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This is a continuation of [[1Y13]] investigating how much the pseudo-SAD structure solution performed in that article can be improved by using both wavelengths separately. | This is a continuation of [[1Y13]] investigating how much the pseudo-SAD structure solution performed in that article can be improved by using both wavelengths separately. | ||
Please note that the "second parts" of both E1 and E2 were not used, in order to be more strictly comparable to the analysis as pseudo-SAD done before. This is XSCALE.INP | Please note that the "second parts" of both E1 and E2 were not used, in order to be more strictly comparable to the analysis as pseudo-SAD done before. | ||
== XSCALE using zero-dose extrapolation == | |||
This is XSCALE.INP as in [[1Y13]], but this time using different output files: | |||
UNIT_CELL_CONSTANTS=103.316 103.316 131.456 90.000 90.000 90.000 | UNIT_CELL_CONSTANTS=103.316 103.316 131.456 90.000 90.000 90.000 | ||
SPACE_GROUP_NUMBER=96 | SPACE_GROUP_NUMBER=96 | ||
OUTPUT_FILE=ip.ahkl | OUTPUT_FILE=ip.ahkl | ||
INPUT_FILE=../e1_1-372/XDS_ASCII.HKL | INPUT_FILE=../e1_1-372/XDS_ASCII.HKL | ||
CRYSTAL_NAME=a | CRYSTAL_NAME=a | ||
OUTPUT_FILE=hrem.ahkl | OUTPUT_FILE=hrem.ahkl | ||
INPUT_FILE=../e2_1-369/XDS_ASCII.HKL | INPUT_FILE=../e2_1-369/XDS_ASCII.HKL | ||
CRYSTAL_NAME=a | CRYSTAL_NAME=a | ||
Note the use of "CRYSTAL_NAME=a" for both wavelengths. It might make sense to use different CRYSTAL_NAMEs for different heavy-atom soaks, but in this case clearly the slopes should be the same, and not depend on wavelength. | |||
The output (XSCALE.LP) is ... | The output (XSCALE.LP) is ... | ||
| Line 197: | Line 204: | ||
== hkl2map == | |||
=== SHELXC === | |||
[[File:1y13-dad-chi2-vs-resol.png]] | |||
[[File:1y13-dad-I-sigI-vs-resol.png]] | |||
[[File:1y13-dad-ddp-sigI-vs-resol.png]] | |||
[[File:1y13-dad-self-anom-cc.png]] | |||
[[File:1y13-dad-anom-cc.png]] | |||
=== SHELXD === | |||
Again we use only 3.3A data for the substructure, and have SHELXD look for 3 sites: | |||
[[File:1y13-dad-ccall-ccweak.png]] | |||
[[File:1y13-dad-histogram.png]] | |||
[[File:1y13-dad-occ.png]] | |||
This works beautifully and with a high success rate - when treating the data as pseudo-SAD, there was only 1 correct solution out of 100 trials. | |||
=== SHELXE === | |||
has no problem phasing the data: | |||
[[File:1y13-dad-contrast.png]] | |||
These are the last lines of the output of SHELXE run from hkl2map: | |||
... | |||
<wt> = 0.300, Contrast = 0.622, Connect. = 0.775 for dens.mod. cycle 40 | <wt> = 0.300, Contrast = 0.622, Connect. = 0.775 for dens.mod. cycle 40 | ||
Estimated mean FOM and mapCC as a function of resolution | Estimated mean FOM and mapCC as a function of resolution | ||
d inf - 4.62 - 3.64 - 3.17 - 2.88 - 2.67 - 2.51 - 2.38 - 2.27 - 2.18 - 2.11 | d inf - 4.62 - 3.64 - 3.17 - 2.88 - 2.67 - 2.51 - 2.38 - 2.27 - 2.18 - 2.11 | ||
| Line 220: | Line 247: | ||
<mapCC> 0.822 0.875 0.853 0.821 0.785 0.755 0.764 0.766 0.698 0.696 | <mapCC> 0.822 0.875 0.853 0.821 0.785 0.755 0.764 0.766 0.698 0.696 | ||
N 4207 4230 4223 4138 4187 4208 4292 4410 4320 3702 | N 4207 4230 4223 4138 4187 4208 4292 4410 4320 3702 | ||
Estimated mean FOM = 0.521 Pseudo-free CC = 56.08 % | Estimated mean FOM = 0.521 Pseudo-free CC = 56.08 % | ||
Density (in map sigma units) at input heavy atom sites | Density (in map sigma units) at input heavy atom sites | ||
Site x y z occ*Z density | Site x y z occ*Z density | ||
1 0.2269 0.7540 0.1175 34.0000 49.55 | 1 0.2269 0.7540 0.1175 34.0000 49.55 | ||
| Line 231: | Line 258: | ||
4 0.1805 0.5336 0.2183 13.8686 23.17 | 4 0.1805 0.5336 0.2183 13.8686 23.17 | ||
5 0.2199 0.7550 0.0807 4.1582 4.40 | 5 0.2199 0.7550 0.0807 4.1582 4.40 | ||
Site x y z h(sig) near old near new | Site x y z h(sig) near old near new | ||
1 0.2271 0.7550 0.1178 49.8 1/0.11 12/4.93 11/9.01 8/13.52 5/19.89 | 1 0.2271 0.7550 0.1178 49.8 1/0.11 12/4.93 11/9.01 8/13.52 5/19.89 | ||
