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As one of the most stunning biological nanostructures, the single-diamond (SD) surface discovered in beetles and weevils exoskeletons possesses the widest complete photonic bandgap known to date and is renowned as the "holy grail" of photonic materials. However, the synthesis of SD is difficult due to its thermodynamical instability compared to the energetically favoured bicontinuous double diamond and other easily formed lattices; thus, the artificial fabrication of SD has long been a formidable challenge. Herein, we report a bottom-up approach to fabricate SD titania networks via a one-pot cooperative assembly scenario employing the diblock copolymer poly(ethylene oxide)-block-polystyrene as a soft template and titanium diisopropoxide bis(acetylacetonate) as an inorganic precursor in a mixed solvent, in which the SD scaffold was obtained by kinetically controlled nucleation and growth in the skeletal channels of the diamond minimal surface formed by the polymer matrix. Electron crystallography investigations revealed the formation of tetrahedrally connected SD frameworks with the space group Fd [Formula: see text] m in a polycrystalline anatase form. A photonic bandgap calculation showed that the resulting SD structure has a wide and complete bandgap. This work solves the complex synthetic enigmas and offers a frontier in hyperbolic surfaces, biorelevant materials, next-generation optical devices, etc.
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Surface layers (S-layers) are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by cryo-electron microscopy, finding the crystalline regularity of the S-layer extended into the layers below (outer membrane, periplasm, and inner membrane). The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity yet allowing a certain degree of free space. The presented results suggest how S-layers, at least in some species, are mesoscale assemblies behaving as structural and functional scaffolds essential for the entire cell envelope.
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Deinococcus , Deinococcus/metabolismo , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Membrana Celular/metabolismoRESUMO
Mixed-anion compounds widen the chemical space of attainable materials compared to single anionic compounds, but the exploration of their structural diversity is limited by common synthetic paths. Especially, oxychlorides rely mainly on layered structures, which suffer from low stability during photo(electro)catalytic processes. Herein we report a strategy to design a new polar 3D tetrahedral framework with composition Zn4 Si2 O7 Cl2 . We use a molten salt medium to enable low temperature crystallization of nanowires of this new compound, by relying on tetrahedral building units present in the melt to build the connectivity of the oxychloride. These units are combined with silicon-based connectors from a non-oxidic Zintl phase to enable precise tuning of the oxygen content. This structure brings high chemical and thermal stability, as well as strongly anisotropic hole mobility along the polar axis. These features, associated with the ability to adjust the transport properties by doping, enable to tune water splitting properties for photoelectrocatalytic H2 evolution and water oxidation. This work then paves the way to a new family of mixed-anion solids.
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Nanoestruturas , Cloreto de Sódio/química , Ânions/química , Nanoestruturas/química , Zinco/química , Dióxido de Silício/química , Cloretos/química , Catálise , Eletroquímica/métodosRESUMO
Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing, and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure-function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduce new data archival challenges in terms of data size and complexity in the years to come.
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Microscopia Crioeletrônica/métodos , Bases de Dados de Proteínas , Proteínas/química , Cristalografia por Raios X , Conformação Proteica , Proteínas/ultraestruturaRESUMO
We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.
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Cristalografia por Raios X/métodos , Microtecnologia/métodos , Muramidase/ultraestrutura , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/instrumentação , Elétrons , Íons , Microtecnologia/instrumentação , Muramidase/química , SíncrotronsRESUMO
The precise structural solution of crystals on a mesostructural scale is challenging due to the difficulties in obtaining electron diffraction and the complicated relationship between the crystal structure factors (CSFs) and the conventional underfocus phase-contrast transmission electron microscopy (TEM) images due to the large unit cell and the complex structures. Here, we present the structural investigation of mesostructured crystals via the combination of electron crystallographic Fourier synthesis and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) that only relies on the mass-thickness contrast. The three-dimensional electrostatic potential is reconstructed from the amplitudes and phases extracted from the Fourier transforms of the corresponding HAADF-STEM images and merged into a set of CSFs. This method is verified on silica scaffolds following a shifted double-diamond surface network with space group I41/amd. The results indicate that electron crystallography reconstruction by HAADF-STEM images is more suitable and accurate in determining the structure in comparison with conventional TEM electron crystallography reconstruction. This approach transfers the contrast of mesostructured crystals to images more accurately and the relationship between the Fourier transforms of HAADF-STEM images and the CSFs is more intuitive. It shows great advantages for the structural solution of crystals on the mesostructural scale.
