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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 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] 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. | |||
== | == Dataset E1 == | ||
Use [[generate_XDS.INP]] and run xds once. Based on R-factors in the resulting CORRECT.LP, and an inspection of BKGPIX.cbf, I modified XDS.INP to have | Use [[generate_XDS.INP]] and run [[xds]] once. Based on R-factors in the resulting CORRECT.LP, and an inspection of BKGPIX.cbf, I modified XDS.INP to have | ||
INCLUDE_RESOLUTION_RANGE=40 2.1 ! too weak beyond 2.1 | INCLUDE_RESOLUTION_RANGE=40 2.1 ! too weak beyond 2.1 Å | ||
VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS=8000. 30000. ! raised from 7000 30000 to mask beamstop | VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS=8000. 30000. ! raised from 7000 30000 to mask beamstop | ||
and ran xds again. This is the excerpt from CORRECT.LP : | and ran xds again. | ||
=== What's the problem? === | |||
This is the excerpt from [[CORRECT.LP]] : | |||
SPACE-GROUP UNIT CELL CONSTANTS UNIQUE Rmeas COMPARED LATTICE- | SPACE-GROUP UNIT CELL CONSTANTS UNIQUE Rmeas COMPARED LATTICE- | ||
Line 45: | Line 51: | ||
SPACE GROUP NUMBER 16 | SPACE GROUP NUMBER 16 | ||
So CORRECT chooses an orthorhombic spacegroup | So CORRECT chooses an orthorhombic spacegroup. | ||
The file continues: | The file continues: | ||
Line 52: | Line 58: | ||
a b ISa | a b ISa | ||
6.058E+00 3.027E-04 23.35 | 6.058E+00 3.027E-04 23.35 | ||
... | ... | ||
Line 81: | Line 86: | ||
Some comments: | Some comments: | ||
* the "STANDARD DEVIATION OF SPOT POSITION (PIXELS)" is significantly higher (1.01) than those reported for the 5°-batches in INTEGRATE.LP (about 0.6) . This suggests that the geometry refinement has to deal with inconsistent data. | |||
* CORRECT obviously indicates an orthorhombic spacegroup. | * CORRECT obviously indicates an orthorhombic spacegroup. | ||
* 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 | * 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 | ||
Line 179: | Line 185: | ||
which is much worse than the spacegroup 19 statistics (compare the ISa values - they differ by a factor of 2 !) so there may be something wrong with some assumptions we were making ... | which is much worse than the spacegroup 19 statistics (compare the ISa values - they differ by a factor of 2 !) so there may be something wrong with some assumptions we were making ... | ||
The easiest thing one can do is to inspect INTEGRATE.LP - this lists scale factor, beam divergence and mosaicity for every reflection. There's a [[jiffies|jiffy]] called "scalefactors" which grep's the relevant lines from INTEGRATE.LP. This shows the scale factor (column 3): | === Identifying a possible cause === | ||
The easiest thing one can do is to inspect INTEGRATE.LP - this lists scale factor, beam divergence and mosaicity for every reflection. There's a [[jiffies|jiffy]] called "scalefactors" which grep's the relevant lines from INTEGRATE.LP ("scalefactors > scales.log"). This shows the scale factor (column 3): | |||
