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This reports processing of triclinic hen egg-white lysozyme data @ 0.65Å resolution (PDB id [ | This reports processing of triclinic hen egg-white lysozyme data @ 0.65Å resolution (PDB id [http://www.rcsb.org/pdb/explore/explore.do?structureId=2VB1 2VB1]). Data (sweeps a to h, each comprising 60 to 360 frames of 72MB) were collected by Zbigniew Dauter at APS 19-ID and are available from [http://bl831.als.lbl.gov/example_data_sets/APS/19-ID/2vb1/ here]. Details of data collection, processing and refinement are [http://journals.iucr.org/d/issues/2007/12/00/be5097/index.html published]. | ||
== XDS processing == | == XDS processing == | ||
# use [[generate_XDS.INP]] to obtain a good starting point | # use [[generate_XDS.INP]] to obtain a good starting point | ||
# edit [[XDS.INP]] and change the following: | # edit [[XDS.INP]] and change/add the following: | ||
ORGX=3130 ORGY=3040 ! for ADSC, header values are subject to interpretation; | ORGX=3130 ORGY=3040 ! for ADSC, header values are subject to interpretation; these values from visual inspection | ||
UNTRUSTED_RECTANGLE=1 3160 3000 3070 ! <xmin xmax ymin ymax> to mask shadow of beamstop; XDS-viewer to find out | |||
TRUSTED_REGION=0 1.5 ! we want the whole detector area | TRUSTED_REGION=0 1.5 ! we want the whole detector area | ||
ROTATION_AXIS=-1 0 0 ! at this beamline the spindle goes backwards! | ROTATION_AXIS=-1 0 0 ! at this beamline the spindle goes backwards! | ||
SILICON=34.812736 ! account for theta-dependant absorption in the CCD's phosphor. The correction is only | |||
! significant for hi-res data; 34.812736=32*(value for silicon as printed to CORRECT.LP if SILICON= not given) | |||
MAXIMUM_NUMBER_OF_PROCESSORS=4 ! for fast processing on a machine with many cores, use (e.g. for 16 cores) | |||
MAXIMUM_NUMBER_OF_JOBS=6 ! This "overcommits" the available cores but on the whole this produces results faster (see below). | |||
SPACE_GROUP_NUMBER=1 ! this is known | |||
UNIT_CELL_CONSTANTS= 27.07 31.25 33.76 87.98 108.00 112.11 ! from 2vb1 | |||
FRIEDEL'S_LAW=TRUE ! we're not concerned with the anomalous signal | |||
Then, run "xds_par". It completes after about 5 minutes on a fast machine, and we may inspect (at least) IDXREF.LP and CORRECT.LP (see below), and use "XDS-viewer FRAME.cbf" to get a visual impression of the integration as it applies to the last frame. | |||
By inspecting IDXREF.LP, one should make sure that everything works as it should, i.e. that a large percentage of reflections was actually indexed nicely, e.g.: | |||
... | ... | ||
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STANDARD DEVIATION OF SPINDLE POSITION (DEGREES) 0.12 | STANDARD DEVIATION OF SPINDLE POSITION (DEGREES) 0.12 | ||
=== Optimization === | === Optimization === | ||
The main target of optimization is the | The main target of optimization is the asymptotic (i.e. best) I/sigma (ISa) (Diederichs (2010) [http://dx.doi.org/10.1107/S0907444910014836 Acta Cryst. D 66, 733-40]) as printed out by CORRECT (and XSCALE). A higher ISa should mean better data. | ||
