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Diamond Light Source Ltd, Harwell Science & Innovation Campus

4 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Xin Meng 1, Gongpu Zhao 1, Peijun Zhang 1
1Department of Structural Biology, University of Pittsburgh School of Medicine

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

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Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
Sangmi Jun 1, Gongpu Zhao 1, Jiying Ning 1, Gregory A. Gibson 2, Simon C. Watkins 2, Peijun Zhang 1
1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

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Biochemistry

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source
Ramona Duman 1, Christian M. Orr 1, Vitaliy Mykhaylyk 1, Kamel El Omari 1, Robert Pocock 1, Vinay Grama 1, Armin Wagner 1
1Diamond Light Source Ltd, Harwell Science & Innovation Campus

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

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Biology

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging
Yuewen Sheng 1, Kyle Morris 1, Julika Radecke *1, Peijun Zhang *1,2,3
1Electron Bio-Imaging Centre, Diamond Light Source Ltd, Harwell Science & Innovation Campus, 2Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, 3Chinese Academy of Medical Sciences Oxford Institute, University of Oxford

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 Å resolution.

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