Electron Crystallography Of Two-dimensional Crystals Research Articles

Studying membrane proteins with MicroED.

The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.

Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age.

Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the feasibility of atomic resolution structure determination using cryo-EM. Atomic resolution single-particle analysis, without the need for crystals, now promises to resolve problems in structural biology that were intractable just a few years ago.

Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.

Systematic Comparison of Molecular Conformations of H+,K+-ATPase Reveals an Important Contribution of the A-M2 Linker for the Luminal Gating

Gastric H(+),K(+)-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K(+)-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H(+),K(+)-ATPase is consistent with that observed in sarcoplasmic reticulum Ca(2+)-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.

A pipeline for comprehensive and automated processing of electron diffraction data in IPLT

Electron crystallography of two-dimensional crystals allows the structural study of membrane proteins in their native environment, the lipid bilayer. Determining the structure of a membrane protein at near-atomic resolution by electron crystallography remains, however, a very labor-intense and time-consuming task. To simplify and accelerate the data processing aspect of electron crystallography, we implemented a pipeline for the processing of electron diffraction data using the Image Processing Library and Toolbox (IPLT), which provides a modular, flexible, integrated, and extendable cross-platform, open-source framework for image processing. The diffraction data processing pipeline is organized as several independent modules implemented in Python. The modules can be accessed either from a graphical user interface or through a command line interface, thus meeting the needs of both novice and expert users. The low-level image processing algorithms are implemented in C++ to achieve optimal processing performance, and their interface is exported to Python using a wrapper. For enhanced performance, the Python processing modules are complemented with a central data managing facility that provides a caching infrastructure. The validity of our data processing algorithms was verified by processing a set of aquaporin-0 diffraction patterns with the IPLT pipeline and comparing the resulting merged data set with that obtained by processing the same diffraction patterns with the classical set of MRC programs.

Automatic recovery of missing amplitudes and phases in tilt-limited electron crystallography of two-dimensional crystals

Electron crystallography of 2D protein crystals provides a powerful tool for the determination of membrane protein structure. In this method, data is acquired in the Fourier domain as randomly sampled, uncoupled, amplitudes and phases. Due to physical constraints on specimen tilting, those Fourier data show a vast un-sampled "missing cone" of information, producing resolution loss in the direction perpendicular to the membrane plane. Based on the flexible language of projection onto sets, we provide a full solution for these problems with a projective constraint optimization algorithm that, for sufficiently oversampled data, produces complete recovery of unmeasured data in the missing cone. We apply this method to an experimental data set of Bacteriorhodopsin and show that, in addition to producing superior results compared to traditional reconstruction methods, full, reproducible, recovery of the missing cone from noisy data is possible. Finally, we present an automatic implementation of the refinement routine as open source, freely distributed, software that will be included in our 2dx software package.

Conformational rearrangement of gastric H+,K+-ATPase induced by an acid suppressant

Acid-related gastric diseases are associated with disorder of digestive tract acidification. The gastric proton pump, H+,K+-ATPase, exports H+ in exchange for luminal K+ to generate a highly acidic environment in the stomach, and is a main target for acid suppressants. Here, we report the three-dimensional structure of gastric H+,K+-ATPase with bound SCH28080, a representative K+-competitive acid blocker, at 7 Å resolution based on electron crystallography of two-dimensional crystals. The density of the bound SCH28080 is found near transmembrane (TM) helices 4, 5 and 6, in the luminal cavity. The SCH28080-binding site is formed by the rearrangement of TM helices, which is in turn transmitted to the cytoplasmic domains, resulting in a luminal-open conformation. These results represent the first structural evidence for a binding site of an acid suppressant on H+,K+-ATPase, and the conformational change induced by this class of drugs.

A Novel Ratchet Mechanism of Gastric H<sup>+</sup>, K<sup>+</sup>-ATPase Revealed by Electron Crystallography of Two-dimensional Crystals

Acid secretion by the stomach results in a pH of about 1. This highly acidic environment is essential for digestion and also acts as a first barrier against bacterial and viral infections. Conversely, too much acid secretion causes gastric ulcer. The mechanism by which this massive proton gradient is generated is of considerable biomedical interest. In this review, we introduce the first molecular model for this remarkable biological phenomenon. The structure of H(+),K(+)-ATPase at 6.5 A resolution was determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the N-terminal tail of the beta-subunit functions as a "ratchet", preventing inefficient transport and reverse-flow of protons. We can thus provide a mechanistic explanation for how the H(+),K(+)-ATPase can generate a million-fold proton gradient across the gastric parietal cell membrane, the highest cation gradient known in any mammalian tissue.

