Sub-2\u2009\xc5 Ewald curvature corrected structure of an AAV2 capsid variant

Yong Zi Tan,Mavis Agbandje-Mckenna,Mario Mietzsch,Joshua A Hull,Joshua Grieger,Robert Mckenna,R Jude Samulski,Dmitry Lyumkis,Sriram Aiyer,Timothy S Baker

doi:10.1038/s41467-018-06076-6

Abstract

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.

Highlights

Single-particle cryogenic electron microscopy provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities
We studied a variant of adeno-associated virus (AAV) serotype 2 containing a single amino-acid substitution, L336C
AAV was suited to our cryogenic electron microscopy (cryo-EM) studies because: (1) it is relatively small for a virus and can be packed across cryo-EM grid holes in reasonably thin ice; (2) it can be stably assembled into homogeneous virus-like particles (VLPs) devoid of genomic material; and (3) it has icosahedral symmetry, which increases the number of asymmetric subunits in the dataset by 60-fold for each particle imaged

Summary

Introduction

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. 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. We sought to address a number of factors limiting the resolution of structure determination by single-particle cryo-EM In these analyses, we studied a variant of adeno-associated virus (AAV) serotype 2 containing a single amino-acid substitution, L336C. We apply several technical advances in single-particle cryo-EM to the structure determination of the AAV2L336C capsid variant, and we quantitatively evaluate the benefits of each, including an experimental demonstration of correcting for the curvature of the Ewald sphere. The results and described methods provide a feasible path toward more routinely obtaining structures at sub-2 Å resolution in single-particle cryo-EM

Methods

Results

Conclusion