Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging

By W. Hagen, W. Wan, John A. G. Briggs. Journal of Structural Biology, 2017

721 citations recorded by the source corpus.

Cryo-electron tomography (cryoET) allows 3D structural information to be obtained from cells and other biological samples in their close-to-native state. In combination with subtomogram averaging, detailed structures of repeating features can be resolved. CryoET data is collected as a series of images of the sample from different tilt angles; this is performed by physically rotating the sample in the microscope between each image. The angles at which the images are collected, and the order in which they are collected, together are called the tilt-scheme. Here we describe a “dose-symmetric tilt-scheme” that begins at low tilt and then alternates between increasingly positive and negative tilts. This tilt-scheme maximizes the amount of high-resolution information maintained in the tomogram for subsequent subtomogram averaging, and may also be advantageous for other applications. We describe implementation of the tilt-scheme in combination with further data-collection refinements including setting thresholds on acceptable drift and improving focus accuracy. Requirements for microscope set-up are introduced, and a macro is provided which automates the application of the tilt-scheme within SerialEM.

Subfields: Radiation, Structural Biology, Surfaces, Coatings and Films

View Paper: https://www.semanticscholar.org/paper/8580681635514dd956bbd6316c42da0a0be716a5

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In Radiation

In Structural Biology

In Surfaces, Coatings and Films

Citations in the corpus

AI-based structure prediction empowers integrative structural analysis of human nuclear pores (2022, methodology): “45 Å (HEK) using an automated dosesymmetric acquisition scheme (74) starting at a given pre-tilt corresponding to the tilt of the FIB-milled lamellae (typically ±13°).”
Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers (2020, methodology): “For both data collections, tilt series images were collected using a dosesymmetric tilt scheme [26] ranging from -60 ̊ to 60 ̊ with 2 ̊ and 3 ̊ intervals for HA-VLPs and HA-NPs, respectively.”
Molecular Architecture of the SARS-CoV-2 Virus (2020, methodology): “361 sets of tilt-series data were collected using the dose-symmetric scheme (Hagen et al., 2017) from 60 to 60 at 3 steps and at various defocus between 1.”