[[File:1y13-e1-scales.png]] | [[File:1y13-e1-scales.png]] | ||
demonstrating that "something happens" between frame 372 and 373 (of course one has to look at the table to find the exact numbers). | |||
'''It should be noted that any abrupt change in conditions during the experiment is going to spoil the resulting data in one way or another. This is most true for a SAD experiment which is supposed to give accurate values for the tiny differences in intensities between Friedel-related reflections.''' | |||
=== A solution === | |||
At this point it is good to look at the data for experiment E2. Here, we find exactly the same problems of bad ISa and high "STANDARD DEVIATION OF SPOT POSITION (PIXELS)" when reducing frames 1-591 in one run of xds. | |||
With this knowledge, we are lead, for E1, to reduce frames 1-372 and 373-592 separately, in spacegroup 96. For E2, we use frames 1-369 and 371-591, respectively. Frame E2-370 has a very high scale factor so we leave it out altogether. | |||
This is also a good time to closely inspect the headers of the frames: | |||
% grep --binary-files=text DATE j1603b3PK_1_E1_37?.img | |||
gives | |||
j1603b3PK_1_E1_370.img:DATE=Sun Jun 27 08:55:51 2004; | |||
j1603b3PK_1_E1_371.img:DATE=Sun Jun 27 08:56:00 2004; | |||
j1603b3PK_1_E1_372.img:DATE=Sun Jun 27 08:56:08 2004; | |||
j1603b3PK_1_E1_373.img:DATE=Sun Jun 27 09:19:45 2004; | |||
j1603b3PK_1_E1_374.img:DATE=Sun Jun 27 09:19:54 2004; | |||
j1603b3PK_1_E1_375.img:DATE=Sun Jun 27 09:20:02 2004; | |||
j1603b3PK_1_E1_376.img:DATE=Sun Jun 27 09:20:10 2004; | |||
j1603b3PK_1_E1_377.img:DATE=Sun Jun 27 09:20:58 2004; | |||
j1603b3PK_1_E1_378.img:DATE=Sun Jun 27 09:21:08 2004; | |||
j1603b3PK_1_E1_379.img:DATE=Sun Jun 27 09:21:17 2004; | |||
and | |||
% grep --binary-files=text DATE j1603b3PK_1_E2_3[67]?.img | |||
gives | |||
j1603b3PK_1_E2_366.img:DATE=Sun Jun 27 08:55:15 2004; | |||
j1603b3PK_1_E2_367.img:DATE=Sun Jun 27 08:55:23 2004; | |||
j1603b3PK_1_E2_368.img:DATE=Sun Jun 27 08:55:32 2004; | |||
j1603b3PK_1_E2_369.img:DATE=Sun Jun 27 08:56:19 2004; | |||
j1603b3PK_1_E2_370.img:DATE=Sun Jun 27 08:56:28 2004; | |||
j1603b3PK_1_E2_371.img:DATE=Sun Jun 27 09:19:26 2004; | |||
j1603b3PK_1_E2_372.img:DATE=Sun Jun 27 09:19:34 2004; | |||
j1603b3PK_1_E2_373.img:DATE=Sun Jun 27 09:20:22 2004; | |||
j1603b3PK_1_E2_374.img:DATE=Sun Jun 27 09:20:30 2004; | |||
j1603b3PK_1_E2_375.img:DATE=Sun Jun 27 09:20:38 2004; | |||
j1603b3PK_1_E2_376.img:DATE=Sun Jun 27 09:20:47 2004; | |||
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 | |||
SPACE_GROUP_NUMBER=96 | |||
OUTPUT_FILE=temp.ahkl | |||
INPUT_FILE=../e1_1-372/XDS_ASCII.HKL | |||
INPUT_FILE=../e1_373-592/XDS_ASCII.HKL | |||
INPUT_FILE=../e2_1-369/XDS_ASCII.HKL | |||
INPUT_FILE=../e2_371-591/XDS_ASCII.HKL | |||
and running [[xscale]], we obtain in XSCALE.LP: | |||
CORRELATIONS BETWEEN INPUT DATA SETS AFTER CORRECTIONS | |||
DATA SETS NUMBER OF COMMON CORRELATION RATIO OF COMMON B-FACTOR | |||
#i #j REFLECTIONS BETWEEN i,j INTENSITIES (i/j) BETWEEN i,j | |||
1 2 15943 0.978 1.0002 0.0106 | |||
1 3 22366 1.000 1.0012 -0.0008 | |||
2 3 15801 0.977 0.9983 0.0557 | |||
1 4 15648 0.979 0.9988 0.0541 | |||