Electron Crystallography as a Technique to Study the Structure on Membrane Proteins in a Lipidic Environment

The native environment of integral membrane proteins is a lipid bilayer. The structure of a membrane protein is thus ideally studied in a lipidic environment. In the first part of this review we describe some membrane protein structures that revealed the surrounding lipids and provide a brief overview of the techniques that can be used to study membrane proteins in a lipidic environment. In the second part of this review we focus on electron crystallography of two-dimensional crystals as potentially the most suitable technique for such studies. We describe the individual steps involved in the electron crystallographic determination of a membrane protein structure and discuss current challenges that need to be overcome to transform electron crystallography into a technique that can be routinely used to analyze the structure of membrane proteins embedded in a lipid bilayer.

Inter-subunit interaction of gastric H+,K+-ATPase prevents reverse reaction of the transport cycle

The gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for generating a million-fold proton gradient across the gastric membrane. We present the structure of gastric H(+),K(+)-ATPase at 6.5 A resolution as determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the beta-subunit N-terminus prevents the reverse reaction from E(2)P to E(1)P, which is likely to be relevant for the generation of a large H(+) gradient in vivo situation.

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 microscopy in structural studies of Photosystem II.

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment-protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II.

Three-dimensional fold of the human AQP1 water channel determined at 4 Å resolution by electron crystallography of two-dimensional crystals embedded in ice

Here, we present a three-dimensional (3D) density map of deglycosylated, human erythrocyte aquaporin 1 (AQP1) determined at 4 Å resolution in plane and ∼7 Å resolution perpendicular to the bilayer. The map was calculated by analyzing images and electron diffraction patterns recorded from tilted (up to 60 °), ice-embedded, frozen-hydrated 2D crystals of AQP1 in lipid bilayer membranes. This map significantly extends the findings related to the folding of the AQP1 polypeptide chain determined by us at a lower, 7 Å by ∼20 Å, resolution. The solvent-accessible volume within a monomer has a vestibular architecture, with a narrow, ∼6.5 Å diameter constriction near the center of the bilayer, where the location of the water-selective channel is postulated to exist. The clearly resolved densities for the transmembrane helices display the protrusions expected for bulky side-chains. The density in the interior of the helix barrel (putative NPA box region) is better resolved compared to our previous map, suggesting clearer linkage to some of the helices, and it may harbor short stretches of α-helix. At the bilayer extremities, densities for some of the inter-helix hydrophilic loops are visible. Consistent with these observed inter-helix connections, possible models for the threading of the AQP1 polypeptide chain are presented. A preferred model is deduced that agrees with the putative locations of a group of aromatic residues in the amino acid sequence and in the 3D density map.

Three-dimensional map of the plasma membrane H+-ATPase in the open conformation.

The H+-ATPase from the plasma membrane of Neurospora crassa is an integral membrane protein of relative molecular mass 100K, which belongs to the P-type ATPase family that includes the plasma membrane Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase. The H+-ATPase pumps protons across the cell's plasma membrane using ATP as an energy source, generating a membrane potential in excess of 200mV. Despite the importance of P-type ATPases in controlling membrane potential and intracellular ion concentrations, little is known about the molecular mechanism they use for ion transport. This is largely due to the difficulty in growing well ordered crystals and the resulting lack of detail in the three-dimensional structure of these large membrane proteins. We have now obtained a three-dimensional map of the H+-ATPase by electron crystallography of two-dimensional crystals grown directly on electron microscope grids. At an in-plane resolution of 8 A, this map reveals ten membrane-spanning alpha-helices in the membrane domain, and four major cytoplasmic domains in the open conformation of the enzyme without bound ligands.

Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins

Electron microscopy has become a powerful technique, along with X-ray crystallography and nuclear magnetic resonance spectroscopy, to study the three-dimensional structure of biological molecules. It has evolved into a number of methods dealing with a wide range of biological samples, with electron crystallography of two-dimensional crystals being so far the only method allowing data collection at near-atomic resolution. In this paper, we review the methodology of electron crystallography and its application to membrane proteins, starting with the pioneering work on bacteriorhodopsin, which led to the first visualization of the secondary structure of a membrane protein in 1975. Since then, improvements in instrumentation, sample preparation, and data analysis have led to atomic models for bacteriorhodopsin and light-harvesting complex II from higher plants. The structures of many more membrane proteins have been studied by electron crystallography and in this review examples are included where a resolution of better than 10 Å has been achieved. Indeed, in some of the given examples an atomic model can be expected in the near future. Finally, a brief outlook is given on current and future developments of electron crystallographic methods.

Atomic model of plant light-harvesting complex by electron crystallography.

The structure of the light-harvesting chlorophyll a/b-protein complex, an integral membrane protein, has been determined at 3.4 A resolution by electron crystallography of two-dimensional crystals. Two of the three membrane-spanning alpha-helices are held together by ion pairs formed by charged residues that also serve as chlorophyll ligands. In the centre of the complex, chlorophyll a is in close contact with chlorophyll b for rapid energy transfer, and with two carotenoids that prevent the formation of toxic singlet oxygen.