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[ccp4bb]: Scaling Problems
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I am currently having difficulty scaling data from a protein-DNA crystal.
Data was collected previously on this crystal form to about 3.2 A. It
processed and scaled as orthorombic with cell dimensions of roughly 39,76,
and 256 A. Data was more recently collected on an anomalous edge in
order to obtain experimental phases, which is where the scaling problems
have arisen. I am using HKL2000 to index, integrate, and scale the data
and all help is welcomed and greatly appreciated.
Statistics from Scalepack on a previous occasion of the same crystal form
at a remote wavelength with little or no anomalous signal.
Shell Lower Upper Average Average Norm. Linear Square
limit Angstrom I error stat. Chi**2 R-fac R-fac
50.00 6.89 4388.1 131.0 114.3 1.885 0.060 0.069
6.89 5.47 1113.3 38.6 34.7 1.390 0.082 0.077
5.47 4.78 847.5 39.7 37.1 1.116 0.103 0.089
4.78 4.34 647.8 41.3 39.6 0.953 0.132 0.109
4.34 4.03 610.6 44.4 42.8 0.950 0.149 0.112
4.03 3.79 368.0 45.8 45.2 0.818 0.222 0.232
3.79 3.60 317.7 50.4 49.9 0.703 0.241 0.212
3.60 3.45 454.3 59.6 58.0 0.636 0.164 0.076
3.45 3.31 622.2 70.4 68.2 0.547 0.097 0.045
3.31 3.20 174.1 72.2 71.9 0.594 0.216 0.132
All reflections 1011.6 59.7 56.3 1.020 0.100 0.075
Spacegroup P2(1)2(1)2(1)
Unit Cell 39.821 76.064 256.236
I/sigma 16.9 (2.4)
Redundancy 6.5
Rejections 0.77
Completeness 97.1% (80.0%)
Molecular replacement attempts were made and a solution obtained. One of
the two complexes in the asymmetric unit showed nice electron density for
both the model used in molecular replacement and sidechains/residues not
included in the search. However, the second protein-DNA complex had only
moderately continuous density for the core of the search model and nearly
no additional features outside of the search.
Recently, data was collected on the Zn anomalous edge in order to obtain
experimental phases exploiting the bound Zn ions in the protein. Indexing
and integration of the data frames was straight-forward and problem free.
However, attempts to scale the data have proved very problematic. Below
is a similar summary of data from Scalepack at the anomalous wavelength.
The error scale factor for Scalepack was set at 1.3, which is standard and
the same as used in the first data collection. Obviously, the chi^2 and
rejections are incredibly high. The detector distance and direct beam
positions used to index the frames is known to be accurate due to other
data on other projects collected before and after this data set. Those
data sets indexed, integrated, and scaled as normal with no problems.
Additionally, data was collected on this crystal form at a remote
wavelength and the data similarly doesn't scale well, so I don't believe
it to be an issue with separation of anomalous pairs.
Shell I/Sigma in resolution shells:
Lower Upper No. of reflections with I / Sigma less than
limit limit 0 1 2 3 5 10 20 >20 total
50.00 6.24 6 10 21 25 42 124 329 2379 2708
6.24 4.96 10 26 40 58 114 299 750 2206 2956
4.96 4.33 42 74 120 190 296 604 1311 1713 3024
4.33 3.94 30 90 147 222 406 881 1719 1279 2998
3.94 3.65 71 166 291 453 761 1424 2379 661 3040
3.65 3.44 74 220 471 733 1202 1963 2555 418 2973
3.44 3.27 176 509 938 1293 1839 2381 2691 208 2899
3.27 3.12 287 917 1536 1900 2293 2594 2805 169 2974
3.12 3.00 505 1390 2039 2331 2545 2759 2889 68 2957
3.00 2.90 772 1890 2447 2647 2815 2971 3040 5 3045
All hkl 1973 5292 8050 9852 12313 16000 20468 9106 29574
Spacegroup P2(1)2(1)2(1)
Unit Cell 39.50 75.70 255.29
I/sigma 23.9 (1.6)
Redundancy 3.0 (separating anomalous pairs)
Rejections 12.56%
Completeness 94.0% (95.3%)
Any suggestions on the source and solution to this problem are greatly
appreciated.
Paul Shaffer
Duke University Medical Center
pls3@duke.edu