Electron crystallography uses cryo-transmission electron microscopy and computer image processing to characterize the structure of membrane proteins. Expressed and purified transmembrane proteins are reconstituted into lipid membranes, where they are artificially arranged into two-dimensional lattices by self-assembly processes. Such 2D crystals are then imaged with high-resolution electron microscopes. The 3D structure of the lipid-membrane embedded proteins can be determined by computer image data processing.
The Center for Cellular Imaging and Nano Analytics (C-CINA) at the University Basel, Switzerland, under the direction of Henning Stahlberg utilizes electron crystallography to study transporters, receptors, and ion channel membrane proteins. Membrane proteins are expressed, purified, and 2D-crystallized in the laboratory, and purified membrane proteins from other members of the TransCure team are studied in Basel. Data collection by electron crystallography is done with transmission electron microscopes. For this, C-CINA operates Switzerland's leading instrument, a FEI Titan Krios, which utilizes an 300kV electron beam to record high-resolution images and diffraction pattern of these frozen hydrated crystals. In addition to electron crystallography of 2D crystals, the Stahlberg group is also utilizing correlative light and electron microscopy (CLEM) and electron tomography, to study the cell-biological context of membrane embedded protein complexes.
Recorded images and diffraction patterns of the membrane proteins are then computationally analyzed, to reconstruct the 3D structure of the proteins.For this task, the Stahlberg group has developed a software tool called 2dx (available at http://2dx.org), which is now widely used throughout the electron crystallography community. 2dx is based on the so-called MRC software programs, and in addition includes several implementations of new algorithms, including a maximum likelihood based single particle processing tool for membrane proteins.
The Stahlberg laboratory is focusing on the Na+/Ca2+ exchanger (NCX), which is the dominant Ca2+-efflux mechanism in the heart that rapidly ejects large amounts of Ca2+ from the cytosol after the muscle contraction phase. Mishandling of cytosolic Ca2+ is often the central cause of both contractile dysfunction and heart arrhythmias that can lead to heart disease, the biggest cause of death in the Western world. In addition, in ischemia and multiple sclerosis NCX may play a damaging role as the ion transporter rather imports than exports Ca2+ under pathological conditions. To better understand the mechanism of Na+/Ca2+ exchange and to ultimately provide a structural basis for the design of more specific drugs that allow to control Ca2+ concentration in the cell or to prevent NCX from operating in reverse-mode, we study the structure of intact NCX.
In addition to structural studies by electron crystallography, the Stahlberg group will also utilize 2D crystals of various membrane proteins for preliminary experiments in preparation of utilization of the Swiss Free Electron Laser (Swiss-FEL), which is being constructed at the Paul Scherrer Institute (PSI). In collaboration with Prof. Gebhard Schertler, PSI, 2D crystals of a potassium channel produced in the Stahlberg lab will be subjected to X-ray diffraction experiments at the PSI, and data analysis software will be developed or adapted for the structural interpretation of these data.
Function in NCCR
- Principal Investigator (PI)
Professor at the Center for Cellular Imaging and Nano Analytics (C-CINA), Biozentrum, University of Basel
- PD (Habilitation) in Biophysics at the University of Basel
- Ph.D. in Structural Biology at the Ecole Polytechnique Fédérale, Lausanne
- M.Sc. in Physics at the Technical University of Berlin
- American Chemical Society
- Microscopy Society of America (MSA)
- Swiss Society for Optics and Microscopy (SSOM)
- German Physical Society (1989-1994)
- W.M.Keck Award (2009)
- UC Davis Chancellor’s Fellow Award (2008-2012)
- NSF CAREER award (2005)