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→Second try: correcting radiation damage at the level of individual reflections
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=== Second try: correcting radiation damage at the level of individual reflections === | === Second try: correcting radiation damage at the level of individual reflections === | ||
Since we noted significant radiation damage we could try to correct that. All we have to do is ask XSCALE to do | Since we noted significant radiation damage we could try to correct that. All we have to do is ask XSCALE to do zero-dose extrapolation: | ||
<pre> | <pre> | ||
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 | ||
| Line 587: | Line 587: | ||
[[File:1y13-raddam-site-occ-raddam.png]] | [[File:1y13-raddam-site-occ-raddam.png]] | ||
[[File:1y13-raddam-contrast-raddam.png]] | [[File:1y13-raddam-contrast-raddam.png]] | ||
== Automatically building almost 3/4 of the main chain == | |||
Based on the sites obtained by SHELXD, we run | |||
shelxe.beta -a -q -h -b -s0.585 -m40 raddam raddam_fa | |||
This already builds a significant number of residues, but also gives an improved list of heavy atom sites - there are actually 6 sites instead of the 5 that SHELXD wrote out (yes, we had asked SHELXD for 3 sites since there are 3 Met residues, but SHELXD as always was smarter than we are). We "mv raddam.hat raddam_fa.res" for another run of SHELXE: | |||
shelxe.beta -a -q -h6 -b -s0.585 -m40 raddam raddam_fa | |||
and get | |||
<pre> | |||
374 residues left after pruning, divided into chains as follows: | |||
A: 43 B: 37 C: 17 D: 19 E: 7 F: 16 G: 16 H: 5 I: 13 | |||
J: 5 K: 9 L: 40 M: 81 N: 12 O: 15 P: 7 Q: 5 R: 8 | |||
S: 13 T: 6 | |||
CC for partial structure against native data = 34.56 % | |||
------------------------------------------------------------------------------ | |||
Global autotracing cycle 4 | |||
<wt> = 0.300, Contrast = 0.484, Connect. = 0.705 for dens.mod. cycle 1 | |||
<wt> = 0.300, Contrast = 0.704, Connect. = 0.780 for dens.mod. cycle 2 | |||
<wt> = 0.300, Contrast = 0.761, Connect. = 0.799 for dens.mod. cycle 3 | |||
<wt> = 0.300, Contrast = 0.795, Connect. = 0.805 for dens.mod. cycle 4 | |||
Pseudo-free CC = 65.99 % | |||
<wt> = 0.300, Contrast = 0.817, Connect. = 0.810 for dens.mod. cycle 5 | |||
<wt> = 0.300, Contrast = 0.834, Connect. = 0.813 for dens.mod. cycle 6 | |||
<wt> = 0.300, Contrast = 0.844, Connect. = 0.816 for dens.mod. cycle 7 | |||
<wt> = 0.300, Contrast = 0.852, Connect. = 0.818 for dens.mod. cycle 8 | |||
<wt> = 0.300, Contrast = 0.856, Connect. = 0.820 for dens.mod. cycle 9 | |||
Pseudo-free CC = 70.07 % | |||
<wt> = 0.300, Contrast = 0.859, Connect. = 0.821 for dens.mod. cycle 10 | |||
<wt> = 0.300, Contrast = 0.860, Connect. = 0.822 for dens.mod. cycle 11 | |||
<wt> = 0.300, Contrast = 0.861, Connect. = 0.822 for dens.mod. cycle 12 | |||
<wt> = 0.300, Contrast = 0.861, Connect. = 0.823 for dens.mod. cycle 13 | |||
<wt> = 0.300, Contrast = 0.861, Connect. = 0.823 for dens.mod. cycle 14 | |||
Pseudo-free CC = 70.84 % | |||
<wt> = 0.300, Contrast = 0.860, Connect. = 0.824 for dens.mod. cycle 15 | |||
<wt> = 0.300, Contrast = 0.860, Connect. = 0.824 for dens.mod. cycle 16 | |||
<wt> = 0.300, Contrast = 0.859, Connect. = 0.824 for dens.mod. cycle 17 | |||
<wt> = 0.300, Contrast = 0.858, Connect. = 0.824 for dens.mod. cycle 18 | |||
<wt> = 0.300, Contrast = 0.857, Connect. = 0.824 for dens.mod. cycle 19 | |||
Pseudo-free CC = 70.93 % | |||
<wt> = 0.300, Contrast = 0.856, Connect. = 0.824 for dens.mod. cycle 20 | |||
<wt> = 0.300, Contrast = 0.856, Connect. = 0.824 for dens.mod. cycle 21 | |||
<wt> = 0.300, Contrast = 0.855, Connect. = 0.824 for dens.mod. cycle 22 | |||
