Re: [ccp4bb] difficult MAD dataset

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Fobs - Fobs
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2009-02-02

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Subject: Re: difficult MAD dataset
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2009-02-02

With a pseudo translation vector like that the SG could be any of the 8
orthorhombic SGs; P222 P21 22 P21212 P212121 P2 21 2 P2 21 21 P 2 2 21

Test them all, and see if any give a dect solution..
Eleanor

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
>
>
>
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Fobs - Fobs
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2009-02-02

Next message:
Subject: Postdoctoral Position in Structural Biology
From: Choel Kim ckim {- at -} BCM {- dot -} EDU
Date: 2009-02-02