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==Thaumatin Br soak: structure solution from either 2-wavelength MAD, or from single wavelength SAD== | ==Thaumatin Br soak: structure solution from either 2-wavelength MAD, or from single wavelength SAD== | ||
Thaumatin is a sweet-tasting protein of 207 residues that crystallizes in P4<sub>1</sub>2<sub>1</sub>2 with a monomer in the asymmetric unit. | |||
Prior to flash cooling in liquid nitrogen at 100 K the crystals were soaked for a few seconds in a solution containing 1 M sodium bromide and 25% (v/v) glycerol. | Prior to flash cooling in liquid nitrogen at 100 K the crystals were soaked for a few seconds in a solution containing 1 M sodium bromide and 25% (v/v) glycerol. | ||
The peak and inflection datasets (360 images each) are available from the [http://www.embl-hamburg.de EMBL Hamburg] website. The XDS data reductions are [ | The peak and inflection datasets (360 images each) are available from the [http://www.embl-hamburg.de/Xray_Tutorial/ EMBL Hamburg] website (alternatively from [http://www.mx.bessy.de/bessy-ws/experiment2.html]). The XDS data reductions and (some of the) phasing calculations are [https://{{SERVERNAME}}/pub/xds-datared/1rqw here]. | ||
Data reduction was performed starting with the [[XDS.INP#MarCDD_225mm_.40_SLS.2C_BL_X06SA|template for the MarCCD 225]] detector, changing | Data reduction was performed starting with the [[XDS.INP#MarCDD_225mm_.40_SLS.2C_BL_X06SA|template for the MarCCD 225]] detector, changing | ||
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== SAD: Peak data alone == | == SAD: Peak data alone == | ||
G. Sheldrick [[ccp4com:SHELX_C/D/E#critical_parameters|recommends]]: "For iodide soaks, a good rule of thumb is to start with a number of iodide sites equal to the number of amino-acids in the asymmetric unit divided by 15." Bromine should behave similarly, so we expect roughly 14 sites. | |||
=== manual structure solution using hkl2map and buccaneer === | === manual structure solution using hkl2map and buccaneer === | ||
The structure was "solved" using the [http://schneider.group.ifom-ieo-campus.it/hkl2map/ hkl2map] GUI. Based on | The structure was "solved" using the [http://schneider.group.ifom-ieo-campus.it/hkl2map/ hkl2map] GUI. Based on the statistics | ||
Resl. Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.5 - 3.0 - 2.6 - 2.4 - 2.2 - 2.0 - 1.80 | |||
N(data) 346 425 519 1144 1119 1964 2834 2163 3043 4326 6351 | |||
Chi-sq 0.71 1.08 0.44 1.03 1.17 1.06 1.06 1.09 1.10 1.08 1.10 | |||
I/sig 47.4 41.8 45.5 50.7 46.1 36.0 22.6 15.4 12.0 8.0 4.1 | |||
%Complete 94.8 98.6 99.0 99.5 99.6 99.6 99.8 99.8 99.8 99.8 99.6 | |||
<d"/sig> 3.13 2.58 2.13 1.90 1.52 1.36 1.16 1.04 0.96 0.91 0.85 | |||
CC(anom) 79.0 78.5 67.1 61.1 45.6 32.4 23.2 15.1 9.0 3.8 -3.6 | |||
[[Image:1rqw-peak-self-anomCC-resolution.png]] [[Image:1rqw-peak-d"sig-resolution.png]] | [[Image:1rqw-peak-self-anomCC-resolution.png]] [[Image:1rqw-peak-d"sig-resolution.png]] | ||
I decided to use 3.3 Å as a suitable cutoff for solving the substructure, and to let SHELXD search for 20 Br atoms | I decided to use 3.3 Å as a suitable cutoff for solving the substructure, and to let SHELXD search for 20 Br atoms. SHELXD then found a convincing solution: | ||
[[Image:1rqw-peak-ccall-ccweak.png]] [[Image:1rqw-peak-histogram-ccall.png]] [[Image:1rqw-peak-occupancy.png]] | [[Image:1rqw-peak-ccall-ccweak.png]] [[Image:1rqw-peak-histogram-ccall.png]] [[Image:1rqw-peak-occupancy.png]] | ||
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=== automatic structure solution using Auto-Rickshaw (http://www.embl-hamburg.de/Auto-Rickshaw/) === | === automatic structure solution using Auto-Rickshaw (http://www.embl-hamburg.de/Auto-Rickshaw/) === | ||
