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Active sites and catalytic activity of heterogeneous catalysts is determined by their surface atomic structures. However, probing the surface structure at an atomic resolution is difficult, especially for solution ensembles of catalytic nanocrystals, which consist of heterogeneous particles with irregular shapes and surfaces. Here, we constructed 3D maps of the coordination number (CN) and generalized CN (CN_) for individual surface atoms of sub-3 nm Pt nanocrystals. Our results reveal that the synthesized Pt nanocrystals are enclosed by islands of atoms with nonuniform shapes that lead to complex surface structures, including a high ratio of low-coordination surface atoms, reduced domain size of low-index facets, and various types of exposed high-index facets. 3D maps of CN_ are directly correlated to catalytic activities assigned to individual surface atoms with distinct local coordination structures, which explains the origin of high catalytic performance of small Pt nanocrystals in important reactions such as oxygen reduction reactions and CO electro-oxidation.
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MOTIVATION: No rigorous statistical tests for detecting point-group symmetry in three-dimensional (3D) charge density maps obtained by electron microscopy (EM) and related techniques have been developed. RESULTS: We propose a method for determining the point-group symmetry of 3D charge density maps obtained by EM and related techniques. Our ab initio algorithm does not depend on atomic coordinates but utilizes the density map directly. We validate the approach for a range of publicly available single-particle cryo-EM datasets. In straightforward cases, our method enables fully automated single-particle 3D reconstruction without having to input an arbitrarily selected point-group symmetry. When pseudo-symmetry is present, our method provides statistics quantifying the degree to which the 3D density agrees with the different point-groups tested. AVAILABILITY AND IMPLEMENTATION: The software is freely available at https://github.com/hael/SIMPLE3.0.
Asunto(s)
Algoritmos , Programas Informáticos , Microscopía por Crioelectrón , Imagenología TridimensionalRESUMEN
Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.
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Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Algoritmos , Microscopía por CrioelectrónRESUMEN
Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a â¼70° opening of the bent and distorted central ß-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane ß-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of ß-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into ß-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted ß-barrel. The intermediate structures of the MACPF domain during refolding into the ß-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
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Membrana Celular/química , Complejo de Ataque a Membrana del Sistema Complemento/química , Proteínas Fúngicas/química , Proteínas Hemolisinas/química , Pleurotus/química , Proteínas Recombinantes de Fusión/química , Animales , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Eritrocitos/química , Eritrocitos/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , OvinosRESUMEN
The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and ß), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.
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Venenos de los Peces/química , Perforina/química , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Colesterol/química , Complejo de Ataque a Membrana del Sistema Complemento/química , Cristalografía por Rayos X , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Solubilidad , Homología Estructural de ProteínaRESUMEN
Pore Forming Toxins (PFTs) represent a key mechanism for permitting the passage of proteins and small molecules across the lipid membrane. These proteins are typically produced as soluble monomers that self-assemble into ring-like oligomeric structures on the membrane surface. Following such assembly PFTs undergo a remarkable conformational change to insert into the lipid membrane. While many different protein families have independently evolved such ability, members of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) superfamily form distinctive giant ß-barrel pores comprised of up to 50 monomers and up to 300Å in diameter. In this review we focus on recent advances in understanding the structure of these giant MACPF/CDC pores as well as the underlying molecular mechanisms leading to their formation. Commonalities and evolved variations of the pore forming mechanism across the superfamily are discussed. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
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Membrana Celular/metabolismo , Evolución Molecular , Perforina/clasificación , Perforina/metabolismo , Animales , Membrana Celular/química , Humanos , Perforina/química , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo â apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.
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Autoinmunidad , Glutamato Descarboxilasa , Homeostasis/inmunología , Simulación de Dinámica Molecular , Neurotransmisores , Ácido gamma-Aminobutírico , Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/química , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/inmunología , Humanos , Neurotransmisores/química , Neurotransmisores/genética , Neurotransmisores/inmunología , Multimerización de Proteína , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/genética , Ácido gamma-Aminobutírico/inmunologíaRESUMEN
Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200-300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three ß-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.
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Proteínas Bacterianas/química , Toxinas Bacterianas/química , Membrana Celular/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Membrana Celular/química , Colesterol , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.
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Enzimas/química , Fagos de Streptococcus/enzimología , Streptococcus equi/virología , Proteínas Virales/química , Cristalografía por Rayos X , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
In situ structures of Platinum (Pt) nanoparticles (NPs) can be determined with graphene liquid cell transmission electron microscopy. Atomic-scale three-dimensional structural information about their physiochemical properties in solution is critical for understanding their chemical function. We here analyze eight atomic-resolution maps of small (<3 nm) colloidal Pt NPs. Their structures are composed of an ordered crystalline core surrounded by surface atoms with comparatively high mobility. 3D reconstructions calculated from cumulative doses of 8500 and 17,000 electrons/pixel, respectively, are characterized in terms of loss of atomic densities and atomic displacements. Less than 5% of the total number of atoms are lost due to dissolution or knock-on damage in five of the structures analyzed, whereas 10-16% are lost in the remaining three. Less than 5% of the atomic positions are displaced due to the increased electron irradiation in all structures. The surface dynamics will play a critical role in the diverse catalytic function of Pt NPs and must be considered in efforts to model Pt NP function computationally.
