Diamond Light Source Ltd, Harwell Science & Innovation Campus

4 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

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.

Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Sangmi Jun¹, Gongpu Zhao¹, Jiying Ning¹, Gregory A. Gibson², Simon C. Watkins², Peijun Zhang¹

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.

Biochemistry

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Ramona Duman¹, Christian M. Orr¹, Vitaliy Mykhaylyk¹, Kamel El Omari¹, Robert Pocock¹, Vinay Grama¹, Armin Wagner¹

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.

Biology

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Yuewen Sheng¹, Kyle Morris¹, 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.