However: ISa also rises if more reflections are thrown out as outliers ("misfits") so it is not considered to be optimization if just WFAC1 is reduced. Please note that the default WFAC1 is 1; this should result in the rejection of about 1% of observations. If you feel that 1% is too much then just increase WFAC1, to, say, 1.5 - that should result in rejection of less than 0.1%. This will slightly increase completeness, but will reduce I/sigma and ISa, and increase R-factors. | However: ISa also rises if more reflections are thrown out as outliers ("misfits") so it is not considered to be optimization if just WFAC1 is reduced. Please note that the default WFAC1 is 1; this should result in the rejection of about 1% of observations. If you feel that 1% is too much then just increase WFAC1, to, say, 1.5 - that should result in rejection of less than (say) 0.1%. This will slightly increase completeness, but will reduce I/sigma and ISa, and increase R-factors. | ||
The following quantities may be tested for their influence on ISa: | The following quantities may be tested for their influence on ISa: | ||
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REFLECTING_RANGE= 0.669 REFLECTING_RANGE_E.S.D.= 0.096 | REFLECTING_RANGE= 0.669 REFLECTING_RANGE_E.S.D.= 0.096 | ||
copy these two lines into XDS.INP | copy these two lines into XDS.INP | ||
* prevent refinement in INTEGRATE: REFINE(INTEGRATE)= ! | |||
== Example: sweep e == | == Example: sweep e == | ||
=== [[XDS.INP]]; as generated by [[generate_XDS.INP]] === | === [[XDS.INP]]; as generated by [[generate_XDS.INP]] === | ||
... | generate_XDS.INP "../../APS/19-ID/2vb1/p1lyso_e.0???.img" | ||
Then include the changes detailed above, resulting in: | |||
<pre> | <pre> | ||
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT | JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT | ||
MAXIMUM_NUMBER_OF_PROCESSORS= | MAXIMUM_NUMBER_OF_PROCESSORS=4 | ||
MAXIMUM_NUMBER_OF_JOBS= | MAXIMUM_NUMBER_OF_JOBS=6 | ||
ORGX= 3130 ORGY= 3040 ! check these values with adxv ! | ORGX= 3130 ORGY= 3040 ! check these values with adxv ! | ||
UNTRUSTED_RECTANGLE=1 3160 3000 3070 ! <xmin xmax ymin ymax> to mask shadow of beamstop; XDS-viewer to find out | |||
DETECTOR_DISTANCE= 99.9954 | DETECTOR_DISTANCE= 99.9954 | ||
OSCILLATION_RANGE= 0.500 | OSCILLATION_RANGE= 0.500 | ||
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! BACKGROUND_RANGE=1 10 ! rather use defaults (first 5 degree of rotation) | ! BACKGROUND_RANGE=1 10 ! rather use defaults (first 5 degree of rotation) | ||
SPACE_GROUP_NUMBER= | SPACE_GROUP_NUMBER=1 ! 0 if unknown | ||
UNIT_CELL_CONSTANTS= | UNIT_CELL_CONSTANTS= 27.07 31.25 33.76 87.98 108.00 112.11 ! PDB 2vb1 | ||
INCLUDE_RESOLUTION_RANGE=50 0 ! after CORRECT, insert high resol limit; re-run CORRECT | INCLUDE_RESOLUTION_RANGE=50 0 ! after CORRECT, insert high resol limit; re-run CORRECT | ||
FRIEDEL'S_LAW=FALSE ! This acts only on the CORRECT step | !FRIEDEL'S_LAW=FALSE ! This acts only on the CORRECT step | ||
! If the anom signal turns out to be, or is known to be, very low or absent, | ! If the anom signal turns out to be, or is known to be, very low or absent, | ||
! use FRIEDEL'S_LAW=TRUE instead (or comment out the line); re-run CORRECT | ! use FRIEDEL'S_LAW=TRUE instead (or comment out the line); re-run CORRECT | ||
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! parameters specifically for this detector and beamline: | ! parameters specifically for this detector and beamline: | ||
DETECTOR= ADSC MINIMUM_VALID_PIXEL_VALUE= 1 OVERLOAD= 65000 | DETECTOR= ADSC MINIMUM_VALID_PIXEL_VALUE= 1 OVERLOAD= 65000 | ||
SENSOR_THICKNESS=0.01 SILICON=34.812736 | |||
NX= 6144 NY= 6144 QX= 0.051294 QY= 0.051294 ! to make CORRECT happy if frames are unavailable | NX= 6144 NY= 6144 QX= 0.051294 QY= 0.051294 ! to make CORRECT happy if frames are unavailable | ||
DIRECTION_OF_DETECTOR_X-AXIS=1 0 0 | DIRECTION_OF_DETECTOR_X-AXIS=1 0 0 | ||
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</pre> | </pre> | ||