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In this study, we successfully solve polymorphs A and B of zeolite EMM-17, which can only crystallize in sub-micrometer-sized crystals while containing complex stacking disorders, from the three-dimensional (3D) electron diffraction (ED) data. This is the first time that the atomic structure of this polymorph has been ab initio solved, and the result reveals a unique 10(12)×10(12)×11-ring channel system. Moreover, we acquire the first atomic-resolution images of EMM-17 using integrated differential phase-contrast scanning transmission electron microscopy. The images allow us to directly observe polymorphs B and C and discover a large number of local structural defects. Based on structural features unraveled from the reciprocal-space 3D ED data and real-space images, we propose a series of energetically feasible local structures in EMM-17. We also demonstrate that the unique porous structure of EMM-17 enables efficient kinetic separation of C6 alkane isomers.
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Microcrystal electron diffraction (MicroED) has recently shown to be a promising technique for structure determination in structural biology and pharmaceutical chemistry. Here, we discuss the unique properties of electrons and motivate its use for diffraction experiments. We review the latest developments in MicroED, and illustrate its applications in macromolecular crystallography, fragment screening and structure guided drug discovery. We discuss the perspectives of MicroED in synthetic chemistry and pharmaceutical development. We anticipate that the rapid advances MicroED showcased here will promote further development of electron crystallography and open up new opportunities for drug discovery.
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Elétrons , Preparações Farmacêuticas , Cristalografia por Raios XRESUMO
A scanning precession electron diffraction system has been integrated with a direct electron detector to allow the collection of improved quality diffraction patterns. This has been used on a two-phase αß titanium alloy (Timetal® 575) for phase and orientation mapping using an existing pattern-matching algorithm and has been compared to the commonly used detector system, which consisted of a high-speed video-camera imaging the small phosphor focusing screen. Noise is appreciably lower with the direct electron detector, and this is especially noticeable further from the diffraction pattern center where the real electron scattering is reduced and both diffraction spots and inelastic scattering between spots are weaker. The results for orientation mapping are a significant improvement in phase and orientation indexing reliability, especially of fine nanoscale laths of α-Ti, where the weak diffracted signal is rather lost in the noise for the optically coupled camera. This was done at a dose of ~19 e−/Å2, and there is clearly a prospect for reducing the current further while still producing indexable patterns. This opens the way for precession diffraction phase and orientation mapping of radiation-sensitive crystalline materials.
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2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10-12â¯Å can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending, refinement over sub-tiles of the images in order to locally refine the sample tilt geometry, implementation of different CTF correction schemes, and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to better than 5â¯Å.
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Cristalografia por Raios X/métodos , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Algoritmos , Microscopia Crioeletrônica/métodos , SoftwareRESUMO
Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.
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Synthesis of new zeolites involving organic molecules relies heavily on the trial-and-error approach, because it is difficult to interpret the determining effects of organics on zeolite crystal symmetry. Here, the intrinsic relationships among the space-filling of organics, included volume of channels, and zeolite crystal symmetry, are systematically demonstrated by experimental and computational means. Under controlled conditions, the "dimer" and "monomer" organics of 1-ethyl-3-methylimidazolium selectively direct different, but related, germanosilicates, the ECNU-16 with a new topology and the existing IM-16 with the UOS topology, respectively. The comprehensive computational study reveals that the zeolite phase selectivity is determined by the unique space-filling behavior of the "dimer" and "monomer" organics, which is closely correlated to their rotation freedom, as well as the included volume of host zeolite channels. The elucidation of this crucial space-filling effect from the fundamental viewpoint will provide new guidelines for the rational design and synthesis of new zeolites in future.
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Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.
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ATPases Transportadoras de Cálcio/química , Catalase/química , Cristalografia por Raios X/métodos , Elétrons , Modelos Moleculares , Animais , Bovinos , Eletricidade EstáticaRESUMO
Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X-ray structures. We determined two molecular structures by employing electron rather than X-ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9â Å resolution from a crystal smaller than 1×2â µm2 . Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X-ray crystallography and is the technique of choice for all unsolved cases in which submicrometer-sized crystals were the limiting factor.
