[phenixbb] Problems with phasing a protein (1300aa)

[Engin Ozkan]

In the recent versions of HKL2000, there is a scale for I. Just change 
that scale, and your I's and sigmas will change accordingly.

Engin

Schubert, Carsten [PRDUS] wrote:
> 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 merging.
>
>     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 regard.
>     Thanks in advance and sorry for such a long mail.
>     Kumar 
>
> ------------------------------------------------------------------------
>
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