From the session website: We often encounter datasets that include weak experimental phases that we must depend on in order to solve our structures. The following two datasets are examples of data collected for the purposes of solving structures by Single-wavelength Anomalous Diffraction (SAD) phasing. The anomalous signal is present, but weak, and therefore care must be taken to preserve the anomalous signal for phasing. This demonstration will educate the user on how to preserve the anomalous differences in a dataset. |
Content:
Let's look at the datasets from the two synchrotron trips separately.
Running
% find_images
shows two datasets: j1603b3PK_1_E1_001.img to j1603b3PK_1_E1_592.img and j1603b3PK_1_E2_001.img to j1603b3PK_1_E2_591.img. A bit more detail can be seen with
% imginfo j1603b3PK_1_E1_001.img j1603b3PK_1_E1_592.img
which gives
################# File = j1603b3PK_1_E1_001.img >>> Image format detected as ADSC ===== Header information: date = 27 Jun 2004 07:04:21 exposure time [seconds] = 5.000 distance [mm] = 210.000 wavelength [A] = 0.979400 Phi-angle (start, end) [degree] = 0.000 0.250 Oscillation-angle in Phi [degree] = 0.250 Omega-angle [degree] = 0.000 2-Theta angle [degree] = -0.003 Pixel size in X [mm] = 0.102400 Pixel size in Y [mm] = 0.102400 Number of pixels in X = 2048 Number of pixels in Y = 2048 Beam centre in X [mm] = 105.100 Beam centre in X [pixel] = 1026.367 Beam centre in Y [mm] = 101.050 Beam centre in Y [pixel] = 986.816 Overload value = 65535 ################# File = j1603b3PK_1_E2_001.img >>> Image format detected as ADSC ===== Header information: date = 27 Jun 2004 07:04:57 exposure time [seconds] = 5.000 distance [mm] = 210.000 wavelength [A] = 0.918400 Phi-angle (start, end) [degree] = 0.000 0.250 Oscillation-angle in Phi [degree] = 0.250 Omega-angle [degree] = 0.000 2-Theta angle [degree] = -0.003 Pixel size in X [mm] = 0.102400 Pixel size in Y [mm] = 0.102400 Number of pixels in X = 2048 Number of pixels in Y = 2048 Beam centre in X [mm] = 105.100 Beam centre in X [pixel] = 1026.367 Beam centre in Y [mm] = 101.050 Beam centre in Y [pixel] = 986.816 Overload value = 65535
So this looks like an inflection and high-energy remote, 2-wavelength MAD on Se dataset. We can get a bit more information about the actual data-collection using
% imgdate.sh -s *.img > img.lis
which shows
# sorted list of: file, Epoch, Date, seconds-to-previous j1603b3PK_1_E1_001.img 1088319861 27 Jun 2004 07:04:21 0 j1603b3PK_1_E1_002.img 1088319869 27 Jun 2004 07:04:29 8 j1603b3PK_1_E1_003.img 1088319877 27 Jun 2004 07:04:37 8 j1603b3PK_1_E1_004.img 1088319886 27 Jun 2004 07:04:46 9 j1603b3PK_1_E2_001.img 1088319897 27 Jun 2004 07:04:57 11 j1603b3PK_1_E2_002.img 1088319905 27 Jun 2004 07:05:05 8 j1603b3PK_1_E2_003.img 1088319913 27 Jun 2004 07:05:13 8 j1603b3PK_1_E2_004.img 1088319922 27 Jun 2004 07:05:22 9 j1603b3PK_1_E1_005.img 1088319933 27 Jun 2004 07:05:33 11 j1603b3PK_1_E1_006.img 1088319941 27 Jun 2004 07:05:41 8 j1603b3PK_1_E1_007.img 1088319949 27 Jun 2004 07:05:49 8 j1603b3PK_1_E1_008.img 1088319957 27 Jun 2004 07:05:57 8 j1603b3PK_1_E2_005.img 1088319968 27 Jun 2004 07:06:08 11 ... j1603b3PK_1_E2_368.img 1088326532 27 Jun 2004 08:55:32 9 j1603b3PK_1_E1_369.img 1088326543 27 Jun 2004 08:55:43 11 j1603b3PK_1_E1_370.img 1088326551 27 Jun 2004 08:55:51 8 j1603b3PK_1_E1_371.img 1088326560 27 Jun 2004 08:56:00 9 j1603b3PK_1_E1_372.img 1088326568 27 Jun 2004 08:56:08 8 j1603b3PK_1_E2_369.img 1088326579 27 Jun 2004 08:56:19 11 j1603b3PK_1_E2_370.img 1088326588 27 Jun 2004 08:56:28 9 j1603b3PK_1_E2_371.img 1088327966 27 Jun 2004 09:19:26 1378 j1603b3PK_1_E2_372.img 1088327974 27 Jun 2004 09:19:34 8 ...
