Award details

Cryo-electron tomography at Leeds University

Reference	BB/C511372/1
Principal Investigator / Supervisor	Professor John Trinick
Co-Investigators / Co-Supervisors	Professor Stephen Baldwin, Professor Jim Deuchars, Professor Michelle Peckham, Professor Neil Ranson, Professor David Rowlands
Institution	University of Leeds
Department	Institute of Membrane & Systems Biology
Funding type	Research
Value (£)	119,000
Status	Completed
Type	Research Grant
Start date	01/02/2005
End date	31/01/2008
Duration	36 months

Abstract

This proposal aims to establish cryo-electron tomography of intact cells, organelles and large supramolecular assemblies at the University of Leeds. Cryo-electron microscopy is now the technique of choice for the study of protein complexes and sub-1 nm resolution is becoming commonplace. However, most electron microscopy of intact tissue and cells relies on the older technique of chemical fixation and staining followed by plastic embedding and sectioning; this methodology generally achieves much poorer resolution, 5-10 nm, which does not allow the shapes of macromolecules to be discerned. As a consequence, high resolution structural data from X-ray crystallography, NMR and electron microscopy cannot be readily integrated into the cellular context. Cryo-electron tomography is a new alternative to plastic sectioning that combines the data from micrographs recorded at different tilt angles to produce a 3D model of a specimen region. Naturally occurring thin layer specimens are minimally perturbed during preparation and are observed unstained and hydrated after rapid freezing to trap the vitreous state of water. Information recovery is limited primarily by the electron radiation damage incurred in the many images (usually greater than 100) in a tilt series, which is recorded automatically under software control. A key element in the cryo-tomography approach is the use of an electron energy filter to remove inelastically scattered electrons. Inelastically scattered electrons are focused before the elastic signal and always predominate in unstained, hydrated specimens, especially in thicker layers where there is multiple scattering of individual electrons. Images recorded using only zero energy loss electrons therefore have a much better signal-to-noise ration. The resolution attainable in 3D tomography models is currently approximately 5 nm. Prospects are good that this will improve to approximately 2 nm where macromolecule shapes and conformational changes can be resolved. Docking of high resolution structures into envelopes obtained by tomography provides the basis for an atomic description of cellular events. In the first instance, the cryo-tomography method will be applied to the following important projects. (I) Structure of the sarcomere in isolated muscle myofibrils. (II) Distribution of actin filaments in filopodia. (III) Mechanism of virus infection. (IV) Exocyst complex and post-Golgi trafficking pathways. (V) Ion channel and neurotransmitter receptor proteins in neuronal presynaptic terminals.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2004 (RE4) [2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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