=== [[CORRECT.LP]] | === [[CORRECT.LP]] 1st pass === | ||
STANDARD DEVIATION OF SPOT POSITION (PIXELS) 0.87 | |||
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES) 0.10 | |||
CRYSTAL MOSAICITY (DEGREES) 0.126 | |||
... | |||
a b ISa | |||
6.630E+00 1.091E-04 37.18 | |||
... | |||
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 | |||
1.77 9195 4841 9501 51.0% 1.5% 1.5% 8708 48.74 2.1% 1.6% 0% 0.000 0 | |||
1.26 29991 15327 16721 91.7% 1.5% 1.6% 29328 45.26 2.1% 1.7% 0% 0.000 0 | |||
1.03 38643 19731 21636 91.2% 1.7% 1.7% 37824 38.67 2.5% 2.1% 0% 0.000 0 | |||
0.89 46156 23404 25561 91.6% 2.3% 2.4% 45504 27.56 3.3% 3.4% 0% 0.000 0 | |||
0.80 51509 26034 28868 90.2% 4.0% 4.0% 50950 17.55 5.6% 7.0% 0% 0.000 0 | |||
0.73 55989 28253 32034 88.2% 7.0% 6.8% 55472 10.98 9.8% 13.2% 0% 0.000 0 | |||
0.68 59733 30115 34776 86.6% 13.1% 13.0% 59236 6.08 18.6% 26.0% 0% 0.000 0 | |||
0.63 35385 18436 37367 49.3% 25.6% 26.9% 33898 2.99 36.3% 52.1% 0% 0.000 0 | |||
0.60 8991 4972 39725 12.5% 51.2% 56.9% 8038 1.34 72.4% 105.0% 0% 0.000 0 | |||
total 335592 171113 246189 69.5% 2.3% 2.4% 328958 19.58 3.3% 7.4% 0% 0.000 0 | |||
NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 343716 | |||
NUMBER OF REJECTED MISFITS 8112 | |||
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 0 | |||
NUMBER OF ACCEPTED OBSERVATIONS 335604 | |||
NUMBER OF UNIQUE ACCEPTED REFLECTIONS 171119 | |||
The number of "misfits" (rejections) is higher than expected (1 %). Either one considers the anomalous signal (of the 6 sulfurs) to be significant, or one simply increases WFAC1 from its default of 1, to (say) 1.2 . | |||
=== [[XDS.INP]]; optimized === | |||
Using the output of "grep _E INTEGRATE.LP|tail -2" edit XDS.INP to have | |||
JOB= INTEGRATE CORRECT | |||
BEAM_DIVERGENCE= 0.428 BEAM_DIVERGENCE_E.S.D.= 0.043 | |||
REFLECTING_RANGE= 0.880 REFLECTING_RANGE_E.S.D.= 0.126 | |||
... | |||
REFINE(INTEGRATE)= ! | |||
Then "cp GXPARM.XDS XPARM.XDS", and then another round of "xds_par". Five minutes later, we get: | |||
=== [[CORRECT.LP]] optimization pass === | |||
This looks a little bit better - less standard deviation, higher ISa, better R-factors, less misfits: | |||
= | STANDARD DEVIATION OF SPOT POSITION (PIXELS) 0.83 | ||
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES) 0.08 | |||
CRYSTAL MOSAICITY (DEGREES) 0.096 | |||
a b ISa | |||
6.439E+00 1.076E-04 37.98 | |||
... | |||
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 | |||
1.77 9149 4817 9501 50.7% 1.5% 1.5% 8664 49.75 2.1% 1.5% 0% 0.000 0 | |||
1.26 30049 15348 16723 91.8% 1.5% 1.6% 29402 46.26 2.1% 1.6% 0% 0.000 0 | |||
1.03 38920 19863 21637 91.8% 1.7% 1.7% 38114 39.61 2.4% 2.0% 0% 0.000 0 | |||
0.89 46381 23508 25562 92.0% 2.2% 2.3% 45746 28.39 3.1% 3.2% 0% 0.000 0 | |||
0.80 51605 26071 28868 90.3% 3.8% 3.8% 51068 18.21 5.3% 6.5% 0% 0.000 0 | |||
0.73 56126 28314 32041 88.4% 6.6% 6.4% 55624 11.45 9.3% 12.3% 0% 0.000 0 | |||
0.68 59735 30093 34771 86.5% 12.6% 12.3% 59284 6.34 17.8% 24.8% 0% 0.000 0 | |||
0.63 35754 18620 37370 49.8% 24.1% 25.5% 34268 3.11 34.1% 48.9% 0% 0.000 0 | |||
0.60 9180 5075 39730 12.8% 48.6% 54.3% 8210 1.40 68.7% 100.5% 0% 0.000 0 | |||
total 336899 171709 246203 69.7% 2.2% 2.3% 330380 20.14 3.2% 6.9% 0% 0.000 0 | |||
NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 344751 | |||
NUMBER OF REJECTED MISFITS 7842 | |||
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 0 | |||
NUMBER OF ACCEPTED OBSERVATIONS 336909 | |||
NUMBER OF UNIQUE ACCEPTED REFLECTIONS 171714 | |||