|
I think you need to first address the issue of radiation damage.
Radiation damage can prevent you from solving the structure even
before it affects Rsym or I/sigma.
I haven't merged data from multiple crystals to solve a MAD structure,
but I'd try it in your case, with inverse beam data collection. You
will have to be conservative with how many frames you keep per
crystal, tossing out frames well before you see the obvious effects of
radiation damage.
I've heard of people using ascorbic acid to radioprotect their crystals.
Were your native crystals also so radiation sensitive? If so, you
could also try mutating away cysteines, which are often the most
sensitive.
Ho
UC Berkeley
--------------------------------------------------------------------------------------------
From: Kumar <[log in to unmask]>
Subject: Problems with phasing a protein (1300aa)
--001485f7c1aada34ff046591e45f
Content-Type: text/plain; charset=ISO-8859-1
Content-Transfer-Encoding: 7bit
Hello CCP4bb 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
|