2 4 14862 0.999 1.0024 -0.0007 | |||
3 4 15524 0.978 0.9999 -0.0015 | |||
which means that e1_1-372 correlates well (1.000) with e2_1-369, and e1_373-59 well (0.999) with e2_371-591, but the crosswise correlations are consistently low (0.978, 0.977, 0.979, 0.978). The adjustment to the error model proves this: | |||
a b ISa ISa0 INPUT DATA SET | |||
6.112E+00 1.429E-03 10.70 22.37 ../e1_1-372/XDS_ASCII.HKL | |||
1.074E+01 1.825E-03 7.14 23.79 ../e1_373-592/XDS_ASCII.HKL | |||
5.707E+00 1.621E-03 10.40 22.82 ../e2_1-369/XDS_ASCII.HKL | |||
8.547E+00 1.796E-03 8.07 24.17 ../e2_371-591/XDS_ASCII.HKL | |||
telling us that "if we merge these datasets together, their error estimates have to be increased a lot". However, if we switch to | |||
UNIT_CELL_CONSTANTS=103.316 103.316 131.456 90.000 90.000 90.000 | |||
SPACE_GROUP_NUMBER=96 | |||
OUTPUT_FILE=firstparts.ahkl | |||
INPUT_FILE=../e1_1-372/XDS_ASCII.HKL | |||
INPUT_FILE=../e2_1-369/XDS_ASCII.HKL | |||
OUTPUT_FILE=secondparts.ahkl | |||
INPUT_FILE=../e1_373-592/XDS_ASCII.HKL | |||
INPUT_FILE=../e2_371-591/XDS_ASCII.HKL | |||
we obtain | |||
a b ISa ISa0 INPUT DATA SET | |||
6.120E+00 3.673E-04 21.09 22.37 ../e1_1-372/XDS_ASCII.HKL | |||
5.713E+00 3.819E-04 21.41 22.82 ../e2_1-369/XDS_ASCII.HKL | |||
5.639E+00 3.151E-04 23.72 23.79 ../e1_373-592/XDS_ASCII.HKL | |||
5.289E+00 3.258E-04 24.09 24.17 ../e2_371-591/XDS_ASCII.HKL | |||
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, 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 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 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 == | |||
Here, we try to learn more about the constituents of "firstparts". | |||
Running "[[xdsstat]] > XDSSTAT.LP" in the e1_1-372 and e2_1-369 directories, we obtain statistics output not available from CORRECT. We open XDSSTAT.LP with the CCP4 program "loggraph", and take a look at [[misfits.pck]], [[rf.pck]], and the other files produced by [[xdsstat]], using [[VIEW]] or [[XDS-Viewer]]: | |||
[[File:e1_1-372-xdsstat1.png]] | |||
Reflections and misfits, by frame - looks normal | |||
[[File:e1_1-372-xdsstat2.png]] | |||
Intensity and sigma by frame - looks normal | |||
[[File:e1_1-372-xdsstat3.png]] | |||
"partiality" and profile agreement, by frame - looks good but it's clear that the profiles at high frame number agree worse with the average profiles, possibly due to radiation damage | |||
[[File:e1_1-372-xdsstat4.png]] | |||
R_meas, by frame, clearly showing good R_meas in the middle of the dataset | |||
[[File:e1_1-372-xdsstat-raddam.png]] | |||
R_d - an R-factor which directly depends on radiation damage. This is calculated as a function of frame number difference and the linear rise indicates significant radiation damage that should be correctable in [[XSCALE]], using the CRYSTAL_NAME keyword. | |||
[[File:e1_1-372-misfits.png]] | |||
misfits mapped on the detector, showing ice rings. | |||
[[File:e1_1-372-rf.png]] | |||
R_meas mapped on the detector, showing elevated R_meas at the location of the ice rings. | |||
== 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 === | |||
