single-particle-cryo-em-data-collection-with-stage-tilt-using-leginon

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure ...

The Salk Institute for Biological Studies

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.

Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant.

A systematic and quantitative evaluation of cryo-EM maps is necessary to judge their quality and to capture all possible sources of error. A single value for global resolution is insufficient to accurately describe the quality of a reconstructed density. We describe the estimation and evaluation of two additional resolution measures, local and directional resolution, using methods based on the Fourier shell correlation (FSC). We apply the protocol to samples that encompass different types of pathologies a user is expected to encounter and provide analyses on how to interpret the output files and resulting maps. Implementation of these tools will facilitate density interpretation and can guide the user in adapting their experiments to improve the quality of cryo-EM maps, and by extension atomic models.

Sriram Aiyer

Laboratory of Genetics

The Salk Institute for Biological Studies

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

Sriram Aiyer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Genetics

The Salk Institute for Biological Studies

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

Laboratory of Genetics