Preprint: Please note that this article has not completed peer review.

Collecting and processing single-particle cryo-EM data with tilts

Dmitry Lyumkis, Yong Zi Tan, Philip Baldwin
DOI: 10.1038/protex.2017.055

Abstract

Here we present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy (cryo-EM) by employing tilts during data collection and quantifying the resulting directional resolution and density isotropy using a 3D Fourier Shell Correlation (FSC) algorithm. These techniques enabled structure determination of the influenza hemagglutinin (HA) trimer, which adopts a highly preferred orientation, to near-atomic resolution. They also improved both isotropy and global resolution for ribosomal biogenesis intermediates, which adopt a moderately preferred orientation. This protocol describes data collection and image processing strategies for employing tilts in single-particle cryo-EM.

Keywords
single-particle cryo-EM, resolution anisotropy, tilts, preferred specimen orientation

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

Acknowledgements

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Preprint: Please note that this article has not completed peer review.

Collecting and processing single-particle cryo-EM data with tilts

Dmitry Lyumkis, Yong Zi Tan, Philip Baldwin

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Abstract

Here we present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy (cryo-EM) by employing tilts during data collection and quantifying the resulting directional resolution and density isotropy using a 3D Fourier Shell Correlation (FSC) algorithm. These techniques enabled structure determination of the influenza hemagglutinin (HA) trimer, which adopts a highly preferred orientation, to near-atomic resolution. They also improved both isotropy and global resolution for ribosomal biogenesis intermediates, which adopt a moderately preferred orientation. This protocol describes data collection and image processing strategies for employing tilts in single-particle cryo-EM.

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

Acknowledgements

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