Hi Napo,
Apart from other things possible (wrong space group is often the
culprit, but it seems like not your case), I would consider a
possibility of crystallizing a different protein than expected.
see "Protein purification and crystallization artifacts: The tale
usually not told"
https://www.ncbi.nlm.nih.gov/pubmed?cmd=search&term=26660914
https://bioreproducibility.org/protein_purification_artifacts/
for examples - we wrote this one after encountering these issues again
and again. This paper provides troubleshooting pointers for researchers
facing difficulties in phasing or model building.
Also, ContaMiner seems to be a great resource:
https://strube.cbrc.kaust.edu.sa/contaminer/submit
Best wishes,
Ivan
With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
https://www.linkedin.com/in/shabalinig/
https://minorlab.org/person/ivan_s/
On 8/29/19 11:28, 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
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:
http://fullonline.org/science/good_part_of_density.png
http://fullonline.org/science/bad_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
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