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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 [ftp://turn5.biologie.uni-konstanz.de//pub/xds-datared/1rqw here].
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 (just a guess!). SHELXD then found a convincing solution:
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 ==
== SAD: Inflection data alone ==
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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).
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).


This is evident from the SHELXC statistics -
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
  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
  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
  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
  %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
  <d"/sig>  2.59  2.18  1.86  1.58  1.29  1.18  1.04  0.95  0.91  0.88  0.81
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[[Image:1rqw-inf-contrast-cycle.png]] [[Image:1rqw-inf-connectivity.png]] [[Image:1rqw-inf-ccmap-resolution.png]]
[[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)!
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