Cryo-electron Microscopy of Membrane Proteins

Abstract

Electron crystallography is used to study membrane proteins in the form of planar, twodimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beaminduced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base. SummaryElectron crystallography studies membrane proteins in form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed...

Overcoming bottlenecks in the membrane protein structural biology pipeline

“…These electron micrographs can be used to pick particles, obtain class averages and back project them into a 3D density map of the membrane protein using imaging process software such as SPIDER, EMAN or IMAGIC [15]. Another method called electron crystallography consists of producing thin layers of planar, two-dimensional (2D) crystals of membrane proteins which are able to scatter electrons [19]. Electron crystallography can therefore generate structures in a near-tonative environmental state [20].…”

Membrane protein structural biology using X-ray free electron lasers

“…This arrangement is closer to physiological conditions than a 3D crystal and provides accessibility to both sides of the protein for ligand interaction. Due to their small size, 2D-crystals suffer from severe radiation damage at synchrotrons and have therefore previously been examined using electron diffraction at cryogenic temperatures [58][59][60]. The main bottlenecks so far are technical difficulties in preparing well ordered 2D crystals, missing possibilities for correcting for lattice bending through image processing, the missing cone problem when collecting tilted data [61] and a lack of automation for 2D crystal production.…”

Imaging and structural studies of DNA–protein complexes and membrane ion channels

“…The possibility of mapping DNA-protein or proteinprotein interactions at Å resolution and directly studying the structure of single ion channels in their native environment will make it possible to elucidate gene expression and signal transduction mechanisms. Although the electrophysiology of membranes is very well understood and several experimental and theoretical methods have been developed, 3 a detailed characterization of the cell membrane through high-resolution imaging is still missing. 4 Moreover, cell membranes with their associated proteins are the targets for several extracellular and intracellular molecules as well as for drugs.…”