<span style="font-family: comic sans ms,sans-serif;">Hello Phenixbb members,</span><br style="font-family: comic sans ms,sans-serif;"><br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">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. </span><br style="font-family: comic sans ms,sans-serif;">
<br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">'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.</span><br style="font-family: comic sans ms,sans-serif;">
<br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">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).</span><br style="font-family: comic sans ms,sans-serif;">
<font size="2"><span style="font-family: courier new,monospace;">Shell Lower Upper Average � � �Average � � Norm. Linear Square</span><br style="font-family: courier new,monospace;"><span style="font-family: courier new,monospace;"> limit � �Angstrom � � � I � error � stat. Chi**2 �R-fac �R-fac</span><br style="font-family: courier new,monospace;">
<span style="font-family: courier new,monospace;"> � � �50.00 � 7.53 � �45.4 � � 1.6 � � 1.3 �1.295 �0.055 �0.047</span><br style="font-family: courier new,monospace;"><span style="font-family: courier new,monospace;"> � � � 7.53 � 5.98 � �11.4 � � 1.3 � � 1.3 �0.672 �0.135 �0.114</span><br style="font-family: courier new,monospace;">
<span style="font-family: courier new,monospace;"> � � � 5.98 � 5.23 � �11.2 � � 1.6 � � 1.6 �0.643 �0.171 �0.152</span><br style="font-family: courier new,monospace;"><span style="font-family: courier new,monospace;"> � � � 5.23 � 4.75 � �16.8 � � 2.0 � � 1.9 �0.736 �0.148 �0.118</span><br style="font-family: courier new,monospace;">
<span style="font-family: courier new,monospace;"> � � � 4.75 � 4.41 � �18.8 � � 2.2 � � 2.2 �0.739 �0.143 �0.132</span><br style="font-family: courier new,monospace;"><span style="font-family: courier new,monospace;"> � � � 4.41 � 4.15 � �14.6 � � 2.4 � � 2.4 �0.653 �0.190 �0.175</span><br style="font-family: courier new,monospace;">
<span style="font-family: courier new,monospace;"> � � � 4.15 � 3.94 � �11.3 � � 2.5 � � 2.5 �0.582 �0.247 �0.226</span><br style="font-family: courier new,monospace;"><span style="font-family: courier new,monospace;"> � � � 3.94 � 3.77 � �10.1 � � 2.8 � � 2.8 �0.511 �0.280 �0.191</span><br style="font-family: courier new,monospace;">
<span style="font-family: courier new,monospace;"> � � � 3.77 � 3.63 � � 8.0 � � 3.1 � � 3.1 �0.450 �0.315 �0.285</span><br style="font-family: courier new,monospace;"><span style="font-family: courier new,monospace;"> � � � 3.63 � 3.50 � � 7.6 � � 3.3 � � 3.2 �0.483 �0.311 �0.270</span><br style="font-family: courier new,monospace;">
<span style="font-family: courier new,monospace;"> �All reflections � � 15.5 � � 2.3 � � 2.2 �0.694 �0.153 �0.106</span></font>
<br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">� </span><br style="font-family: comic sans ms,sans-serif;">
<span style="font-family: comic sans ms,sans-serif;">Now, I want you to help me by answering some of my queries:</span><br style="font-family: comic sans ms,sans-serif;"><br style="font-family: comic sans ms,sans-serif;">
<span style="font-family: comic sans ms,sans-serif;">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?<br><br>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.<br><br>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.</span><br style="font-family: comic sans ms,sans-serif;">
<br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">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).</span><br style="font-family: comic sans ms,sans-serif;">
<br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">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?</span><br style="font-family: comic sans ms,sans-serif;">
<br style="font-family: comic sans ms,sans-serif;"><span style="font-family: courier new,monospace;"><span style="font-family: comic sans ms,sans-serif;">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.</span><br style="font-family: comic sans ms,sans-serif;">
<span style="font-family: comic sans ms,sans-serif;">Thanks in advance and sorry for such a long mail.</span><br style="font-family: comic sans ms,sans-serif;"><span style="font-family: comic sans ms,sans-serif;">Kumar</span></span>