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

Kumar 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
cut-off 5.

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
> 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
>
>
> _______________________________________________
> phenixbb mailing list
> phenixbb at phenix-online.org
> http://www.phenix-online.org/mailman/listinfo/phenixbb
>
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://phenix-online.org/pipermail/phenixbb/attachments/20090320/d4376efb/attachment-0003.htm>


More information about the phenixbb mailing list