Dear Alison,
Christian Dumas of our institute has recently developed and published a
program for HA detection. Being extremely powerful, and easy to use
(http://www.cbs.cnrs.fr/SP/crystal/SUPERFLIP/), it has produced very precise
HA positions for datasets that no other program could treat previously. Even
space group issues may be overcome by simply working in P1. It's frankly
quite an astonishing piece of software.
Good luck
Stefan
==================================================
Stefan T. Arold, PhD
Centre de Biochimie Structurale
CNRS UMR 5048 - UM 1 - INSERM UMR 554
29 rue de Navacelles
34090 MONTPELLIER Cedex - France
email: [log in to unmask]
Phone: +33 (0)4.67.41.77.02
Fax: +33 (0)4.67.41.79.13
==================================================
----- Original Message -----
From: "Alison Li" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Thursday, January 29, 2009 10:37 PM
Subject: [ccp4bb] difficult MAD dataset
> We recently collected a complete 2.5A MAD dataset. However, finding a
> solution has not been as straightfoward for reasons unclear to us. We
> would
> be grateful for any helpful advice or suggestions.
>
> The thin plate shaped crystal was grown from a relatively small protein
> (90
> residues).
>
> The crystal diffracted well with visible and defined reflections up to
> ~2.8A
> range.
>
> With both HKL2000 and mosflm, initial indexing indicated orthorhombic unit
> cell
> with dimension of 52 x 82 x 100.
>
> Systematic absences along a and b axis were observed thus the dataset was
> scaled in P21212 space group.
>
> The unit cell dimension and space group suggested 4 protein chains per
> ASU.
>
> There is a pseudo-translation with 55 % peak at a fractional coordinate of
> 0.5, 0.5, 0.48.
>
> There are 7 methionines per chain. Thus we expect 28 Se per ASU. Mass
> spectroscopy and fluoresence scan both confirmed successful incorporation
> of
> Se-methionine in the crystal.
>
> According to xtriage, anomalous signal extended to 3.0A at least in the
> peak
> dataset (The table of measurability as a function of resolution is shown
> below).
>
> unused: - 43.0868 [ 0/5 ]
> bin 1: 43.0868 - 5.3953 [2751/2902] 0.5838
> bin 2: 5.3953 - 4.2836 [2893/2905] 0.4575
> bin 3: 4.2836 - 3.7425 [2891/2899] 0.2820
> bin 4: 3.7425 - 3.4005 [2888/2896] 0.1898
> bin 5: 3.4005 - 3.1568 [2864/2898] 0.1222
> bin 6: 3.1568 - 2.9708 [2835/2898] 0.0653
> bin 7: 2.9708 - 2.8220 [2777/2906] 0.0458
> bin 8: 2.8220 - 2.6992 [2714/2902] 0.0226
> bin 9: 2.6992 - 2.5953 [2550/2865] 0.0168
> bin 10: 2.5953 - 2.5057 [2307/2914] 0.0071
> unused: 2.5057 - [ 0/0 ]
>
> However, HA search using hkl2map, Autosol, and Autosharp resulted in only
> 3~4 HA sites. When hkl2map was used, most HA sites had poor CC and
> Patterson FOM and did not clearly stand out as they normally should.
>
>
> Structural homologs suggest that the protein has a compact single core
> domain comprised of 4 a-helices. The positions of most HAs are unlikely to
> be
> located in the flexible region.
> If any abnormalies are seen with the dataset, it's during scaling step in
> HKL2000.
> Chi2 is unusually high at lower resolution (Chi2 is >3 from 3.5A as shown
> below) and there is a relatively high percentage of rejections (>1.5 %).
>
> Shell Lower Upper Average Average Norm. Linear Square
> limit Angstrom I error stat. Chi**2 R-fac R-fac
> 50.00 5.38 4299.0 77.3 49.0 7.886 0.076 0.085
> 5.38 4.27 2938.7 52.7 35.0 5.843 0.083 0.090
> 4.27 3.73 2314.2 45.9 33.5 3.935 0.082 0.084
> 3.73 3.39 1245.3 34.4 28.9 2.838 0.101 0.094
> 3.39 3.15 658.6 28.1 25.9 1.957 0.132 0.127
> 3.15 2.96 451.9 27.1 25.7 1.322 0.157 0.138
> 2.96 2.82 307.2 27.1 26.3 1.001 0.201 0.169
> 2.82 2.69 253.1 28.8 28.1 0.866 0.225 0.193
> 2.69 2.59 199.3 31.5 31.0 0.801 0.262 0.233
> 2.59 2.50 159.5 34.7 34.4 0.688 0.292 0.261
> All reflections 1312.9 39.0 31.9 2.748 0.100 0.089
>
> Xtriage also complains that there are abnormal intensities at some
> resolution
> ranges.
>
> Finally, the crystallization requires CdSO4, so we suspect that cadmium
> ions
> are incorporated in the crystal. If so, we suspect there may be weak
> anomalous signal contribution from Cd as well.
>
> In summary, we appear to have a complete dataset that shows strong
> anomalous signal. However it appears that we have overlooked something or
> there is an unusual crystallographic issue that we are not aware of. Any
> suggestions will be very much appreciated.
>
> Alison Li
> Graudate student, Dr. Mark Paetzel's group
> Simon Fraser University, BC, Canada
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