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1Y13: Difference between revisions
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The structure is [http://www.rcsb.org/pdb/explore/explore.do?structureId=1Y13 deposited] in the PDB, solved with SAD and refined at a resolution of 2.2 A in spacegroup P4(3)2(1)2 (#96). | The structure is [http://www.rcsb.org/pdb/explore/explore.do?structureId=1Y13 deposited] in the PDB, solved with SAD and refined at a resolution of 2.2 A in spacegroup P4(3)2(1)2 (#96). | ||
The data for this project were provided by Jürgen Bosch (SGPP) and are linked to [http://bl831.als.lbl.gov/example_data_sets/ACA2011/DPWTP-website/index.html the ACA 2011 workshop website]. | The data for this project were provided by Jürgen Bosch (SGPP) and are linked to [http://bl831.als.lbl.gov/example_data_sets/ACA2011/DPWTP-website/index.html the ACA 2011 workshop website] and [https://{{SERVERNAME}}/pub/xds-datared/1y13/ here]. | ||
There are two high-resolution (2 Å) datasets E1 (wavelength 0.9794Å) and E2 (@ 0.9174Å) collected (with 0.25° increments) at an ALS beamline on June 27, 2004, and a weaker dataset collected earlier at a SSRL beamline. We will only use the former two datasets here. | There are two high-resolution (2 Å) datasets E1 (wavelength 0.9794Å) and E2 (@ 0.9174Å) collected (with 0.25° increments) at an ALS beamline on June 27, 2004, and a weaker dataset collected earlier at a SSRL beamline. We will only use the former two datasets here. | ||
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a b ISa | a b ISa | ||
6.058E+00 3.027E-04 23.35 | 6.058E+00 3.027E-04 23.35 | ||
... | ... | ||
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* the number of MISFITS is higher than 1%. From the first long table (fine-grained in resolution) table in CORRECT.LP we learn that the misfits are due to faint high-resolution ice rings - so this is a problem intrinsic to the data, and not to their mode of processing. | * the number of MISFITS is higher than 1%. From the first long table (fine-grained in resolution) table in CORRECT.LP we learn that the misfits are due to faint high-resolution ice rings - so this is a problem intrinsic to the data, and not to their mode of processing. | ||
To my surprise, pointless does not agree with CORRECT's standpoint: | To my surprise, pointless ("pointless xdsin XDS_ASCII.HKL") does not agree with CORRECT's standpoint: | ||
<pre> | <pre> | ||
Scores for each symmetry element | Scores for each symmetry element | ||
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thus proving that both datasets were interrupted for 20 minutes around frame 370. | thus proving that both datasets were interrupted for 20 minutes around frame 370. | ||
Interestingly, both datasets appear to be collected at the same time, but at different wavelengths (E1 at 0.9794 Å, E2 at 0.9184 Å), and yet the individual parts merge as follows: using the following XSCALE.INP: | |||
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 | ||
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proving that the second parts of datasets E1 and E2 should be treated separately from the first parts. | proving that the second parts of datasets E1 and E2 should be treated separately from the first parts. | ||
Upon inspection of the cell parameters, we find that the cell axes of the second "halfs" are shorter by a factor of 0.9908 when compared with the first parts. This suggests that they were collected at a longer wavelength | Upon inspection of the cell parameters, we find that the cell axes of the second "halfs" are shorter by a factor of 0.9908 when compared with the first parts. This suggests that they were collected at a longer wavelength, or that radiation damage changed the cell parameters during the 20-minute break - usually it makes them longer (Ravelli ''et al.'' (2002), J. Synchrotron Rad. 9, 355-360), but this may be the exception to the rule! Maybe the crystal even was exposed to the beam during that time, in an attempt to try radiation-damage induced phasing (see e.g. Ravelli ''et al'' Structure 11 (2003), 217-220). | ||
The almost-simultaneous DATEs in the headers may be explained by | The almost-simultaneous DATEs in the headers may be explained by a wavelength-switching measuring strategy which alternatingly collects 4 frames at one wavelength as E1, then changes the wavelength and collects 4 frames into E2. | ||
So this little detective work appears to | So this little detective work appears to give us useful information about what happened in the morning of Sunday June 27, 2004 at ALS beamline 821 - but some questions remain. | ||
== Further analysis of datasets E1 and E2 == | == Further analysis of datasets E1 and E2 == | ||
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R_meas mapped on the detector, showing elevated R_meas at the location of the ice rings. | R_meas mapped on the detector, showing elevated R_meas at the location of the ice rings. | ||
== Solving the structure == | == Solving the structure with pseudo-SAD == | ||
