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University of Cambridge

3 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Joel R. Meyerson^1,2, Tommi A. White¹, Donald Bliss³, Amy Moran³, Alberto Bartesaghi¹, Mario J. Borgnia¹, M. Jason V. de la Cruz¹, David Schauder¹, Lisa M. Hartnell¹, Rachna Nandwani^1,4, Moez Dawood⁵, Brianna Kim⁶, Jun Hong Kim⁷, John Sununu⁸, Lisa Yang⁹, Siddhant Bhatia¹⁰, Carolyn Subramaniam¹, Darrell E. Hurt¹¹, Laurent Gaudreault¹², Sriram Subramaniam¹

¹Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, ²The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , ³National Library of Medicine, National Institutes of Health, ⁴Massachusetts Institute of Technology, ⁵William Fremd High School, ⁶University of Virginia , ⁷Duke University , ⁸Yale University, ⁹University of Notre Dame , ¹⁰Washington University in St. Louis , ¹¹Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ¹²Thomas Jefferson High School for Science and Technology

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Neuroscience

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Samsudeen Ponissery Saidu^*1, Michele Dibattista^*1, Hugh R. Matthews², Johannes Reisert¹

¹Monell Chemical Senses Center, ²Department of Physiology, Development & Neuroscience, Physiological Laboratory, University of Cambridge

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Biology

Robust 3D DNA FISH Using Directly Labeled Probes

Daniel J. Bolland¹, Michelle R. King², Wolf Reik^2,3, Anne E. Corcoran¹, Christel Krueger²

¹Nuclear Dynamics Programme, The Babraham Institute, ²Epigenetics Programme, The Babraham Institute, ³Centre for Trophoblast Research, University of Cambridge

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.