We can see:
Running
% find_images
reports 90 images: J11C05b3_12_001.img to J11C05b3_12_090.img. With
% imginfo J11C05b3_12_001.img
we get some more information
################# File = J11C05b3_12_001.img >>> Image format detected as ADSC ===== Header information: exposure time [seconds] = 83.630 distance [mm] = 450.000 wavelength [A] = 0.979245 Phi-angle (start, end) [degree] = 209.000 210.000 Oscillation-angle in Phi [degree] = 1.000 Omega-angle [degree] = 0.000 Pixel size in X [mm] = 0.102588 Pixel size in Y [mm] = 0.102588 Number of pixels in X = 3072 Number of pixels in Y = 3072 Beam centre in X [mm] = 157.500 Beam centre in X [pixel] = 1535.267 Beam centre in Y [mm] = 157.500 Beam centre in Y [pixel] = 1535.267
Unfortunately, the image header doesn't record the collection date - so we can't analyse it in the same way as above (in order to find potential problems).
First let's check the ALS dataset:
Image | Full image | Centre region | Upper-left |
j1603b3PK_1_E1_001 | |||
j1603b3PK_1_E1_061 | |||
j1603b3PK_1_E1_121 | |||
j1603b3PK_1_E1_181 | |||
j1603b3PK_1_E1_241 | |||
j1603b3PK_1_E1_301 | |||
j1603b3PK_1_E1_361 | |||
j1603b3PK_1_E1_421 | |||
j1603b3PK_1_E1_481 | |||
j1603b3PK_1_E1_541 | |||
j1603b3PK_1_E2_001 | |||
j1603b3PK_1_E2_061 | |||
j1603b3PK_1_E2_121 | |||
j1603b3PK_1_E2_181 | |||
j1603b3PK_1_E2_241 | |||
j1603b3PK_1_E2_301 | |||
j1603b3PK_1_E2_361 | |||
j1603b3PK_1_E2_421 | |||
j1603b3PK_1_E2_481 | |||
j1603b3PK_1_E2_541 |
The beam centre is a bit off-centre. This can often cause problems if the visible beam centre and the header value are not in sync. So let's quickly check that:
% adxv j1603b3PK_1_E1_001.img
will show the image
and the control panel
We will zoom in (selecting the "100%" button in the control panel)
and move the mouse to the visible beam centre
We can read off the pixel values as (1026,1060). How does that relate to the header values (1026.367,986.816) from above?
% beam8.sh 1026.367 986.816 2048
shows
Convention Beam centre ----------------------------- x, y = 1026.37 986.82 -x, y = 1021.63 986.82 x,-y = 1026.37 1061.18 -x,-y = 1021.63 1061.18 y, x = 986.82 1026.37 -y, x = 1061.18 1026.37 y,-x = 986.82 1021.63 -y,-x = 1061.18 1021.63
So the header seem to follow the (x,-y) convention.
