Approaches to Membrane Protein Crystallization Workshop

Friday, March 26th, 9:10 am - 4:00 pm.
Room 134 (Tiered Classroom), The Telus Centre
University of Alberta, Edmonton.

The Alberta Synchrotron Institute is pleased to present this short workshop on techniques and applications of membrane protein crystallization.

Continental breakfast and lunch will be provided for participants.
Registration is free, but space for the workshop is limited.
Please email to register or for more information.

Workshop Announcement (PDF file) | Workshop Program | Talk Abstracts

Workshop Program

Further details will be provided as they become available

Talk Abstracts

"The multiple homologue approach to membrane protein crystallography"

Randal Bass

Amgen Corporation

A major stumbling block in membrane protein x-ray crystallography has been the production of diffraction-quality crystals. An approach pioneered in the Rees lab at Caltech has been the identification, cloning, expression, and detergent screening of multiple homologues within a targeted class of membrane proteins. Just as Kendrew sampled multiple species of whale meats in an attempt to obtain crystals of myoglobin, members of the Rees lab use molecular biology to obtain the same genetic sampling of members of a given protein family. Subsequent to identification and cloning of the homologues, extensive expression and detergent screening experiments are carried out to identify those proteins that have the highest likelihood of successful crystallization. These techniques, along with modifications made to data processing of the resulting diffracting crystals will be discussed.

Randal Bass' Lecture Notes (Powerpoint Presentation)

"Edmonton-Vancouver-Jena-Stanford: a long journey to the NarGHI crystal structure"

Michela G. Bertero, Richard A. Rothery, Monica K. Palak, Cynthia Hou, Daniel Lim, Francis Blasco, Joel H. Weiner, and Natalie C.J. Strynadka

Department of Biochemistry, University of British Columbia
Membrane Protein Research Group, University of Alberta
Laboratoire de Chimie Bactérienne, CNRS, Marseille, France

In order to adapt to different environmental challenges, the facultative anaerobe Escherichia coli is able to assemble specific and modular respiratory chains by synthesis of appropriate dehydrogenases and reductases. Under anaerobic conditions in the presence of nitrate, E. coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (Nitrate Reductase A; NarGHI), which reduces nitrate to nitrite and forms part of a proton-motive force generating redox loop. We have determined the crystal structure of NarGHI to 1.9Å resolution. Our structure consists of a heterotrimeric complex of approximately 220kDa. The catalytic and electron transfer subunits (NarG and NarH, respectively) are localized in the cytoplasm and anchored to the membrane by NarI, a novel diheme cytochrome b with five transmembrane helices. Experimental approaches to achieve high-level overexpression of this complex, purification from the membranes in an active and monodisperse form, successful crystallization and phasing will be discussed.

Michela Bertero's Lecture Notes (PDF format)

"The ups and downs of three-dimensional crystallization for gradient-driven transporters: Case studies of GlpT and AE1 three-dimensional crystallization."

M. Joanne Lemieux

Department of Biochemistry, University of Alberta

Gradient-driven transporters, with their high proportion of membrane embedded segments, have been notoriously difficult to crystallize. In this presentation, the protocol for GlpT crystallization leading to its structural determination will be discussed. In addition, problems encountered during GlpT crystal optimization and strategies used to overcome them will be discussed. Cell culture, protein purification and lipid content will be considered. For comparison, the crystallization of another transporter, AE1, will be discussed in the context of overall factors contributing to crystallization of integral membrane proteins.

Joanne Lemieux's Lecture Notes (Powerpoint Presentation)

"Case studies in membrane protein crystallization & related endeavors"

Michael C. Wiener

Department of Molecular Physiology and Biological Physics, University of Virginia

The focus of my science is the structural biology of integral membrane proteins. We have worked extensively on a number of microbial and eukaryotic membrane proteins, with efforts in expression, purification, crystallization, structure determination as well as other biochemical, biophysical and functional studies. Also, we have significant efforts underway in various aspects of methods development with the goal of making the acquisition of membrane protein structures a less risky and difficult undertaking. This workshop presentation will consist of several sections:

1. (perhaps) a very brief introduction to some aspects of membrane protein crystallization
2. discussion of our (past) efforts on four proteins:
   - human aquaporin-1 (hAQP1)
   - E. coli aquaporin Z (AqpZ)
   - E. coli ClC chloride channel (YadQ)
   - E. coli outer membrane cobalamin transporter BtuB
3. our (ongoing) efforts in crystallization screen development

We obtained crystals of all of the proteins mentioned above; however, only BtuB yielded crystals of sufficient quality for structure determination. Each of these proteins presented different challenges and different results; discussion of them provides an entrée into many of the methods used in the production and crystallization of membrane proteins.

Michael Wiener's Lecture Notes (Powerpoint Presentation)