cisTEM: User-friendly software for single-particle image processing

Abstract

7We have developed new open-source software called cisTEM (computational imaging system 8 for transmission electron microscopy) for the processing of data for high-resolution electron 9 cryo-microscopy and single-particle averaging. cisTEM features a graphical user interface that is 10 used to submit jobs, monitor their progress, and display results. It implements a full processing 11 pipeline including movie processing, image defocus determination, automatic particle picking, 12 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and 13high-resolution refinement and reconstruction. Some of these steps implement newly-developed 14 algorithms; others were adapted from previously published algorithms. The software is 15 optimized to enable processing of typical datasets (2000 micrographs, 200k -300k particles) on 16 a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated 17 processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that 18 can be adapted for most computing environments.

Structure of a rabies virus polymerase complex from electron cryo-microscopy

“…S1A, B) as described in Methods. We created an initial 3D reference de novo using the ab-initio module in cisTEM (Grant et al, 2018) from dose-fractionated movies obtained at 200 kV acceleration, and obtained a 6.7 Å resolution map from 22,000 particles after classification and refinement using RELION (Scheres, 2012) (Fig. S1C).…”

“…We picked 43,000 particles by hand from 316 motion-corrected movies using EMAN2.1 (Ludtke, 2016). A de novo 3D reference was obtained using the ab-initio procedure in cisTEM (Grant et al, 2018) following preliminary 2D classification, and the hand subsequently flipped to yield a reference bearing strong resemblance to VSV L-P. Final 2D and 3D classification using the flipped reference were then carried out in RELION 2.1 (Kimanius et al, 2016), yielding a 6.7Å (medium-resolution) map from 23,000 particles.…”

Reconstruction of Average Subtracted Tubular Regions (RASTR) Enables Structure Determination of Tubular Filaments by Cryo-EM

“…Post-processing and validation: A variety of aligning and classifying programs were used. cisTEM 41 was used for most of the 3D alignment and refinement, and cryoSPARC 42 for the majority of 2D alignment and classification. RELION 43 was used as an alternative for 2D classification and 3D refinement.…”

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition

“…Mask diameter used for template picking was 300 Å. A total of 215,554 particles were extracted with 432×432 pixel boxes from full-size images, and the stack and metadata file were exported to cisTEM (49) and used to generate the 2D classes shown in Fig. 6.…”

“…His-tagged RmtC proteins were detected by immunoblotting with a rabbit anti-6×His antibody (α6×His; Proteintech; 10001-0-AP) overnight at 4 ˚C. Blots were probed for with 432×432 pixel boxes from full-size images, and the stack and metadata file were exported to cisTEM (49) and used to generate the 2D classes shown in Fig. 6.…”