Let's look at the XSCALE statistics for the merged-together "firstparts": | |||
NOTE: Friedel pairs are treated as different reflections. | |||
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION | |||
RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano | |||
LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr | |||
9.40 6122 844 883 95.6% 2.9% 3.5% 6111 54.76 3.2% 1.4% 79% 2.137 313 | |||
6.64 12037 1611 1621 99.4% 2.9% 3.6% 12035 51.54 3.1% 1.5% 80% 2.259 684 | |||
5.43 15348 2065 2086 99.0% 3.5% 3.7% 15347 47.79 3.7% 1.7% 78% 2.294 908 | |||
4.70 18714 2487 2498 99.6% 3.0% 3.7% 18711 49.55 3.2% 1.5% 72% 1.712 1120 | |||
4.20 21104 2797 2821 99.1% 3.1% 3.7% 21102 47.24 3.3% 1.7% 72% 1.727 1271 | |||
3.84 23316 3095 3117 99.3% 3.8% 4.0% 23313 42.74 4.1% 2.1% 65% 1.617 1420 | |||
3.55 25693 3345 3366 99.4% 4.4% 4.5% 25693 37.93 4.7% 2.6% 50% 1.411 1548 | |||
3.32 28017 3633 3653 99.5% 5.2% 5.2% 28015 32.89 5.6% 3.6% 40% 1.335 1687 | |||
3.13 30266 3842 3848 99.8% 7.2% 7.2% 30264 25.87 7.7% 4.8% 36% 1.158 1797 | |||
2.97 32595 4114 4118 99.9% 10.4% 10.4% 32594 19.26 11.1% 7.7% 30% 1.068 1925 | |||
2.83 34384 4315 4320 99.9% 14.3% 14.8% 34382 14.88 15.3% 10.3% 20% 0.937 2031 | |||
2.71 35654 4475 4478 99.9% 18.3% 19.1% 35652 12.13 19.5% 13.1% 15% 0.891 2110 | |||
2.61 37307 4705 4710 99.9% 27.5% 28.8% 37304 8.44 29.4% 19.8% 11% 0.834 2224 | |||
2.51 38997 4893 4896 99.9% 35.5% 38.0% 38997 6.78 38.0% 26.0% 10% 0.817 2318 | |||
2.43 40036 5026 5027 100.0% 51.3% 55.1% 40032 4.92 54.8% 38.0% 2% 0.738 2387 | |||
2.35 39975 5180 5222 99.2% 71.3% 68.9% 39967 3.78 76.4% 52.7% 21% 0.887 2446 | |||
2.28 42041 5385 5423 99.3% 93.7% 93.1% 42037 2.90 100.3% 66.7% 11% 0.798 2548 | |||
2.21 43012 5538 5541 99.9% 85.7% 88.3% 43011 2.87 91.8% 58.8% 10% 0.818 2644 | |||
2.16 42610 5701 5703 100.0% 113.6% 120.7% 42607 2.13 122.0% 85.4% 4% 0.722 2724 | |||
2.10 38996 5634 5912 95.3% 146.1% 153.9% 38944 1.50 157.8% 122.7% 3% 0.711 2639 | |||
total 606224 78685 79243 99.3% 6.7% 7.2% 606118 16.88 7.2% 12.0% 29% 1.055 36744 | |||
The anomalous correlation is good at low resolution, though not outstanding. At high resolution it rises again but this is presumably due to the ice rings. | |||
I like to use [[ccp4com:hkl2map|hkl2map]] which runs [[ccp4com:SHELX C/D/E|SHELXC]], [[ccp4com:SHELX C/D/E|SHELXD]] and [[ccp4com:SHELX C/D/E|SHELXE]] from its GUI. Before doing so, we have to run XDSCONV with the following XDSCONV.INP: | |||
INPUT_FILE=firstparts.hkl | |||
OUTPUT_FILE=temp.hkl SHELX | |||
First, the shelxc output which shows that these data are quite good: | |||
[[File:e1+e2_firstparts-i-sigi-resol.png]] [[File:e1+e2_firstparts-self-anomcc.png]] | |||
And then we show the result of 100 trials at substructure solution of shelxd, trying to find 3 Se atoms at 30 - 3.3Å resolution (I also tried 3.0 3.1 3.2 3.4 3.5 Å but 3.3 Å was best). | |||
[[File:e1+e2_firstparts-ccall-ccweak.png]] [[File:e1+e2_firstparts-occ-vs-peak.png]] | |||
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 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: | |||
<pre> | |||
UNIT_CELL_CONSTANTS=103.316 103.316 131.456 90.000 90.000 90.000 | |||
