = 1 (0) Number of solutions stored (deleted) after final selection = 1 (0) ------------ *Thanks a lot. Yu * 2011/3/15 Francis E Reyes

Go back to your xtriage log file... Post the section that looks similar to the one below...

The full list of Patterson peaks is:

x y z height p-value(height) ( 0.500, 0.000, 0.233 ) : 26.198 (2.753e-03) ( 0.000, 0.338, 0.000 ) : 5.699 (7.800e-01)

If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible:

space group operator unit cell of reference setting C 2 2 21 (-a,c,2*b) x+1/2, y, z+1/4 (55.47, 73.64, 81.46, 90.00, 90.00, 90.00)

Systematic absences

Also, post the results of the rotation function.. What is the RFZ score and how did it compare to any other rotation solutions.

F

On Mar 15, 2011, at 1:13 PM, Zhang yu wrote:

...

It indicates that pseudo translational symmetry is present in the dataset. I didn't do anything when I first try MR. Is that possible I merged two lattice together during data process, or should I reprocess the data to P1?

I attached the scale log file,

Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 10.29 1030.9 76.3 20.0 1.095 0.105 0.118 10.29 8.18 791.3 52.2 18.1 1.108 0.093 0.101 8.18 7.15 350.8 23.5 11.3 1.160 0.108 0.106 7.15 6.49 195.8 17.1 11.4 1.090 0.138 0.131 6.49 6.03 143.7 15.7 12.1 1.029 0.167 0.154 6.03 5.67 130.8 15.6 13.2 1.101 0.187 0.173 5.67 5.39 129.9 16.8 14.6 1.074 0.198 0.184 5.39 5.16 136.0 17.9 15.6 1.078 0.202 0.186 5.16 4.96 138.3 19.2 16.8 1.044 0.207 0.192 4.96 4.79 150.9 20.7 18.0 1.082 0.209 0.191 4.79 4.64 151.3 21.1 18.8 1.140 0.225 0.210 4.64 4.50 143.1 22.4 20.3 1.070 0.236 0.226 4.50 4.39 127.5 22.5 21.1 1.027 0.262 0.251 4.39 4.28 117.7 22.8 22.3 1.051 0.291 0.279 4.28 4.18 105.8 24.2 23.7 0.988 0.322 0.317 4.18 4.09 93.0 25.0 24.9 0.947 0.365 0.371 4.09 4.01 84.9 25.3 25.2 0.884 0.409 0.403 4.01 3.94 76.6 27.4 27.3 0.843 0.457 0.490 3.94 3.87 66.5 27.5 27.5 0.778 0.505 0.559 3.87 3.80 59.3 28.9 28.8 0.766 0.575 0.696 All reflections 222.9 26.7 19.5 1.037 0.165 0.128

2011/3/15 Francis E Reyes This is probably the most revealing to me.. What did you do about this when you did the MR?

On Mar 15, 2011, at 9:45 AM, Zhang yu wrote:

The analyses of the Patterson function reveals a significant off-origin peak that is 56.00 % of the origin peak, indicating pseudo translational symmetry.

--------------------------------------------- Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder

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Yes, I rescaled my data to C2 (186.38, 103.16, 295.88, 90.00, 98.79, 90.00), and AutoMR found a solution. After refinement, the R went down to ~0.38, and the electron density fits well with my model except some flexible parts. Although there is a small crash between symmetry related molecules, due to the flexible feature of that domain, I don't think it is a problem. But the index confused me a lot. When I try to re-index my space group in HKL2000. The initial Index always suggests P1 (103.32, 185.24, 291.75, 97.55, 90.18, 90.48 ) Distortion Index 0.00% P2 (186.38, 103.16, 295.88, 90.00, 98.79, 90.00) Distortion Index 0.22% (they are basically the same) while HKL2000 couldn't index the data to correct C2, the only C2 it found is C2 (592.23, 103.32, 185.24, 90.00, 97.52, 90.00) Distortion index 4.11% the unit cell looks twice bigger than current one, and it has quite unacceptable distortion index. It couldn't be refined. (Extremely high chi square and mosaicity) So, I just rescaled the integrated data (P2) to C2. The statistics of scale is OK, and there is no obvious violation. Could someone tell me why HKL2000 couldn't find the correct C2 SP? Yu 2011/3/16 Ed Pozharski

On Tue, 2011-03-15 at 19:49 -0400, Zhang yu wrote:

...

