2. The camera is controlled by EMMenu4 with two pre-set camera recording settings. One may be a full-chip readout with
subsampling by 2, the second a full-chip readout with subsampling by 4; both in rolling shutter mode.
3. Computing infrastructure is essential for both data collection
and processing. Here, we describe software operating in a
Linux environment on a server system powered by 40 processing threads (Intel
® Xeon
® CPU E5-2680 v2 @ 2.80 GHz)
with 256 GB of RAM, connected to a storage server with
400 TB of redundant (RAID6) NFS storage.
2.6 Data Reduction
and Structure
Determination
Software
1. Data parsing and visualization software: Software for conversion of diffraction movies to crystallographic format diffraction
images is limited. We describe conversion of Tietz Video Image
Processing Systems (TVIPS) files to SMV format using a custom program: tvips2smv (https://cryoem.ucla.edu/pages/
MicroED) [13]. Diffraction image visualization can be
achieved using the Adxv program (www.scripps.edu/tainer/
arvai/adxv.html) [24].
2. Data processing programs (see Note 3): Indexing, integration,
merging, and scaling can be achieved through a variety of
software programs. We describe use of the XDS package
(http://xds.mpimf-heidelberg.mpg.de/) [25].
3. Ab initio structure determination is achieved using various
direct methods programs. We describe use of the SHELX-97
suite, programs SHELXD and SHELXT (http://shelx.uni-ac.
gwdg.de/) [26].
4. X-ray crystallographic refinement programs suitable for processing electron diffraction data must have the capacity to use
electron scattering factors. Among those described here are
the SHELX-97 suite [26]; Phenix suite (https://www.phenixonline.org/) [27]; CCP4 suite: REFMAC (http://www.ccp4.
ac.uk/) [28].
5. Programs for dataset visualization, model building, and structural analysis include the ViewHKL program in the CCP4 suite
for dataset visualization [29], the Coot model building program (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/
coot/) [30] and PyMOL (https://pymol.org/2/) for model
building and structure analysis [31].
3 Methods
This section provides guidance on the experimental workflow of
electron diffraction of peptide nano- and microcrystals, from sample preparation to structure determination (Figs. 1 and 2).
332
Chih-Te Zee et al.
subsampling by 2, the second a full-chip readout with subsampling by 4; both in rolling shutter mode.
3. Computing infrastructure is essential for both data collection
and processing. Here, we describe software operating in a
Linux environment on a server system powered by 40 processing threads (Intel
® Xeon
® CPU E5-2680 v2 @ 2.80 GHz)
with 256 GB of RAM, connected to a storage server with
400 TB of redundant (RAID6) NFS storage.
2.6 Data Reduction
and Structure
Determination
Software
1. Data parsing and visualization software: Software for conversion of diffraction movies to crystallographic format diffraction
images is limited. We describe conversion of Tietz Video Image
Processing Systems (TVIPS) files to SMV format using a custom program: tvips2smv (https://cryoem.ucla.edu/pages/
MicroED) [13]. Diffraction image visualization can be
achieved using the Adxv program (www.scripps.edu/tainer/
arvai/adxv.html) [24].
2. Data processing programs (see Note 3): Indexing, integration,
merging, and scaling can be achieved through a variety of
software programs. We describe use of the XDS package
(http://xds.mpimf-heidelberg.mpg.de/) [25].
3. Ab initio structure determination is achieved using various
direct methods programs. We describe use of the SHELX-97
suite, programs SHELXD and SHELXT (http://shelx.uni-ac.
gwdg.de/) [26].
4. X-ray crystallographic refinement programs suitable for processing electron diffraction data must have the capacity to use
electron scattering factors. Among those described here are
the SHELX-97 suite [26]; Phenix suite (https://www.phenixonline.org/) [27]; CCP4 suite: REFMAC (http://www.ccp4.
ac.uk/) [28].
5. Programs for dataset visualization, model building, and structural analysis include the ViewHKL program in the CCP4 suite
for dataset visualization [29], the Coot model building program (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/
coot/) [30] and PyMOL (https://pymol.org/2/) for model
building and structure analysis [31].
3 Methods
This section provides guidance on the experimental workflow of
electron diffraction of peptide nano- and microcrystals, from sample preparation to structure determination (Figs. 1 and 2).
332
Chih-Te Zee et al.