It appears reasonable to discard the "second parts" since they are strongly influenced by radiation damage. Then, we could | |||
# merge together (into one output file) the two first parts of E1 and E2, thus obtaining a single pseudo-SAD dataset. The reason for doing this is that the anomalous signal of both datasets is so strong, and their (isomorphous) difference is weak (after all, the correlation coefficient is 1.000 !) | |||
# keep the first parts of E1 (inflection, according to the documentation) and E2 (high-enery remote) separate, and treat them as MAD (or rather, DAD). | |||
=== First try === | === First try === | ||
Let's look at the XSCALE statistics for "firstparts": | Let's look at the XSCALE statistics for the merged-together "firstparts": | ||
NOTE: Friedel pairs are treated as different reflections. | NOTE: Friedel pairs are treated as different reflections. | ||
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This looks reasonable although the absolute value of CCall is so low that there is little hope that the structure can be solved with this amount of information. And indeed, SHELXE did not show a difference between the two hands (in fact we even know that the "original hand" is the correct one since the inverted had would correspond to spacegroup #92 !). | This looks reasonable although the absolute value of CCall is so low that there is little hope that the structure can be solved with this amount of information. And indeed, SHELXE did not show a difference between the two hands (in fact we even know that the "original hand" is the correct one since the inverted had would correspond to spacegroup #92 !). | ||
=== Second try: correcting radiation damage | === Second try: correcting radiation damage by 0-dose extrapolation === | ||
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: | 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: | ||
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CRYSTAL_NAME=a | CRYSTAL_NAME=a | ||
</pre> | </pre> | ||
As a result we obtain: | As a result we obtain in XSCALE.LP: | ||
<pre> | <pre> | ||
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</pre> | </pre> | ||
We | We note that the "CORRELATION OF COMMON DECAY-FACTORS BETWEEN INPUT DATA SETS" are really high which confirms the hypothesis that this is a valid procedure to perform. | ||
Comparison of the last table with that of the previous paragraph, i.e. without zero-dose extrapolation, shows that the I/sigma, the anomalous correlation coefficients and the SigAno are significantly higher. Does this translate into better structure solution? It does: | Comparison of the last table with that of the previous paragraph, i.e. without zero-dose extrapolation, shows that the I/sigma, the anomalous correlation coefficients and the SigAno are significantly higher. Does this translate into better structure solution? It does: | ||
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[[File:1y13-raddam-contrast-raddam.png]] | [[File:1y13-raddam-contrast-raddam.png]] | ||
== Automatically building | === Automatically building the main chain of 452 out of 519 residues === | ||
Based on the sites obtained by SHELXD, we run | Based on the sites obtained by SHELXD, we run | ||
shelxe.beta -a -q -h -b -s0.585 -m40 raddam raddam_fa | 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: | 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 | shelxe.beta -a -q -h6 -b -s0.585 -m40 -n3 raddam raddam_fa | ||
and get | and get | ||
<pre> | <pre> | ||
452 residues left after pruning, divided into chains as follows: | |||
A: | A: 15 B: 5 C: 22 D: 22 E: 27 F: 62 G: 263 H: 36 | ||
CC for partial structure against native data = | CC for partial structure against native data = 39.83 % | ||
------------------------------------------------------------------------------ | ------------------------------------------------------------------------------ | ||
| Line 607: | Line 606: | ||
Global autotracing cycle 4 | Global autotracing cycle 4 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.447, Connect. = 0.705 for dens.mod. cycle 1 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.660, Connect. = 0.781 for dens.mod. cycle 2 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.723, Connect. = 0.801 for dens.mod. cycle 3 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.762, Connect. = 0.807 for dens.mod. cycle 4 | ||
Pseudo-free CC = | Pseudo-free CC = 64.88 % | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.785, Connect. = 0.810 for dens.mod. cycle 5 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.806, Connect. = 0.813 for dens.mod. cycle 6 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.820, Connect. = 0.815 for dens.mod. cycle 7 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.831, Connect. = 0.817 for dens.mod. cycle 8 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.839, Connect. = 0.819 for dens.mod. cycle 9 | ||
