Cryo-electron microscopy

Mission

The health of cells and organisms depends critically on a spatially defined interplay of proteins, RNA and/or DNA in large complexes that focus and enhance specific functions at certain sites in the cell. Our goal is to understand the structural basis by which these complexes are formed and how their architecture contributes to their mechanisms of action. Many of these complexes are scarce, flexible or compositionally heterogeneous. These properties are important for their function but render them difficult targets for structural studies by X-ray crystallography. Such complexes can often be studied by electron cryo-microscopy, a true single molecule approach with the potential to determine structures and quantitatively assess functionally important temporal and compositional heterogeneity, generating near-atomic resolution models for subsequent studies of molecular function. When coupled with additional high-resolution studies of smaller building blocks and domains plus the mapping of functional interactions by mutagenesis and biochemical analysis, complex function can ultimately be understood at the molecular level. The facility provides an environment for effective structure determination of biological macromolecular assemblies.

Equipment

FEI Tecnai F20 - Mausi Installed in June 2010, the facility provides state of the art equipment. A FEI F20 electron microscope (200 kV, field emission gun) equipped with an 8k x 8k CMOS camera allows efficient collection of high-resolution structural data. This is the first 8k camera that is mounted to an F20 electron microscope. The camera allows rapid data collection for high-resolution image processing projects.

A Gatan-Cold stage and various freezing devices (vitrobot, controlled environment freezing apparatus) are available for cryo-microscopy.

For sample preparation glow discharge and carbon coater is shared with the conventional EM-facility, which includes a Phillips CM120 Biotwin electron microscope.

Image processing is supported by seven workstations (dual Quad, 8 GB RAM) that are connected to a blade centre with 8 nodes (64 cores) and a fast 20 terabyte file-server. Common Image processing software is installed (Spider, IMAGIC, EMAN, XMIPP, MRC-programs, ...).

The microscope is situated in Rutherford G.01, the EM prep labs and Biotwin EM is next door in G.04, and computer workstations are in Darwin 2.201 and in the COIL facility, Swann building.

People

The facility is headed by Fabio Nudelman, Support is offered on a collaborative basis by experienced users.

Funding Model

The Facility was funded by The Wellcome Trust (equipment grant WT087658) and by SULSA. Maintanance of the facility is funded via facilities charges.

We are actively seeking new collaborative projects that might benefit from structural studies by cryo-EM. The EM facility staff can offer advice in sample preparation, imaging, image processing and project management on a collaborative basis (contact ).