Electron crystallography of proteins in membranes

Electron crystallography has played a vital role in advancing our understanding of proteins in membranes since the 'fluid mosaic model' was proposed in 1972. It is now an established technique to reveal the structures of proteins in their natural bilayer environment and makes possible the study of biological mechanisms through freeze-trapping of transitional states. Thus, images and diffraction patterns of well-ordered, planar and tubular protein-lipid crystals are yielding atomic models, which tell us how the proteins in situ are designed and carry out their membrane-specific tasks. Recent methodological advances and the inclusion of tomographic and cryo-sectioning techniques are enabling detailed information to be obtained from increasingly smaller and more disordered membrane assemblies, extending the potential of this approach.     

Electron crystallography of aquaporins

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.     

Electron crystallography reveals the structure of metarhodopsin I

Rhodopsin is the prototypical G protein-coupled receptor, responsible for detection of dim light in vision. Upon absorption of a photon, rhodopsin undergoes structural changes, characterised by distinct photointermediates. Currently, only the ground-state structure has been described. We have determined a density map of a photostationary state highly enriched in metarhodopsin I, to a resolution of 5.5 A in the membrane plane, by electron crystallography. The map shows density for helix 8, the cytoplasmic loops, the extracellular plug, all tryptophan residues, an ordered cholesterol molecule and the beta-ionone ring. Comparison of this map with X-ray structures of the ground state reveals that metarhodopsin I formation does not involve large rigid-body movements of helices, but there is a rearrangement close to the bend of helix 6, at the level of the retinal chromophore. There is no gradual build-up of the large conformational change known to accompany metarhodopsin II formation. The protein remains in a conformation similar to that of the ground state until late in the photobleaching process.     

Electron crystallography of bacteriorhodopsin with millisecond time resolution

The goal of time-resolved crystallographic experiments is to capture dynamic "snapshots" of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light-induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5-A resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge-frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin.     

Electron crystallography of membrane proteins: two-dimensional crystallization and screening by electron microscopy

Structural and functional information of membrane proteins at ever-increasing resolution is being obtained by electron crystallography. While a large amount of work on the development of methods for electron microscopy and image processing has resulted in tremendous advances in terms of speed of data collection and resolution, general guidelines for crystallization are first starting to emerge. Yet two-dimensional crystallization itself will always remain the limiting factor of this powerful approach in structural biology. Two-dimensional crystallization through detergent removal by dialysis is the most widely used technique. Four main factors need to be considered for the dialysis method: the protein preparation, the detergent, the lipid added as well as any constituent lipid, and the buffer conditions. Equally important is proper and careful screening to identify two-dimensional crystals.     

Electron crystallography - the waking beauty of structural biology

Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes.     

Electron diffraction of membranes

Summary Electron diffraction conducted on myelin membranes, photosynthetic and photoreceptor membranes yielded spot diffraction patterns indicating an ordered state of membranes; the interplanar spacings being of the order of Å units. It was observed, too, that a membrane specimen accommodates different space structures. Based on these findings it is suggested that membrane functions-like active transport-are exercised through phase transitions; the lattice acts as a coupling agent.     

Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.     

Cellulose and glass fiber affinity membranes for the chromatographic separation of biomolecules

Macroporous cellulose and glass membranes were prepared from filter paper and glass fiber filter, respectively. To enhance their stability, the cellulose membranes were crosslinked with epichlorohydrin, and the glass membranes were crosslinked with glutaraldehyde or organic bifunctional silanes. Several pathways for the modification, activation, and ligand immobilization were used and compared. For cellulose membranes, the diazotization method provided the best results, whereas the glutaraldehyde method provided the best performance for glass membranes, regarding both their stability and ligand immobilization capacity. The characterization of the membranes was made by using a triazine dye, bovine serum albumin, and trypsin as test ligands. The membrane morphologies and the uniformities of ligand distribution across the membrane cartridges were investigated. Numerous affinity ligands were immobilized onto the membranes, and the prepared affinity membranes have been used to separate or purify concanavalin A, peroxidase, protease inhibitors, globulin, fibronectin, and other biomolecules.     

Electron crystallography of the scrapie prion protein complexed with heavy metals

The insolubility of the disease-causing isoform of the prion protein (PrP^Sc) has prevented studies of its three-dimensional structure at atomic resolution. Electron crystallography of two-dimensional crystals of N-terminally truncated PrP^Sc (PrP 27-30) and a miniprion (PrP^Sc106) provided the first insights at intermediate resolution on the molecular architecture of the prion. Here, we report on the structure of PrP 27-30 and PrP^Sc106 negatively stained with heavy metals. The interactions of the heavy metals with the crystal lattice were governed by tertiary and quaternary structural elements of the protein as well as the charge and size of the heavy metal salts. Staining with molybdate anions revealed three prominent densities near the center of the trimer that forms the unit cell, coinciding with the location of the β-helix that was proposed for the structure of PrP^Sc. Differential staining also confirmed the location of the internal deletion of PrP^Sc106 at or near these densities.     

Electron tomography of plant thylakoid membranes

For more than half a century, electron microscopy has been a main tool for investigating the complex ultrastructure and organization of chloroplast thylakoid membranes, but, even today, the three-dimensional relationship between stroma and grana thylakoids, and the arrangement of the membrane protein complexes within them are not fully understood. Electron cryo-tomography (cryo-ET) is a powerful new technique for visualizing cellular structures, especially membranes, in three dimensions. By this technique, large membrane protein complexes, such as the photosystem II supercomplex or the chloroplast ATP synthase, can be visualized directly in the thylakoid membrane at molecular (4-5 nm) resolution. This short review compares recent advances by cryo-ET of plant thylakoid membranes with earlier results obtained by conventional electron microscopy.     

Electron crystallography of human blood coagulation factor VIII bound to phospholipid monolayers

Coagulation factor VIII binds to negatively charged platelets prior to assembly with the serine protease, factor IXa, to form the factor X-activating enzyme (FX-ase) complex. The macromolecular organization of membrane-bound factor VIII has been studied by electron crystallography for the first time. For this purpose two-dimensional crystals of human factor VIII were grown onto phosphatidylserine-containing phospholipid monolayers, under near to physiological conditions (pH and salt concentration). Electron crystallographic analysis revealed that the factor VIII molecules were organized as monomers onto the lipid layer, with unit cell dimensions: a = 81.5A, b = 67.2 A, gamma = 66.5 degrees, P1 symmetry. Based on a homology-derived molecular model of the factor VIII (FVIII) A domains, the FVIII projection structure solved at 15-A resolution presents the A1, A2, and A3 domain heterotrimer tilted approximately 65 degrees relative to the membrane plane. The A1 domain is projecting on top of the A3, C1, and C2 domains and with the A2 domain protruding partially between A1 and A3. This organization of factor VIII allows the factor IXa protease and epidermal growth factor-like domain binding sites (localized in the A2 and A3 domains, respectively) to be situated at the appropriate position for the binding of factor IXa. The conformation of the lipid-bound FVIII is therefore very close to that for the activated factor VIIIa predicted in the FX-ase complex.