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

Schubert, Carsten [PRDUS] CSCHUBER at its.jnj.com
Fri Mar 20 12:53:15 PDT 2009

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.

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