Skip to content

Processing phenoxypyridone MicroED data

Jump to bottom
keitaroyam edited this page Aug 22, 2021 · 3 revisions

Raw data

  • Available in Zenodo. DOI
  • Collected on Talos Arctica G2 (200 kV)
  • Ceta (indirect CMOS) detector
  • Images collected using Velox software (.emd format)
  • Stage tilt controlled using SerialEM

Preparation

Here I use DIALS-2.1.0. Please download and install DIALS first.

To process emd files using DIALS, you need to install the dxtbx class. Please download FormatEMD.py and execute

dxtbx.install_format -g FormatEMD.py

Specify -u instead of -g if you do not have permission to DIALS installation directory.

See also here for the tips for electron diffraction data processing.

Processing phenoxypyridone data using DIALS+KAMO

1. Generate a mask

See rac-COTE

2. Prepare metadata

See rac-COTE

3. Indexing and integration

4. Merging

A-form

#!/bin/sh
# settings
dmin=0.65 # resolution
anomalous=false # true or false
lstin=formerge.lst # list of XDS_ASCII.HKL files
use_ramdisk=true # set false if there is few memory or few space in /tmp
# _______/setting

kamo.multi_merge \
        workdir=blend_${dmin}A_framecc_b+B \
        lstin=${lstin} d_min=${dmin} anomalous=${anomalous} \
        space_group=None reference.data=None \
        program=xscale xscale.reference=bmin xscale.degrees_per_batch=1 \
        reject_method=framecc+lpstats rejection.lpstats.stats=em.b+bfactor \
        clustering=blend blend.min_cmpl=90 blend.min_redun=4 blend.max_LCV=None blend.max_aLCV=None \
        max_clusters=None xscale.use_tmpdir_if_available=${use_ramdisk} \
        batch.engine=sge batch.par_run=merging batch.nproc_each=8 nproc=8 batch.sge_pe_name=smp

In the largest cluster (33 crystals):

 SPACE_GROUP_NUMBER=    3
 UNIT_CELL_CONSTANTS=     7.72    14.21     9.03  90.000 104.921  90.000

 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  CC(1/2)  Anomal  SigAno   Nano
   LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

     1.95        7112     147       149       98.7%       9.5%      7.5%     7112   69.58      9.6%    99.9*   -38    0.833     121
     1.38       14266     271       273       99.3%      15.7%     12.2%    14266   46.34     15.8%    99.9*   -33    0.876     242
     1.13       17379     318       318      100.0%      22.9%     20.5%    17379   30.85     23.2%    99.8*   -18    0.928     294
     0.98       21448     388       390       99.5%      29.5%     32.5%    21448   21.16     29.8%    99.7*    -9    0.786     359
     0.87       26218     474       474      100.0%      56.1%     80.9%    26218    9.98     56.6%    98.5*   -12    0.659     444
     0.80       25082     454       454      100.0%      84.0%    140.2%    25082    6.23     84.8%    97.0*   -11    0.549     428
     0.74       29400     530       532       99.6%     145.7%    266.0%    29400    3.52    147.1%    93.5*    -5    0.522     502
     0.69       25912     590       593       99.5%     212.1%    400.5%    25912    2.25    214.6%    87.3*    -2    0.501     562
     0.65       17333     604       600      100.7%     385.3%    709.6%    17333    1.11    392.2%    68.7*     2    0.438     577
    total      184150    3776      3783       99.8%      50.0%     76.6%   184150   13.83     50.6%    99.6*   -14    0.621    3529

B-form

#!/bin/sh
# settings
dmin=0.80 # resolution
anomalous=false # true or false
lstin=formerge.lst # list of XDS_ASCII.HKL files
use_ramdisk=true # set false if there is few memory or few space in /tmp
# _______/setting

kamo.multi_merge \
        workdir=blend_${dmin}A_framecc_b+B \
        lstin=${lstin} d_min=${dmin} anomalous=${anomalous} \
        space_group=None reference.data=None \
        program=xscale xscale.reference=bmin xscale.degrees_per_batch=1 \
        reject_method=framecc+lpstats rejection.lpstats.stats=em.b+bfactor \
        clustering=blend blend.min_cmpl=90 blend.min_redun=4 blend.max_LCV=None blend.max_aLCV=None \
        max_clusters=None xscale.use_tmpdir_if_available=${use_ramdisk} \
        batch.engine=sge batch.par_run=merging batch.nproc_each=8 nproc=8 batch.sge_pe_name=smp

In the largest cluster (16 crystals):

 SPACE_GROUP_NUMBER=    3
 UNIT_CELL_CONSTANTS=    10.89     4.45    21.28  90.000 100.214  90.000

 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  CC(1/2)  Anomal  SigAno   Nano
   LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

     2.40        1658      98       105       93.3%       9.3%      7.1%     1657   36.75      9.5%    99.8*   -14    1.041      43
     1.70        3705     176       177       99.4%      22.1%     16.0%     3703   23.99     22.6%    99.4*   -27    0.970     110
     1.39        4692     208       209       99.5%      43.2%     42.4%     4691   15.09     44.1%    98.0*   -33    0.756     146
     1.20        5753     255       257       99.2%      72.3%     89.8%     5752    9.40     73.9%    98.2*   -42    0.562     182
     1.07        6219     270       271       99.6%     106.8%    152.2%     6218    7.39    109.0%    97.3*   -23    0.613     204
     0.98        7527     306       307       99.7%     211.9%    334.8%     7525    4.64    216.0%    94.6*   -24    0.481     238
     0.91        6499     289       289      100.0%    1004.6%   1741.7%     6498    2.08   1025.1%    84.1*   -19    0.417     224
     0.85        8946     363       363      100.0%    1466.7%   2577.6%     8944    1.64   1494.8%    56.7*   -28    0.378     292
     0.80        9361     391       392       99.7%     -99.9%    -99.9%     9359    0.00    -99.9%    63.4*    -9    0.363     311
    total       54360    2356      2370       99.4%     247.6%    415.1%    54347    7.76    252.6%    98.4*   -27    0.526    1750
Clone this wiki locally