<span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: arial; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px;">We merged .x files only. Only in this case, we have seen such a low value of average I (in all the datasets). We used HKL 2000 with refinement sigma cut-off 5.</span><br>
<br><div class="gmail_quote">2009/3/20 Schubert, Carsten [PRDUS] <span dir="ltr"><<a href="mailto:CSCHUBER@its.jnj.com">CSCHUBER@its.jnj.com</a>></span><br><blockquote class="gmail_quote" style="border-left: 1px solid rgb(204, 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;">
<div>
<div><span><font color="#0000ff" face="Cambria">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? </font></span></div><div><div></div><div class="h5">
<blockquote>
<div dir="ltr" align="left"><font size="2" face="Tahoma">-----Original Message-----<br><b>From:</b>
<a href="mailto:phenixbb-bounces@phenix-online.org" target="_blank">phenixbb-bounces@phenix-online.org</a>
[mailto:<a href="mailto:phenixbb-bounces@phenix-online.org" target="_blank">phenixbb-bounces@phenix-online.org</a>]<b>On Behalf Of
</b>Kumar<br><b>Sent:</b> Friday, March 20, 2009 3:42 PM<br><b>To:</b>
<a href="mailto:phenixbb@phenix-online.org" target="_blank">phenixbb@phenix-online.org</a><br><b>Subject:</b> [phenixbb] Problems with phasing
a protein (1300aa)<br><br></font></div><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>
</blockquote></div></div></div>
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<br></blockquote></div><br>