2 Materials
2.1 Software
and Hardware
In this work, we refer to the software package FOCUS (http://
www.focus-em.org) [22] and a version of the FREALIGN package
[39] extended with additional features useful for the processing of
2D crystal data, hereby called frealign-2dx to avoid confusion with
the original implementation (http://github.com/C-CINA/
frealign-2dx) [38]. Both, FOCUS and frealign-2dx are freely available as open-source software and run on Linux-based operating
systems.
A typical computing workstation to carry out the data processing steps described ahead will have the following hardware components (see Note 1):
1. 2Â 12-core CPU or better.
2. 256 GB RAM.
3. >50 TB HD storage.
4. 1 TB SSD storage (used as a fast “scratch” disk).
5. 2Â NVIDIA GTX 1080 GPU card or better (see Note 2).
With this setup, it is possible to carry out real-time data preprocessing (e.g., drift correction and CTF estimation, see Subheading 3.1), classical 2D crystal processing (see Subheading 3.2), and
single particle refinements (Subheading 3.3.1 onwards). However,
for the processing of large datasets, in particular with large box
sizes, a high-performance computing (HPC) cluster may be
required.
3 Methods
We here describe the computational steps required to process 2D
crystal data with single particle software. For details and protocols
on the growth of 2D crystals and their sample preparation for TEM
imaging, please refer to [40–43]. After exporting the data from
FOCUS [22], we will use frealign-2dx, an extended version of the
FREALIGN package [39] (see Supplementary Notes 1 and 2 of
[38]) although any other single particle analysis package can also be
used, in principle (see Note 3).
3.1 Data Acquisition
and Initial Processing
of Movies
To ensure a good spectral SNR (SSNR) in the high-resolution
range, we recommend the acquisition of movies at a microscope
magnification corresponding to a pixel size <1 A ˚ at the sample
level, to benefit from the improved detective quantum efficiency
(DQE) of DEDs at lower detector resolutions [29]. Total exposures in the range of 40–50 e
À
/A ˚ 2 per movie are known to work
well. Multiple 2D crystals should be imaged, applying tilt angles
270
Ricardo Righetto and Henning Stahlberg
2.1 Software
and Hardware
In this work, we refer to the software package FOCUS (http://
www.focus-em.org) [22] and a version of the FREALIGN package
[39] extended with additional features useful for the processing of
2D crystal data, hereby called frealign-2dx to avoid confusion with
the original implementation (http://github.com/C-CINA/
frealign-2dx) [38]. Both, FOCUS and frealign-2dx are freely available as open-source software and run on Linux-based operating
systems.
A typical computing workstation to carry out the data processing steps described ahead will have the following hardware components (see Note 1):
1. 2Â 12-core CPU or better.
2. 256 GB RAM.
3. >50 TB HD storage.
4. 1 TB SSD storage (used as a fast “scratch” disk).
5. 2Â NVIDIA GTX 1080 GPU card or better (see Note 2).
With this setup, it is possible to carry out real-time data preprocessing (e.g., drift correction and CTF estimation, see Subheading 3.1), classical 2D crystal processing (see Subheading 3.2), and
single particle refinements (Subheading 3.3.1 onwards). However,
for the processing of large datasets, in particular with large box
sizes, a high-performance computing (HPC) cluster may be
required.
3 Methods
We here describe the computational steps required to process 2D
crystal data with single particle software. For details and protocols
on the growth of 2D crystals and their sample preparation for TEM
imaging, please refer to [40–43]. After exporting the data from
FOCUS [22], we will use frealign-2dx, an extended version of the
FREALIGN package [39] (see Supplementary Notes 1 and 2 of
[38]) although any other single particle analysis package can also be
used, in principle (see Note 3).
3.1 Data Acquisition
and Initial Processing
of Movies
To ensure a good spectral SNR (SSNR) in the high-resolution
range, we recommend the acquisition of movies at a microscope
magnification corresponding to a pixel size <1 A ˚ at the sample
level, to benefit from the improved detective quantum efficiency
(DQE) of DEDs at lower detector resolutions [29]. Total exposures in the range of 40–50 e
À
/A ˚ 2 per movie are known to work
well. Multiple 2D crystals should be imaged, applying tilt angles
270
Ricardo Righetto and Henning Stahlberg