This uses Santosh Panjikar's script DPS2AR.csh: | This uses Santosh Panjikar's script [http://www.embl-hamburg.de/Auto-Rickshaw/DPS2AR DPS2AR.csh] version 1.04: | ||
DPS2AR.csh datafile1 thau-peak-I.mtz keeps world ver completeversion nres 207 nsm 20 masu 1 meth SAD SG P41212 \ | DPS2AR.csh datafile1 thau-peak-I.mtz keeps world ver completeversion nres 207 nsm 20 masu 1 meth SAD SG P41212 \ | ||
email kay.diederichs@uni-konstanz.de sequencefile 1rqw.seq | email kay.diederichs@uni-konstanz.de sequencefile 1rqw.seq | ||
thau-peak-I.mtz was obtained in two steps: | thau-peak-I.mtz was obtained in two steps: | ||
a) generating temp.hkl from XDS_ASCII.HKL with the following XDSCONV.INP: | a) generating temp.hkl from XDS_ASCII.HKL with the following XDSCONV.INP: | ||
INPUT_FILE=XDS_ASCII.HKL | INPUT_FILE=XDS_ASCII.HKL | ||
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FSYVLDKPTT VTCPGSSNYR VTFCPTA | FSYVLDKPTT VTCPGSSNYR VTFCPTA | ||
The result of this is a model that only lacks residues 1, 82, 83, 207, and which has a "core RMS" (from coot's SSM superpose) of 0.14 Å against 1rqw (which is a 1.05 Å structure). | The result of this (see the [http://webapps.embl-hamburg.de/lresult/421317/LOG/viewlog/result.html Auto-Rickshaw logfile]) is a model that only lacks residues 1, 82, 83, 207, and which has a "core RMS" (from coot's SSM superpose) of 0.14 Å against 1rqw (which is a 1.05 Å structure). | ||
It couldn't be simpler than that. Thanks, Santosh! | It couldn't be simpler than that. Thanks, Santosh! | ||
== SAD: Inflection data alone == | |||
This is of course much more difficult, because the anomalous data are weaker (and as this is the second dataset collected from the crystal maybe there is already a little bit of radiation damage). | |||
A lot of care was put into the XDS data reduction - many of the ideas that can be found in this wiki were employed to get as good data as possible. | |||
The result is that the anomalous signal is useful - this is evident from the SHELXC statistics: | |||
Resl. Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.5 - 3.0 - 2.6 - 2.4 - 2.2 - 2.0 - 1.80 | |||
N(data) 346 427 519 1142 1123 1972 2830 2173 3043 4340 6339 | |||
I/sig 54.8 48.4 52.4 56.6 51.2 40.6 26.0 18.1 13.9 9.4 4.8 | |||
%Complete 94.8 98.6 99.0 99.5 99.6 99.6 99.8 99.8 99.8 99.8 99.8 | |||
<d"/sig> 2.59 2.18 1.86 1.58 1.29 1.18 1.04 0.95 0.91 0.88 0.81 | |||
[[Image:1rqw-inf-d"-sig.png]] [[Image:1rqw-inf-i-sig.png]] | |||
This time I tried to solve the substructure at 3.6 Å resolution, again 20 sites - | |||
[[Image:1rqw-inf-ccall-ccweak.png]] [[Image:1rqw-inf-ccall-patfom.png]] [[Image:1rqw-inf-ccall-try.png]] | |||
So there are indeed much less good trials: | |||
[[Image:1rqw-inf-histogram-ccall.png]] [[Image:1rqw-inf-occupancy-peak.png]] | |||
SHELXE was run with 53.5% solvent: | |||
[[Image:1rqw-inf-contrast-cycle.png]] [[Image:1rqw-inf-connectivity.png]] [[Image:1rqw-inf-ccmap-resolution.png]] | |||
The structure can be solved from here, with Arp/Warp and probably also with buccaneer/refmac. | |||
=== automatic structure solution using Auto-Rickshaw (http://www.embl-hamburg.de/Auto-Rickshaw/) === | |||
In the same way as with the peak data, Auto-Rickshaw was started. Please see the [http://webapps.embl-hamburg.de/lresult/542521/LOG/viewlog/result.html Auto-Rickshaw logfile]. | |||
The structure could be solved but Arp/Warp had a difficult start - take a look at the [[1rqw_arp_warp_log|Arp/Warp logfile]]! The final model lacks residues 1, 85, 205, 206, 207 and the "core RMS" of SSM superpose, against 1rqw, is 0.15 Å (better than I would expect). | |||
It should also be noted that the structure can ''not'' be solved when searching for 15 or 28 sites - rather, 20 appears to be the magic number. | |||
Once again, great job, Santosh (and of course those people who wrote the programs that Auto-Rickshaw uses)! | |||