Pseudo-free CC = | Pseudo-free CC = 69.74 % | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.845, Connect. = 0.820 for dens.mod. cycle 10 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.849, Connect. = 0.821 for dens.mod. cycle 11 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.851, Connect. = 0.822 for dens.mod. cycle 12 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.853, Connect. = 0.823 for dens.mod. cycle 13 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.854, Connect. = 0.823 for dens.mod. cycle 14 | ||
Pseudo-free CC = 70. | Pseudo-free CC = 70.80 % | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 15 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.855, Connect. = 0.824 for dens.mod. cycle 16 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.855, Connect. = 0.824 for dens.mod. cycle 17 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 18 | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 19 | ||
Pseudo-free CC = | Pseudo-free CC = 71.03 % | ||
<wt> = 0.300, Contrast = 0. | <wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 20 | ||
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 | ||
<FOM> 0. | <FOM> 0.736 0.786 0.768 0.721 0.701 0.681 0.618 0.595 0.587 0.540 | ||
<mapCC> 0. | <mapCC> 0.862 0.932 0.946 0.934 0.924 0.924 0.922 0.913 0.882 0.858 | ||
N 4206 4227 4214 4135 4185 4207 4292 4406 4320 3702 | N 4206 4227 4214 4135 4185 4207 4292 4406 4320 3702 | ||
Estimated mean FOM = 0. | Estimated mean FOM = 0.674 Pseudo-free CC = 71.18 % | ||
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. | 1 0.2276 0.7578 0.1189 34.0000 29.98 | ||
2 0. | 2 0.1568 0.6345 0.3049 32.2898 30.44 | ||
3 0. | 3 0.1767 0.5344 0.2160 32.2388 29.67 | ||
4 0.3059 0. | 4 0.3059 0.4535 0.1297 26.0746 23.51 | ||
5 0. | 5 0.0280 0.8243 0.1410 22.7324 21.02 | ||
6 0. | 6 0.0383 0.9748 0.0492 21.5050 21.18 | ||
Site x y z h(sig) near old near new | Site x y z h(sig) near old near new | ||
1 0. | 1 0.1569 0.6345 0.3048 30.4 2/0.02 9/13.36 3/15.73 2/19.52 7/22.13 | ||
2 0. | 2 0.2278 0.7578 0.1188 30.0 1/0.02 1/19.52 6/21.97 7/22.48 9/25.02 | ||
3 0.1767 0. | 3 0.1767 0.5345 0.2158 29.7 3/0.03 9/2.90 1/15.73 4/19.45 2/26.88 | ||
4 0. | 4 0.3060 0.4536 0.1292 23.5 4/0.07 3/19.45 9/21.16 8/26.49 5/26.83 | ||
5 0. | 5 0.0382 0.9748 0.0490 21.2 6/0.02 8/2.63 8/15.66 5/15.88 6/19.80 | ||
6 0. | 6 0.0278 0.8240 0.1416 21.1 5/0.08 5/19.80 8/21.59 7/21.87 2/21.97 | ||
7 0. | 7 0.1854 0.9571 0.1787 -5.0 5/21.86 6/21.87 1/22.13 2/22.48 8/22.57 | ||
8 0. | 8 0.0427 0.9993 0.0530 -5.0 6/2.62 5/2.63 8/15.31 5/15.66 6/21.59 | ||
9 0. | 9 0.1787 0.5611 0.2228 -4.7 3/2.91 3/2.90 1/13.36 4/21.16 2/25.02 | ||
</pre> | </pre> | ||
At this point the structure is obviously solved, and we could use | At this point the structure is obviously solved, and we could use buccaneer or Arp/wArp to add side chains and the rest of the model. 3-fold NCS surely helps! | ||
== Could we do better? == | === Could we do better? === | ||
Yes, of course (as always). I can think of | Yes, of course (as always). I can think of four things to try: | ||
* an [[optimization]] round of running xds for the two datasets | * 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. | * using a negative offset for STARTING_DOSE in XSCALE.INP, as documented in the [[XSCALE]] wiki article. | ||
* | * use MERGE=TRUE in XDSCONV.INP. I tried it and this gives 20 solutions with CCall+CCweak > 25 out of 1000 trials, whereas MERGE=FALSE (the default) gives only 4 solutions! Update Sep 2011: the [[ccp4com:SHELX_C/D/E#Obtaining_the_SHELX_programs|beta-test version]] of SHELXC should have a fix for this. | ||
== better phases from DAD (Double Anomalous Dispersion) == | |||
The reason why pseudo-SAD is described here first is that, historically, I did it first since I thought that the wavelength could not realistically be changed within 3 seconds, and I therefore thought that the headers were wrong and this would not actually be a two-wavelength experiment. Along these lines, I interpreted the correlation coefficient of 1.0 between the E1 and E2 first parts as indicating that no isomorphous difference exists. | |||
In a discussion with Gerard Bricogne and Clemens Vonrhein after the ACA2011 workshop it turned out that my theory, which claims that E1 and E2 are actually the same wavelength, is wrong. This was investigated by looking at the difference map (obtained using phenix.fobs_minus_fobs_map) of E1 and E2 (taking the first parts in each case) phased with the 1y13 model, which shows three strong (14-19 sigma) peaks. The fact that the 1-370 pieces merge so well seems to be a consequence of the fact that the anomalous signal of the two wavelengths is so similar, and the dispersive difference between the wavelengths does not significantly decrease the high correlation coefficient in data scaling. | |||
Thus even better phasing would be obtained by keeping the wavelengths separate and doing MAD (in fact DAD) - but zero-dose extrapolation could and should be done in the same way. I've therefore continued the analysis in [[1Y13-DAD]]. | |||