Electron microscopy (EM) is gaining substantial momentum in the realm of structural biology techniques for membrane proteins and macromolecular complexes. This technique uses a beam of electrons that are transmitted through a thin specimen and the resulting images collected on digital cameras. Computers are then utilized to reconstruct the two-dimensional image projections of the biological specimen into a three-dimensional volume.
CCMSB houses state-of-the-art electron microscopes in newly renovated space (JEOL JEM2200FS, FEI Tecnai F20, FEI Tecnai Spirit). The microscopes are used for collecting images suitable for single particle reconstruction algorithms and for collecting tilt-series to generate tomograms by electron tomography. The facility is set up to be used with negative stained or flash-frozen (cryo-EM; cryo-tomography) samples.
The primary users of the EM CCMSB facility are Dr. Phoebe Stewart, Dr. Jason Mears, Dr. Vera Moiseenkova-Bell, Dr. Derek Taylor and Dr. Krzysztof Palczewsk. We encourage investigators to contact EM CCMSB members to obtain assistance and support for pilot projects, and to inquire about possible collaborations. In addition, EM facility assisted use and training can be provided with hourly fees by the EM facility manager.
Single-particle EM can provide structural information for a large variety of biological molecules without the need to produce crystals. Proteins from 200 kDa to several MDa in size can be analyzed. Very little sample is required for this technique. For cryo-EM the specimen, typically a protein complex, is embedded in vitreous ice, and is kept at cryogenic temperatures while images are recorded by the electron microscope. After completing the imaging, single-particle reconstruction methods are used to solve the structure of the protein. Current resolution for single particle cryo-EM is in the 4-25 Å range.
Cryo-electron tomography can unveil the detailed 3D structures of cellular and subcellular macromolecular objects. High pressure-freezing technique for cells and tissue can provide exceptional preservation of 100µm large samples in the native state. Single cells can be vitrified by plunging them into liquid ethane. This allows studying macromolecular complexes in their cellular environment, which provides a deep insight into cellular processes at the molecular level. 2D images (projections) of the sample collected at different angles allow the 3D structure to be reconstructed by the back-projection method. Resolution for cryo-electron tomography is in the 30-100 Å range.
To exploit the capacity of the electron microscope to acquire amplitude and phase information at 3-5 Å resolution for crystallographic measurements, highly ordered 2D crystals are imperative. They should exhibit lateral dimensions of several microns over which the crystallinity should be perfect. Both images (projections) and electron diffraction patterns of crystals kept at low temperature are recorded at minimum dose. Datasets are processed to yield the atomic structure of the crystallized protein. Electron crystallography is of special interest for membrane proteins that are crystallized in the presence of lipids, which reconstitutes the proteins in their native environment, the lipid bilayer. Hence, membrane proteins can be crystallized in an active state, and the structure of different functional conformations can be assessed.
Sudheer Molugu, PhD
EM Facility Manager
Cleveland Center for Membrane and Structural Biology
1819 E 101st St
Cleveland, Ohio 44106-7099