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

Francis E Reyes Francis.Reyes at Colorado.EDU
Fri Mar 20 12:48:46 PDT 2009


I only say this because it happened to me recently (as opposed to  
having extensive success), but since you have a native dataset have  
you tried a MIRAS or SIRAS?  I recently was able to solve a structure  
(though nowhere near your size) that didn't solve with SAD but did  
solve with SIRAS.

Cheers

FR

On Mar 20, 2009, at 1:41 PM, Kumar wrote:

> 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

---------------------------------------------
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder

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