SPACE_GROUP_NUMBER=96 | |||
OUTPUT_FILE=temp.ahkl | |||
INPUT_FILE=../e1_1-372/XDS_ASCII.HKL | |||
CRYSTAL_NAME=a | |||
INPUT_FILE=../e2_1-369/XDS_ASCII.HKL | |||
CRYSTAL_NAME=a | |||
</pre> | |||
As a result we obtain in XSCALE.LP: | |||
<pre> | |||
****************************************************************************** | |||
RESULTS FROM ZERO-DOSE EXTRAPOLATION OF REFLECTION INTENSITIES | |||
for reference on this subject see: | |||
K. Diederichs, S. McSweeney & R.B.G. Ravelli, Acta Cryst. D59, 903-909(2003). | |||
"Zero-dose extrapolation as part of macromolecular synchrotron data reduction" | |||
****************************************************************************** | |||
Radiation damage can lead to localized modifications of the structure. | |||
To correct for this effect, XSCALE modifies the intensity measurements | |||
I(h,i) by individual correction factors, | |||
exp{-b(h)*dose(h,i)} | |||
where h,i denotes the i-th observation with unique reflection indices | |||
h, and dose(h,i) the X-ray dose accumulated by the crystal when the | |||
reflection was recorded. Assuming a constant dose for each image | |||
(dose_rate), the accumulated dose when recording image_number(i), on | |||
which I(h,i) was observed, is then | |||
dose(h,i) = starting_dose + dose_rate * (image_number(i)-first_image) | |||
The decay factor b(h) is determined from the assumption that symmetry | |||
related reflections in a data set taken from the same crystal should | |||
have the same intensity after correction. Moreover, b(h) is assumed to | |||
be the same for Friedel-pairs and independent of the X-ray wavelength. | |||
To avoid overfitting the data, XSCALE starts with the hypothesis that | |||
b(h)=0 and rejects this assumption if its probability is below 10.0%. | |||
CORRELATION OF COMMON DECAY-FACTORS BETWEEN INPUT DATA SETS | |||
----------------------------------------------------------- | |||
First INPUT_FILE= ../e2_1-369/XDS_ASCII.HKL | |||
CRYSTAL_NAME= a | |||
Second INPUT_FILE= ../e1_1-372/XDS_ASCII.HKL | |||
CRYSTAL_NAME= a | |||
RESOLUTION NUMBER CORRELATION | |||
LIMIT OF PAIRS FACTOR | |||
9.40 210 0.955 | |||
6.64 441 0.955 | |||
5.43 587 0.940 | |||
4.70 692 0.969 | |||
4.20 750 0.949 | |||
3.84 836 0.920 | |||
3.55 809 0.942 | |||
3.32 775 0.925 | |||
3.13 663 0.888 | |||
2.97 557 0.837 | |||
2.83 375 0.681 | |||
2.71 302 0.812 | |||
2.61 212 0.625 | |||
2.51 163 0.508 | |||
2.43 95 0.291 | |||
2.35 139 0.722 | |||
2.28 110 0.688 | |||
2.21 91 0.734 | |||
2.16 88 0.561 | |||
2.10 54 0.126 | |||
total 7949 0.788 | |||
X-RAY DOSE PARAMETERS USED FOR EACH INPUT DATA SET | |||
-------------------------------------------------- | |||
CRYSTAL_NAME= a | |||
STARTING_DOSE DOSE_RATE NAME OF INPUT FILE | |||
initial refined initial refined | |||
0.000E+00 8.557E+00 1.000E+00 1.000E+00 ../e1_1-372/XDS_ASCII.HKL | |||
0.000E+00 0.000E+00 1.000E+00 1.024E+00 ../e2_1-369/XDS_ASCII.HKL | |||
STATISTICS OF 0-DOSE CORRECTED DATA FROM EACH CRYSTAL | |||
----------------------------------------------------- | |||
NUNIQUE = Number of unique reflections with enough symmetry- | |||
