[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
gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D
8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://phenix-online.org/pipermail/phenixbb/attachments/20090320/d8676ea6/attachment-0003.htm>
More information about the phenixbb
mailing list