The images of the single scan dataset:
Image | Full image | Centre region | Upper-left |
1 | |||
31 | |||
61 |
We first process the ALS dataset:
We've already noticed that the beam centre recorded in the header needs to be converted in order to match the actual image. So we could tell autoPROC about that by using
% process BeamCentreFrom="header:x,-y" -d 01 | tee 01.lis
However, we also noticed that the diffraction seems to get weaker towards the end of data collection. So lets switch on a series of options that try and take care of such potential issues (apart from beam centre convention and loss of diffraction power, it also tries and deals with ice-rings):
% process -M automatic -d 01 | tee 01.lis
This reads a so-called macro (named "automatic") - for a list of macros please run process -M list.
which gives for the inflection wavelength spacegroup P4212
Summary data for Project: Test Crystal: A Dataset: 0.979400 Overall InnerShell OuterShell --------------------------------------------------------------------------- Low resolution limit 33.345 33.345 2.437 High resolution limit 2.429 11.134 2.429 Rmerge 0.081 0.055 0.442 Ranom 0.079 0.052 0.400 Rmeas (within I+/I-) 0.086 0.057 0.480 Rmeas (all I+ & I-) 0.085 0.059 0.481 Rpim (within I+/I-) 0.036 0.023 0.261 Rpim (all I+ & I-) 0.026 0.019 0.188 Total number of observations 265518 2369 1637 Total number unique 26953 321 254 Mean(I)/sd(I) 20.0 31.3 4.2 Completeness 98.6 95.3 98.8 Multiplicity 9.9 7.4 6.4 Anomalous completeness 98.4 95.0 99.6 Anomalous multiplicity 5.3 5.4 3.3
and for the high-energy remote spacegroup P41212
Summary data for Project: Test Crystal: A Dataset: 0.918400 Overall InnerShell OuterShell --------------------------------------------------------------------------- Low resolution limit 33.345 33.345 2.480 High resolution limit 2.473 11.134 2.473 Rmerge 0.082 0.057 0.478 Ranom 0.080 0.055 0.459 Rmeas (within I+/I-) 0.088 0.061 0.544 Rmeas (all I+ & I-) 0.086 0.061 0.519 Rpim (within I+/I-) 0.036 0.025 0.289 Rpim (all I+ & I-) 0.026 0.020 0.199 Total number of observations 251499 2404 1152 Total number unique 25542 324 175 Mean(I)/sd(I) 19.7 30.4 4.1 Completeness 98.6 96.1 98.3 Multiplicity 9.8 7.4 6.6 Anomalous completeness 98.3 97.2 100.0
The final files:
We can run with all defaults
% process -d 01 | tee 01.lis
to get spacegroup P41212 and
Summary data for Project: Test Crystal: A Dataset: 0.97925 Overall InnerShell OuterShell --------------------------------------------------------------------------- Low resolution limit 131.822 131.822 3.458 High resolution limit 3.447 15.990 3.447 Rmerge 0.103 0.037 0.392 Ranom 0.096 0.032 0.385 Rmeas (within I+/I-) 0.112 0.036 0.447 Rmeas (all I+ & I-) 0.112 0.041 0.426 Rpim (within I+/I-) 0.058 0.018 0.223 Rpim (all I+ & I-) 0.044 0.018 0.160 Total number of observations 62137 658 512 Total number unique 9595 128 79 Mean(I)/sd(I) 18.0 40.2 4.6 Completeness 97.8 97.7 81.4 Multiplicity 6.5 5.1 6.5 Anomalous completeness 93.2 94.1 68.3 Anomalous multiplicity 3.6 3.3 3.9
and files:
Can those already be used for solving the structure? See the autoSHARP tutorial.
The POINTLESS step in determining the most likely spacegroup comes up with P4212 (ALS infl) and P41212 (ALS hrem and SSRL data). Although there is still some checking to be done, it seems most likely that we have P41212 (or the enantiomorph P43212).
Work in progress