related observations to determine a decay factor b(h) | |||
N0-DOSE = Number of 0-dose extrapolated unique reflections | |||
NERROR = Number of unique extrapolated reflections expected | |||
to be overfitted. A large ratio of N0-DOSE/NERROR | |||
justifies the data correction as carried out here. | |||
S_corr = mean value of Sigma(I) for 0-dose extrapolated data | |||
S_norm = mean value of Sigma(I) for the same data but | |||
without 0-dose extrapolation. | |||
NFREE = degrees of freedom for calculating S_corr | |||
CRYSTAL_NAME= a | |||
RESOLUTION NUNIQUE N0-DOSE N0-DOSE/ S_corr/ NFREE | |||
LIMIT NERROR S_norm | |||
9.40 496 378 68.0 0.543 3180 | |||
6.64 908 703 78.9 0.554 6245 | |||
5.43 1140 894 77.0 0.574 8064 | |||
4.70 1351 1040 77.4 0.599 9671 | |||
4.20 1518 1133 69.9 0.620 10585 | |||
3.84 1665 1187 73.9 0.630 11129 | |||
3.55 1787 1220 65.1 0.671 11917 | |||
3.32 1941 1289 58.1 0.690 12728 | |||
3.13 2042 1172 49.8 0.717 11877 | |||
2.97 2182 1103 48.1 0.750 11498 | |||
2.83 2281 911 40.1 0.798 9662 | |||
2.71 2352 812 34.2 0.825 8611 | |||
2.61 2467 702 34.1 0.848 7383 | |||
2.51 2566 627 31.5 0.875 6595 | |||
2.43 2624 499 31.2 0.895 5295 | |||
2.35 2709 629 31.6 0.888 6240 | |||
2.28 2821 603 28.5 0.893 6147 | |||
2.21 2880 560 32.4 0.905 5758 | |||
2.16 2959 448 30.3 0.907 4394 | |||
2.10 2860 413 29.9 0.924 3745 | |||
total 41549 16323 46.8 0.739 160724 | |||
****************************************************************************** | |||
SCALING FACTORS FOR Sigma(I) AS FUNCTION OF RESOLUTION | |||
****************************************************************************** | |||
SCALING FACTORS FOR Sigma(I) FOR DATA SET ../e1_1-372/XDS_ASCII.HKL | |||
RESOLUTION (ANGSTROM) | |||
10.33 6.12 4.76 4.03 3.56 3.23 2.97 2.76 2.60 2.46 2.34 2.23 2.14 | |||
FACTOR 0.94 0.96 0.88 0.93 0.99 0.98 0.99 0.99 0.99 0.98 1.10 1.00 0.99 | |||
SCALING FACTORS FOR Sigma(I) FOR DATA SET ../e2_1-369/XDS_ASCII.HKL | |||
RESOLUTION (ANGSTROM) | |||
10.32 6.11 4.76 4.03 3.56 3.22 2.97 2.76 2.60 2.46 2.34 2.23 2.14 | |||
FACTOR 0.96 0.98 0.89 0.94 1.01 1.01 1.02 1.01 1.00 0.99 1.11 1.02 0.98 | |||
****************************************************************************** | |||
STATISTICS OF SCALED OUTPUT DATA SET : temp.ahkl | |||
FILE TYPE: XDS_ASCII MERGE=FALSE FRIEDEL'S_LAW=FALSE | |||
1270 OUT OF 607179 REFLECTIONS REJECTED | |||
605909 REFLECTIONS ON OUTPUT FILE | |||
****************************************************************************** | |||
DEFINITIONS: | |||
R-FACTOR | |||
observed = (SUM(ABS(I(h,i)-I(h))))/(SUM(I(h,i))) | |||
expected = expected R-FACTOR derived from Sigma(I) | |||
COMPARED = number of reflections used for calculating R-FACTOR | |||
I/SIGMA = mean of intensity/Sigma(I) of unique reflections | |||
(after merging symmetry-related observations) | |||
Sigma(I) = standard deviation of reflection intensity I | |||
estimated from sample statistics | |||
R-meas = redundancy independent R-factor (intensities) | |||
Rmrgd-F = quality of amplitudes (F) in the scaled data set | |||
For definition of R-meas and Rmrgd-F see | |||
Diederichs & Karplus (1997), Nature Struct. Biol. 4, 269-275. | |||
