Hi Alison,
At how many (and which) wavelengths were data collected?
How much data at each wavelength?
Which option was used in scaling with HKL2000 ("anomalous", "scale
anomalous" or neither)?
Any indication of radiation damage during data collection (i.e.
disappearing higher resolution spots) or during scaling (i.e. increasing
B-factor)?
Which maps were used for heavy atom search?
Let's answer these for starters.
N.
Ruslan Sanishvili (Nukri), Ph.D.
GM/CA-CAT, Bld. 436, D007
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439
Tel: (630)252-0665
Fax: (630)252-0667
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> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Alison Li
> Sent: Thursday, January 29, 2009 3:38 PM
> To: [log in to unmask]
> 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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