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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 statistics | The structure was "solved" using the [http://schneider.group.ifom-ieo-campus.it/hkl2map/ hkl2map] GUI. Based on the statistics | ||
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[[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 version | 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 (see | 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 == | == SAD: Inflection data alone == | ||
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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/) === | ||
In the same way as with the peak data, Auto-Rickshaw was started. Please see the | 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). | 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). | ||