Anomal = mean correlation factor between two random subsets | |||
Corr of anomalous intensity differences | |||
SigAno = mean anomalous difference in units of its estimated | |||
standard deviation (|F(+)-F(-)|/Sigma). F(+), F(-) | |||
are structure factor estimates obtained from the | |||
merged intensity observations in each parity class. | |||
Nano = Number of unique reflections used to calculate | |||
Anomal_Corr & SigAno. At least two observations | |||
for each (+ and -) parity are required. | |||
NOTE: Friedel pairs are treated as different reflections. | |||
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION | |||
RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano | |||
LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr | |||
9.40 6095 844 883 95.6% 2.0% 2.6% 6084 73.41 2.1% 0.9% 87% 2.706 313 | |||
6.64 12006 1611 1621 99.4% 2.0% 2.8% 12004 68.81 2.1% 1.0% 84% 2.555 684 | |||
5.43 15339 2065 2086 99.0% 2.2% 2.8% 15338 63.28 2.4% 1.2% 82% 2.409 908 | |||
4.70 18697 2486 2498 99.5% 1.9% 2.6% 18694 70.84 2.1% 1.0% 75% 1.855 1120 | |||
4.20 21080 2796 2821 99.1% 2.0% 2.7% 21078 66.87 2.1% 1.1% 67% 1.727 1270 | |||
3.84 23300 3094 3117 99.3% 2.5% 3.0% 23297 58.10 2.7% 1.5% 64% 1.551 1420 | |||
3.55 25676 3344 3366 99.3% 3.1% 3.6% 25676 48.56 3.4% 1.9% 50% 1.326 1548 | |||
3.32 28013 3633 3653 99.5% 3.9% 4.3% 28011 41.76 4.1% 2.8% 37% 1.244 1687 | |||
3.13 30254 3841 3848 99.8% 5.7% 6.0% 30252 32.18 6.1% 4.1% 35% 1.125 1796 | |||
2.97 32595 4114 4118 99.9% 8.8% 9.1% 32594 23.53 9.4% 6.8% 26% 1.038 1925 | |||
2.83 34368 4313 4320 99.8% 12.8% 13.3% 34366 17.65 13.6% 9.5% 21% 0.989 2030 | |||
2.71 35627 4472 4478 99.9% 16.9% 17.4% 35625 14.15 18.1% 12.2% 18% 0.965 2108 | |||
2.61 37300 4704 4710 99.9% 25.8% 26.4% 37297 9.70 27.6% 19.3% 16% 0.930 2223 | |||
2.51 38975 4890 4896 99.9% 33.8% 34.9% 38975 7.68 36.1% 24.1% 14% 0.888 2315 | |||
2.43 39971 5019 5027 99.8% 49.1% 50.8% 39967 5.47 52.5% 37.2% 8% 0.810 2380 | |||
2.35 39968 5179 5222 99.2% 67.9% 67.5% 39960 4.07 72.7% 50.4% 25% 0.927 2445 | |||
2.28 42067 5388 5423 99.4% 89.9% 94.3% 42063 3.03 96.2% 63.5% 16% 0.796 2548 | |||
2.21 43011 5538 5541 99.9% 82.3% 83.3% 43010 3.16 88.1% 57.9% 14% 0.871 2644 | |||
2.16 42577 5697 5703 99.9% 108.5% 112.2% 42574 2.37 116.6% 83.1% 3% 0.760 2720 | |||
2.10 38988 5633 5912 95.3% 142.1% 144.2% 38936 1.67 153.5% 119.2% 6% 0.772 2638 | |||
total 605907 78661 79243 99.3% 5.5% 6.1% 605801 21.72 5.9% 11.3% 27% 1.095 36722 | |||
</pre> | |||
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: | |||
[[File:1y13-raddam-ccall-ccweak-raddam.png]] | |||
[[File:1y13-raddam-site-occ-raddam.png]] | |||
[[File:1y13-raddam-contrast-raddam.png]] | |||
=== Automatically building the main chain of 452 out of 519 residues === | |||
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 -n3 raddam raddam_fa | |||
and get | |||
<pre> | |||
452 residues left after pruning, divided into chains as follows: | |||
A: 15 B: 5 C: 22 D: 22 E: 27 F: 62 G: 263 H: 36 | |||
CC for partial structure against native data = 39.83 % | |||
------------------------------------------------------------------------------ | |||