If the observed pseudo translationals are crystallographic the following spacegroups and unit cells are possible:

space group operator unit cell of reference setting

C 1 2 1 (a-1/4,b-1/4,c) x+1/2, y+1/2, z (186.38, 103.16, 295.88, 90.00, 98.79, 90.00)

I presume that you tried to process your data in C2 and it did not work?

-- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs

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On Wed, 2011-03-16 at 09:55 -0600, Francis E Reyes wrote:

If you have a strong pseudotranslation (PST), you may see alternating weak/strong reflections along the axes.

Well, in C2 there is no pseudotranslation, right?

Watch the integration in P2, do you notice that some (weak) reflections (usually in between lattice lines) are missed?

Shouldn't it be the other way around - with half of the spots covering absent reflections? -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs

On Wed, 2011-03-16 at 11:32 -0400, Zhang yu wrote:

Could someone tell me why HKL2000 couldn't find the correct C2 SP?

Who knows. In my experience, autoindexing in denzo is sometimes finicky. Most tricks alter the set of peaks used for indexing, by restricting resolution, increasing/decreasing their number, even changing the spot radius. Since denzo picked a much larger unit cell, it may have been swayed by spots from a satellite crystal. In this case, the Patterson clearly indicated which space group is right, but you may have avoided the problem by running your images by another indexing program. LabelIT is, imho, a particularly robuts tool. I am sure mosflm and/or xds could have worked too. Wish you to find the DNA in your maps, Ed. -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs

Can you just rescale the data in C2 if you did the integration in P2? I know HKL2000(at least the gui will) will flag you for trying to switch bravais lattices. i feel like this is not exactly what you meant if got good scaling statistics. As for HKL2000 not auto-finding your C-centered monoclinic space group, you can play around with the number of peaks(add more or use less), the peak size, and resolution limits. Some combination has worked for us when we had a tricky crystals with alternating weak and heavy rows. -Todd -----Original Message----- From: [email protected] on behalf of Zhang yu Sent: Wed 3/16/2011 10:32 AM To: PHENIX user mailing list Subject: Re: [phenixbb] Only one solution in AutoMR with very negative LLG So, I just rescaled the integrated data (P2) to C2. The statistics of scale is OK, and there is no obvious violation. Yu

Yeah, the protein is huge, around 450KD. Changing the ASU content did help a little bit. By searching two copies in ASU, I got a solution with better LLG #+ #* Initial LLG Initial R Refined LLG Refined R Unique Tmplt 1 1 4885.54 61.38 4885.54 61.38 YES But the Rfactor are still high and stuck during refinement. Regarding the possible effect of space group, I reindexed the dataset from P2 to P21. AutoMR also gave similar solutions with high R values. 2011/3/15 Ed Pozharski

On Tue, 2011-03-15 at 11:45 -0400, Zhang yu wrote:

...

Fast Rotation Function Table: 1 ------------------------------- #SET Top (Z) Second (Z) Third (Z) 1 -1129.09 32.53 --- --- --- --- ---- ---------- ----- ---------- ----- ---------- -----

Well, your Z-score is very promising. What size protein/DNA you have? The likely asu content is over a megadalton, so unless the protein is huge, you probably have more than one copy - this would explain bad initial R-values. Patterson suggests that too. You probably need to play with the estimate of asu content a bit to get rid of negative LLG. What happens if you run a search looking for the second copy with the first one in place?

-- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs

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