| Line 240: | Line 267: | ||
6 0.0384 0.9752 0.0526 8.8 3/19.51 6/16.61 7/19.04 3/19.52 8/22.99 | 6 0.0384 0.9752 0.0526 8.8 3/19.51 6/16.61 7/19.04 3/19.52 8/22.99 | ||
[[ | At this point, I copied the "dad.hat" file with its updated substructure (which has all 6 sites) to "dad_fa.res", thus overwriting the coordinates found by SHELXD (which has 4 correct, and one wrong sites). Then I used the beta version with the same command as in [[1Y13]]: | ||
shelxe.beta -a -q -h6 -b -s0.585 -m40 -n3 dad dad_fa | |||
indeed giving 3 chains with around 155 residues, each | |||
... | |||
0 groups of atoms closer than 2.4A (e.g. disulfides) fused together for NCS | |||
3-fold NCS found, mode 2, mean deviation for all 6 input atoms = 0.142 A | |||
Overall CC between Eobs (from delF) and Ecalc (from heavy atoms) = 12.58% | |||
... | |||
... | |||
Applying NCS and splicing-in transformed chains that fit density | |||
465 residues left after pruning, divided into chains as follows: | |||
A: 150 B: 159 C: 156 | |||
CC for partial structure against native data = 42.18 % | |||
... | |||
<wt> = 0.300, Contrast = 0.825, Connect. = 0.821 for dens.mod. cycle 40 | <wt> = 0.300, Contrast = 0.825, Connect. = 0.821 for dens.mod. cycle 40 | ||
Estimated mean FOM and mapCC as a function of resolution | Estimated mean FOM and mapCC as a function of resolution | ||
d inf - 4.62 - 3.64 - 3.17 - 2.88 - 2.67 - 2.51 - 2.38 - 2.27 - 2.18 - 2.11 | d inf - 4.62 - 3.64 - 3.17 - 2.88 - 2.67 - 2.51 - 2.38 - 2.27 - 2.18 - 2.11 | ||
| Line 264: | Line 293: | ||
<mapCC> 0.846 0.898 0.932 0.930 0.921 0.929 0.931 0.925 0.889 0.873 | <mapCC> 0.846 0.898 0.932 0.930 0.921 0.929 0.931 0.925 0.889 0.873 | ||
N 4207 4230 4223 4138 4187 4208 4292 4410 4320 3702 | N 4207 4230 4223 4138 4187 4208 4292 4410 4320 3702 | ||
Estimated mean FOM = 0.675 Pseudo-free CC = 71.89 % | Estimated mean FOM = 0.675 Pseudo-free CC = 71.89 % | ||
Density (in map sigma units) at input heavy atom sites | Density (in map sigma units) at input heavy atom sites | ||
Site x y z occ*Z density | Site x y z occ*Z density | ||
1 0.2271 0.7550 0.1178 34.0000 42.57 | 1 0.2271 0.7550 0.1178 34.0000 42.57 | ||
| Line 276: | Line 305: | ||
5 0.1570 0.6337 0.3039 7.9390 22.32 | 5 0.1570 0.6337 0.3039 7.9390 22.32 | ||
6 0.0384 0.9752 0.0526 6.0078 14.61 | 6 0.0384 0.9752 0.0526 6.0078 14.61 | ||
Site x y z h(sig) near old near new | Site x y z h(sig) near old near new | ||
1 0.2276 0.7565 0.1184 42.8 1/0.18 7/2.75 8/3.22 5/19.63 3/21.97 | 1 0.2276 0.7565 0.1184 42.8 1/0.18 7/2.75 8/3.22 5/19.63 3/21.97 | ||
| Line 286: | Line 315: | ||
7 0.2484 0.7678 0.1089 -5.5 1/2.82 1/2.75 8/5.73 5/18.33 3/23.88 | 7 0.2484 0.7678 0.1089 -5.5 1/2.82 1/2.75 8/5.73 5/18.33 3/23.88 | ||
8 0.2095 0.7314 0.1210 -5.3 1/3.07 1/3.22 7/5.73 3/21.21 5/22.10 | 8 0.2095 0.7314 0.1210 -5.3 1/3.07 1/3.22 7/5.73 3/21.21 5/22.10 | ||
== What do we learn? == | |||
In no particular order: | |||
* That the dispersive signal helps a lot in substructure solution: 27 successful trial in 100 using DAD, instead of 1 using pseudo-SAD. | |||
* That the correlation coefficient between two wavelengths of a MAD experiment can be better than 0.9995 if there is no difference in radiation damage (in other words, the dispersive signal does not seem to significantly lower the correlation). | |||
* That zero-dose extrapolation helps a lot, and works very well: if it is not done, we obtain only 5 correct solutions out of 100, and the highest CCall / CCweak is 17.85 / 12.33 instead of 36.34 / 25.24 (I don't show the plots here). | |||
* That the wavelength change only takes 3 seconds at this beamline, which makes such an experiment really attractive. | |||
== Availability of data == | |||
The directory [https://{{SERVERNAME}}/pub/xds-datared/1y13] has tarballs of the raw data. The XDS-processed data are available at [https://{{SERVERNAME}}/pub/xds-datared/1y13/e1_1-372_XDS_ASCII.HKL.bz2] and [https://{{SERVERNAME}}/pub/xds-datared/1y13/e2_1-369_XDS_ASCII.HKL.bz2]. | |||