[phenixbb] Problems with phasing a protein (1300aa)
ewaldxray at gmail.com
Fri Mar 20 13:00:08 PDT 2009
We merged .x files only. Only in this case, we have seen such a low value
of average I (in all the datasets). We used HKL 2000 with refinement sigma
2009/3/20 Schubert, Carsten [PRDUS] <CSCHUBER at its.jnj.com>
> You are very likely to be right to be worried about your average I. I
> have never seen stats like that. Did you merge on the basis of .x files as
> is recommended or based on final .sca files of your individual batches?
> -----Original Message-----
> *From:* phenixbb-bounces at phenix-online.org [mailto:
> phenixbb-bounces at phenix-online.org]*On Behalf Of *Kumar
> *Sent:* Friday, March 20, 2009 3:42 PM
> *To:* phenixbb at phenix-online.org
> *Subject:* [phenixbb] Problems with phasing a protein (1300aa)
> Hello Phenixbb members,
> I have been trying to obtain phases for a protein which contain ~1300aa. We
> have obtained native data to a resolution of 3.3A (Space group I222 or
> I212121). But we are having tough time phasing it.
> 'Se' labeled crystals diffracts maximally up to 3.5 to 4 A and dies very
> quickly on most of the beamlines. We have scanned at Se wavelength and it
> gives very strong signal as it contain ~45 Se in AU (1300 aa). It is
> difficult to collect a complete dataset (maximally we get 50-60 % completion
> with Rmerge ~15) out of one crystal on regular beamline. At microfocus
> beamline (APS), we were able to collect data in 3-4 batches and merge them
> to get a complete dataset (Rmerge ~18-20) out of one crystal. We used data
> collected on microfocus beamline (at peak wavelength) for locating heavy
> atom position using SHELXD, Solve and Phenix.hyss. SOlve and Phenix.hyss
> find very few heavy atom sites 1-5 whereas SHELX-CDE lists many but shows no
> difference in original and inverted (contrast and connectivity). Our phasing
> attempts with datasets obtained after merging two incomplete dataset from
> two different crystal has also been disappointing.
> My another worry is absolute value of average intensity, which seems to be
> quite low in most of the datasets. Below I have pasted last table of
> scale.log (HKL2000).
> Shell Lower Upper Average Average Norm. Linear Square
> limit Angstrom I error stat. Chi**2 R-fac R-fac
> 50.00 7.53 45.4 1.6 1.3 1.295 0.055 0.047
> 7.53 5.98 11.4 1.3 1.3 0.672 0.135 0.114
> 5.98 5.23 11.2 1.6 1.6 0.643 0.171 0.152
> 5.23 4.75 16.8 2.0 1.9 0.736 0.148 0.118
> 4.75 4.41 18.8 2.2 2.2 0.739 0.143 0.132
> 4.41 4.15 14.6 2.4 2.4 0.653 0.190 0.175
> 4.15 3.94 11.3 2.5 2.5 0.582 0.247 0.226
> 3.94 3.77 10.1 2.8 2.8 0.511 0.280 0.191
> 3.77 3.63 8.0 3.1 3.1 0.450 0.315 0.285
> 3.63 3.50 7.6 3.3 3.2 0.483 0.311 0.270
> All reflections 15.5 2.3 2.2 0.694 0.153 0.106
> Now, I want you to help me by answering some of my queries:
> 1. Is it possible to get MAD/SAD phasing done from a dataset having more
> than 15% Rmerge and resolution in the range of 4 - 4.5 Ang?
> 2. Will a complete data set obtained from merging various batches(30-40
> frames each) from one or more than one crystal will have proper anomalous
> signal for phasing? I am worried as weak anomalous signal may get lost while
> 3. Will such a low value of average Intensities (as shown above from HKL
> scale log file) will be good enough for MAD/SAD phasing or I really need to
> improve crystal quality for stronger diffraction.
> 4. For MAD/SAD phasing, till what resolution we need to have anomalous
> signal ? Many of my datasets shows anomalous signal maximally up to 6-8 A
> (calculated using Phenix.xtriage).
> 5. Since I have low resolution (3.5 to 4 A)data, relatively high Rmerge
> (14-15%), lower value of average intensity, anomalous signal up to 6 A or
> so..... which programs will be more useful for heavy atom location and to
> prevent false positives from being selected?
> We have been also trying our luck with heavy atom soak but that also has
> not been very encouraging. I would appreciate any suggestions in this
> Thanks in advance and sorry for such a long mail.
> phenixbb mailing list
> phenixbb at phenix-online.org
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