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Re: [ccp4bb]: Scaling Problems
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Suggestions:
1) Are you sure your space group is P2(1)2(1)2(1) and not P2(1)2(1)2?
See the posting to the bulletin board from Marilyn Yoder's group on
Feb. 4, 2003 concerning this possibility.
2) Are you sure that you have two complexes in the unit cell? Do you
see peaks in a self-rotation map and/or native Patterson map?
3) Are you positive that your second crystal was orthorhombic and not
monoclinic?
3) It would be nice to see the resolution vs. R-factor plot for the
Zn anomalous data set, to compare it with the results from the first
dataset.
Diana
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>*** CCP4 home page http://www.ccp4.ac.uk ***
>
>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
--
* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Assistant Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd. Tel: +1 214 648 9760
Y4.330a Fax: +1 214 648 8954
Dallas, TX 75390-9038, U.S.A. Email: Diana.Tomchick@UTSouthwestern.edu