Membrane Protein Structure by Cryo Transmission Electron Microscopy

We study the structure and function of biological membranes and of membrane proteins, using transmission electron microscopy of frozen hydrated samples.

Membrane proteins are central to health and disease. These transmembrane polypeptide molecules function in the biological membranes of our cells, and serve among other roles as channels, transporters, and receptors. While databases contain an impressive amount of structural knowledge about soluble proteins, our knowledge about the structure and function of membrane proteins, especially human membrane proteins, is frustratingly sparse. It is, however, mostly membrane proteins, that are the targets of todays drugs. Knowledge about the structure and function of membrane proteins is of highest importance for pharmacological research.

Membrane proteins have to be expressed and purified. We are focusing our efforts on the human sodium proton exchanger hNHE1, which plays a central role in cellular pH and salt regulation, and is thereby important in cardiomyophathy, and solid tumors. We also collaborate with the Membrane Protein Expression Center (MPEC) and are part of the Center for Structures of Membrane Proteins (CSMP). While MPEC expresses and purifies membrane proteins, CSMP focuses on the structure determination of prokaryotic membrane proteins. Importantly, CSMP combines the methods of structure determination by X-ray, NMR, Mass-Spec, cryo-EM and Bioinformatics, see http://csmp.ucsf.edu/pi.htm. Other collaborations include the laboratories of Joe Mindell, NIH, and Crina Nimigean, UCD.

We study the structure of purified proteins and membrane proteins by single particle transmission electron microscopy, and perform two-dimensional membrane protein crystallization using mostly dialysis methods for computer-controlled detergent removal.

Structural data are collected using the electron beam. We utilize three Transmission Electron Microscopes (TEMs), a Philips EM410LS, a JEOL JEM-1230, and a JEOL JEM-2100FEG/STEM, which are equipped with 1k, 2k and 4k CCD cameras respectively. We expect delivery of another two TEMs, a JEOL JEM-3000SFF in December 2007, and of another JEOL JEM-2100F-CsCorr-STEM/TEM in Spring 2008. We also utilize Scanning Transmission Electron Microscopy (STEM), which uses a focused electron probe that is scanned over the biological samples. We investigate the usage of STEM technology for the imaging of frozen hydrated cryo-EM samples, and collaborate with the laboratory of Nigel Browning at UC Davis and LLNL.

Recorded micrographs are digitized with our Heidelberg Primescan scanner, or are recorded on the CCD cameras directly. These data are then computer image processed. To this end, we develop the 2dx software package that allows the efficient processing of a large number of 2D crystal images of membrane proteins. 2dx is developed as open-source GPL software. It features a user-friendly graphical user interface, and offers optionally the fully automatic processing of 2D crystal images. We recently implemented a Maximum Likelihood module for the 2dx package, in collaboration with Niko Grigorieff. We distribute this software in the workshop on electron crystallography, which we organize at UC Davis in collaboration with Ben Hankamer.

Cluster on Chromosome Mechanics

We also study the structure and function of proteins involved in chromosome mechanics. In collaboration with the laboratories of Jon Scholey, Wolf D. Heyer, Steve Kowalczykowski, and Alex Mogilner, we investigate the role of these soluble proteins during DNA maintenance and repair, and chromosome segregation.

In collaboration with