Hi Allison,
How sure are you of your spacegroup? Have you tried P212121? It is a much more common spacegroup. How did you look for your systematic absences? It is possible that the systematic absences are also present along the third axis even though all the reflections have not been measured. Scalepack would not show "absences" for the third axis at the end of the logfile if none of the supposed-to-be absent reflections were measured so it could mislead you.
good luck,
Eric
__________________________
Eric Larson, PhD
MSGPP Consortium
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195
On Thu, 29 Jan 2009, Alison Li wrote:
> 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
>
|