David L. Stokes

Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microscopy.

Desmosomes are a complex assembly of protein molecules that form at the cell surface and mediate cell–cell adhesion. Much is known about the composition of desmosomes and there is an established consensus for the location of and interactions between constituent proteins within the assembly. Furthermore, X-ray crystallography has determined atomic structures of isolated domains from several constituent proteins. Nevertheless, there is a lack of understanding about the architecture of the intact assembly and the physical principles behind the adhesive strength of desmosomes therefore remain vague. We have used electron tomography to address this problem. In previous work, we investigated the in situ structure of desmosomes from newborn mouse skin preserved by freeze-substitution and imaged in resin-embedded thin sections. In our present work, we have isolated desmosomes from cow snout and imaged them in the frozen unstained state. Although not definitive, the resulting images provide support for the irregular groupings of cadherin molecules seen previously in mouse skin.

▪ Abstract Active transport of cations is achieved by a large family of ATP-dependent ion pumps, known as P-type ATPases. Various members of this family have been targets of structural and functional investigations for over four decades. Recently, atomic structures have been determined for Ca²⁺-ATPase by X-ray crystallography, which not only reveal the architecture of these molecules but also offer the opportunity to understand the structural mechanisms by which the energy of ATP is coupled to calcium transport across the membrane. This energy coupling is accomplished by large-scale conformational changes. The transmembrane domain undergoes plastic deformations under the influence of calcium binding at the transport site. Cytoplasmic domains undergo dramatic rigid-body movements that deliver substrates to the catalytic site and that establish new domain interfaces. By comparing various structures and correlating functional data, we can now begin to associate the chemical changes constituting the reaction cycle with structural changes in these domains.

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic‐reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca²⁺‐ATPase effects reuptake in an ATP‐dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca²⁺‐ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca²⁺‐ATPase at 8‐Å resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP‐binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30‐Å resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.