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The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) is a structurally and functionally complex system that mediates sugar uptake in bacteria. Besides several soluble subunits, the glucose-specific PTS includes the integral membrane protein IICB that couples the transmembrane transport of glucose to its phosphorylation. Here, we used electron crystallography of sugar-embedded tubular crystals of the glucose-specific IIC transport domain from Escherichia coli (ecIICglc) to visualize the structure of the transporter in the presence and absence of its substrate. Using an in vivo transport assay and binding competition experiments, we first established that, while it transports d-glucose, ecIICglc does not bind l-glucose. We then determined the projection structure of ecIICglc from tubular crystals embedded in d- and l-glucose and found a subtle conformational change. From comparison of the ecIICglc projection maps with crystal structures of other IIC transporters, we can deduce that the transporter adopts an inward-facing conformation, and that the maps in the presence and absence of the substrate reflect the transporter before and after release of the transported glucose into the cytoplasm. The transition associated with substrate release appears to require a subtle structural rearrangement in the region that includes hairpin 1.
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Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Facilitadoras de Transporte de Glucose/química , Proteínas de Membrana Transportadoras/química , Cristalografia , Elétrons , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 ß-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.
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Proteínas de Membrana/química , Cristalização , Detergentes/química , Concentração de Íons de Hidrogênio , Lipídeos/químicaRESUMO
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.
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ATPase Trocadora de Hidrogênio-Potássio/química , Ativação do Canal Iônico , Sus scrofa , Animais , Antiulcerosos/química , Domínio Catalítico , Microscopia Crioeletrônica , ATPase Trocadora de Hidrogênio-Potássio/fisiologia , Imidazóis/química , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Inibidores da Bomba de Prótons/químicaRESUMO
BACKGROUND: The mechanisms underlying water transport through aquaporin (AQP) have been debated for two decades. The water permeation phenomenon of AQP seems inexplicable because the Grotthuss mechanism does not allow for simultaneous fast water permeability and inhibition of proton transfer through the hydrogen bonds of water molecules. SCOPE OF REVIEW: The AQP1 structure determined by electron crystallography provided the first insights into the proton exclusion mechanism despite fast water permeation. Although several studies have provided clues about the mechanism based on the AQP structure, each proposed mechanism remains incomplete. The present review is focused on AQP function and structure solved by electron crystallography in an attempt to fill the gaps between the findings in the absence and presence of lipids. MAJOR CONCLUSIONS: Many AQP structures can be superimposed regardless of the determination method. The AQP fold is preserved even under conditions lacking lipids, but the water arrangement in the channel pore differs. The differences might be explained by dipole moments formed by the two short helices in the lipid bilayer. In addition, structure analyses of double-layered two-dimensional crystals of AQP suggest an array formation and cell adhesive function. GENERAL SIGNIFICANCE: Electron crystallography findings not only have contributed to resolve some of the water permeation mechanisms, but have also elucidated the multiple functions of AQPs in the membrane. The roles of AQPs in the brain remain obscure, but their multiple activities might be important in the regulation of brain and other biological functions. This article is part of a Special Issue entitled Aquaporins.
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Aquaporinas/química , Cristalografia/métodos , Elétrons , Modelos Moleculares , Conformação Proteica , Água/químicaRESUMO
Electron crystallography of two-dimensional (2D) crystals determines the structure of membrane proteins in the lipid bilayer by imaging with cryo-electron microscopy and image processing. Membrane proteins can be packed in regular 2D arrays by their reconstitution in the presence of lipids at low lipid to protein weight-to-weight ratio. The crystal quality depends on the protein purity and homogeneity, its stability, and on the crystallization conditions. A 2D crystal presents the membrane protein in a functional and fully lipidated state. Electron crystallography determines the 3D structure even of small membrane proteins up to atomic resolution, but 3D density maps have a better resolution in the membrane plane than in the vertical direction. This problem can be partly eliminated by applying an iterative algorithm that exploits additional known constraints about the 2D crystal. 2D electron crystallography is particularly attractive for the structural analysis of membrane proteins that are too small for single particle analyses and too unstable to form 3D crystals. With the recent introduction of direct electron detector cameras, the routine determination of the atomic 3D structure of membrane-embedded membrane proteins is in reach.
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Lipoilação , Lipídeos de Membrana/química , Proteínas de Membrana/ultraestrutura , Cristalografia/métodosRESUMO
Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The "gold standard Fourier shell correlation" resolution of our final reconstruction reaches 13 Å, which is significantly better than the previously obtained 17 Å resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.