Global autotracing cycle 4 | |||
<wt> = 0.300, Contrast = 0.447, Connect. = 0.705 for dens.mod. cycle 1 | |||
<wt> = 0.300, Contrast = 0.660, Connect. = 0.781 for dens.mod. cycle 2 | |||
<wt> = 0.300, Contrast = 0.723, Connect. = 0.801 for dens.mod. cycle 3 | |||
<wt> = 0.300, Contrast = 0.762, Connect. = 0.807 for dens.mod. cycle 4 | |||
Pseudo-free CC = 64.88 % | |||
<wt> = 0.300, Contrast = 0.785, Connect. = 0.810 for dens.mod. cycle 5 | |||
<wt> = 0.300, Contrast = 0.806, Connect. = 0.813 for dens.mod. cycle 6 | |||
<wt> = 0.300, Contrast = 0.820, Connect. = 0.815 for dens.mod. cycle 7 | |||
<wt> = 0.300, Contrast = 0.831, Connect. = 0.817 for dens.mod. cycle 8 | |||
<wt> = 0.300, Contrast = 0.839, Connect. = 0.819 for dens.mod. cycle 9 | |||
Pseudo-free CC = 69.74 % | |||
<wt> = 0.300, Contrast = 0.845, Connect. = 0.820 for dens.mod. cycle 10 | |||
<wt> = 0.300, Contrast = 0.849, Connect. = 0.821 for dens.mod. cycle 11 | |||
<wt> = 0.300, Contrast = 0.851, Connect. = 0.822 for dens.mod. cycle 12 | |||
<wt> = 0.300, Contrast = 0.853, Connect. = 0.823 for dens.mod. cycle 13 | |||
<wt> = 0.300, Contrast = 0.854, Connect. = 0.823 for dens.mod. cycle 14 | |||
Pseudo-free CC = 70.80 % | |||
<wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 15 | |||
<wt> = 0.300, Contrast = 0.855, Connect. = 0.824 for dens.mod. cycle 16 | |||
<wt> = 0.300, Contrast = 0.855, Connect. = 0.824 for dens.mod. cycle 17 | |||
<wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 18 | |||
<wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 19 | |||
Pseudo-free CC = 71.03 % | |||
<wt> = 0.300, Contrast = 0.854, Connect. = 0.824 for dens.mod. cycle 20 | |||
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> 0.736 0.786 0.768 0.721 0.701 0.681 0.618 0.595 0.587 0.540 | |||
<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 | |||
Estimated mean FOM = 0.674 Pseudo-free CC = 71.18 % | |||
Density (in map sigma units) at input heavy atom sites | |||
Site x y z occ*Z density | |||
1 0.2276 0.7578 0.1189 34.0000 29.98 | |||
2 0.1568 0.6345 0.3049 32.2898 30.44 | |||
3 0.1767 0.5344 0.2160 32.2388 29.67 | |||
4 0.3059 0.4535 0.1297 26.0746 23.51 | |||
5 0.0280 0.8243 0.1410 22.7324 21.02 | |||
6 0.0383 0.9748 0.0492 21.5050 21.18 | |||
Site x y z h(sig) near old near new | |||
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 0.2278 0.7578 0.1188 30.0 1/0.02 1/19.52 6/21.97 7/22.48 9/25.02 | |||
3 0.1767 0.5345 0.2158 29.7 3/0.03 9/2.90 1/15.73 4/19.45 2/26.88 | |||
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 0.0382 0.9748 0.0490 21.2 6/0.02 8/2.63 8/15.66 5/15.88 6/19.80 | |||
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 0.1854 0.9571 0.1787 -5.0 5/21.86 6/21.87 1/22.13 2/22.48 8/22.57 | |||
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 0.1787 0.5611 0.2228 -4.7 3/2.91 3/2.90 1/13.36 4/21.16 2/25.02 | |||
</pre> | |||
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? === | |||
Yes, of course (as always). I can think of four 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. | |||
* 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]]. |