<wt> = 0.300, Contrast = 0.854, Connect. = 0.825 for dens.mod. cycle 23 | |||
<wt> = 0.300, Contrast = 0.853, Connect. = 0.824 for dens.mod. cycle 24 | |||
Pseudo-free CC = 70.85 % | |||
<wt> = 0.300, Contrast = 0.853, Connect. = 0.824 for dens.mod. cycle 25 | |||
<wt> = 0.300, Contrast = 0.852, Connect. = 0.824 for dens.mod. cycle 26 | |||
<wt> = 0.300, Contrast = 0.851, Connect. = 0.825 for dens.mod. cycle 27 | |||
<wt> = 0.300, Contrast = 0.850, Connect. = 0.824 for dens.mod. cycle 28 | |||
<wt> = 0.300, Contrast = 0.850, Connect. = 0.824 for dens.mod. cycle 29 | |||
Pseudo-free CC = 70.69 % | |||
<wt> = 0.300, Contrast = 0.849, Connect. = 0.824 for dens.mod. cycle 30 | |||
<wt> = 0.300, Contrast = 0.849, Connect. = 0.825 for dens.mod. cycle 31 | |||
<wt> = 0.300, Contrast = 0.848, Connect. = 0.824 for dens.mod. cycle 32 | |||
<wt> = 0.300, Contrast = 0.848, Connect. = 0.824 for dens.mod. cycle 33 | |||
<wt> = 0.300, Contrast = 0.847, Connect. = 0.824 for dens.mod. cycle 34 | |||
Pseudo-free CC = 70.51 % | |||
<wt> = 0.300, Contrast = 0.847, Connect. = 0.824 for dens.mod. cycle 35 | |||
<wt> = 0.300, Contrast = 0.846, Connect. = 0.824 for dens.mod. cycle 36 | |||
<wt> = 0.300, Contrast = 0.846, Connect. = 0.824 for dens.mod. cycle 37 | |||
<wt> = 0.300, Contrast = 0.845, Connect. = 0.824 for dens.mod. cycle 38 | |||
<wt> = 0.300, Contrast = 0.845, Connect. = 0.824 for dens.mod. cycle 39 | |||
Pseudo-free CC = 70.35 % | |||
<wt> = 0.300, Contrast = 0.844, Connect. = 0.824 for dens.mod. cycle 40 | |||
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 | |||
<FOM> 0.712 0.774 0.761 0.710 0.694 0.682 0.622 0.598 0.582 0.534 | |||
<mapCC> 0.841 0.923 0.941 0.927 0.920 0.926 0.926 0.915 0.877 0.853 | |||
N 4206 4227 4214 4135 4185 4207 4292 4406 4320 3702 | |||
Estimated mean FOM = 0.668 Pseudo-free CC = 70.35 % | |||
Density (in map sigma units) at input heavy atom sites | |||
Site x y z occ*Z density | |||
1 0.2273 0.7578 0.1190 34.0000 30.83 | |||
2 0.1770 0.5343 0.2167 29.6922 29.13 | |||
3 0.1568 0.6341 0.3057 27.3088 29.14 | |||
4 0.3059 0.4523 0.1308 25.2552 23.34 | |||
5 0.0287 0.8253 0.1396 20.5870 20.24 | |||
6 0.0389 0.9744 0.0501 14.1270 19.44 | |||
Site x y z h(sig) near old near new | |||
1 0.2276 0.7578 0.1189 30.8 1/0.04 8/17.51 10/19.34 2/19.52 5/21.94 | |||
2 0.1568 0.6345 0.3049 29.3 3/0.11 8/3.01 3/15.74 1/19.52 7/22.22 | |||
3 0.1767 0.5344 0.2160 29.3 2/0.10 2/15.74 8/16.49 4/19.41 1/26.90 | |||
4 0.3059 0.4535 0.1297 23.7 4/0.19 3/19.41 9/26.54 6/26.81 6/28.31 | |||
5 0.0280 0.8243 0.1410 20.6 5/0.23 6/19.72 9/21.55 7/21.87 1/21.94 | |||
6 0.0383 0.9748 0.0492 19.5 6/0.15 9/2.58 9/15.62 6/15.91 5/19.72 | |||
7 0.1847 0.9579 0.1793 -5.2 5/21.79 5/21.87 2/22.22 1/22.59 9/22.61 | |||
8 0.1842 0.6442 0.3069 -4.9 3/3.02 2/3.01 3/16.49 1/17.51 10/22.77 | |||
9 0.0430 0.9990 0.0523 -4.8 6/2.59 6/2.58 9/15.17 6/15.62 5/21.55 | |||
10 0.3437 0.8355 0.0209 4.6 1/19.38 1/19.34 8/22.77 2/23.02 5/27.73 | |||
</pre> | |||
At this point the structure is obviously solved, and we could use buccanneer or Arp/wArp to add side chains and the rest of the model. 3-fold NCS surely helps! | |||
== Could we do better? == | |||
Yes, of course (as always). I can think of three things to try: | |||
* an [[optimization]] round of running xds for the two datasets | |||
* using a negative offset for STARTING_DOSE in XSCALE.INP, as documented in the [[XSCALE]] wiki article. | |||
* adding the "secondparts" data assuming this is a longer wavelength | |||
But this time we learn that one has to take special care of the data in particular when they were measured by someone else who does not tell us everything we need to know. Second, zero-dose extrapolation made the day. | |||