RESUMEN
MOTIVATION: The ß-barrel is a ubiquitous fold that is deployed to accomplish a wide variety of biological functions including membrane-embedded pores. Key influences of ß-barrel lumen diameter include the number of ß-strands (n) and the degree of shear (S), the latter value measuring the extent to which the ß-sheet is tilted within the ß-barrel. Notably, it has previously been reported that the shear value for small antiparallel ß-barrels (n≤24) typically ranges between n and 2n. Conversely, it has been suggested that the ß-strands in giant antiparallel ß-barrels, such as those formed by pore forming cholesterol-dependent cytolysins (CDC), are parallel relative to the axis of the ß-barrel, i.e. S=0. The S=0 arrangement, however, has never been observed in crystal structures of small ß-barrels. Therefore, the structural basis for how CDCs form a ß-barrel and span a membrane remains to be understood. RESULTS: Through comparison of molecular models with experimental data, we are able to identify how giant CDC ß-barrels utilize a 'near parallel' arrangement of ß-strands where S=n/2. Furthermore, we show how side-chain packing within the ß-barrel lumen is an important limiting factor with respect to the possible shear values for small ß-barrels (n≤24 ß-strands). In contrast, our models reveal no such limitation restricts the shear value of giant ß-barrels (n>24 ß-strands). Giant ß-barrels can thus access a different architecture compared with smaller ß-barrels.
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Toxinas Bacterianas/química , Proteínas Citotóxicas Formadoras de Poros/química , Estructura Secundaria de Proteína , Proteínas Bacterianas/química , Modelos Moleculares , Estreptolisinas/químicaRESUMEN
The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2ß loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.
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Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Modelos Químicos , Modelos Moleculares , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Sitios de Unión , Simulación por Computador , Mapeo Epitopo , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Unión Proteica , Conformación ProteicaRESUMEN
Determining the 3D atomic structures of multi-element nanoparticles in their native liquid environment is crucial to understanding their physicochemical properties. Graphene liquid cell (GLC) TEM offers a platform to directly investigate nanoparticles in their solution phase. Moreover, exploiting high-resolution TEM images of single rotating nanoparticles in GLCs, 3D atomic structures of nanoparticles are reconstructed by a method called "Brownian one-particle reconstruction". We here introduce a 3D atomic structure determination method for multi-element nanoparticle systems. The method, which is based on low-pass filtration and initial 3D model generation customized for different types of multi-element systems, enables reconstruction of high-resolution 3D Coulomb density maps for ordered and disordered multi-element systems and classification of the heteroatom type. Using high-resolution image datasets obtained from TEM simulations of PbSe, CdSe, and FePt nanoparticles that are structurally relaxed with first-principles calculations in the graphene liquid cell, we show that the types and positions of the constituent atoms are precisely determined with root mean square displacement values less than 24 pm. Our study suggests that it is possible to investigate the 3D atomic structures of synthesized multi-element nanoparticles in liquid phase.
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Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a "one-particle Brownian 3D reconstruction method" based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, we introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo-electron microscopy. Our developments are made available through the open-source software package SINGLE.
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We here introduce the third major release of the SIMPLE (Single-particle IMage Processing Linux Engine) open-source software package for analysis of cryogenic transmission electron microscopy (cryo-EM) movies of single-particles (Single-Particle Analysis, SPA). Development of SIMPLE 3.0 has been focused on real-time data processing using minimal CPU computing resources to allow easy and cost-efficient scaling of processing as data rates escalate. Our stream SPA tool implements the steps of anisotropic motion correction and CTF estimation, rapid template-based particle identification and 2D clustering with automatic class rejection. SIMPLE 3.0 additionally features an easy-to-use web-based graphical user interface (GUI) that can be run on any device (workstation, laptop, tablet or phone) and supports a remote multi-user environment over the network. The new project-based execution model automatically records the executed workflow and represents it as a flow diagram in the GUI. This facilitates meta-data handling and greatly simplifies usage. Using SIMPLE 3.0, it is possible to automatically obtain a clean SP data set amenable to high-resolution 3D reconstruction directly upon completion of the data acquisition, without the need for extensive image processing post collection. Only minimal standard CPU computing resources are required to keep up with a rate of â¼300 Gatan K3 direct electron detector movies per hour. SIMPLE 3.0 is available for download from simplecryoem.com.
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Precise three-dimensional (3D) atomic structure determination of individual nanocrystals is a prerequisite for understanding and predicting their physical properties. Nanocrystals from the same synthesis batch display what are often presumed to be small but possibly important differences in size, lattice distortions, and defects, which can only be understood by structural characterization with high spatial 3D resolution. We solved the structures of individual colloidal platinum nanocrystals by developing atomic-resolution 3D liquid-cell electron microscopy to reveal critical intrinsic heterogeneity of ligand-protected platinum nanocrystals in solution, including structural degeneracies, lattice parameter deviations, internal defects, and strain. These differences in structure lead to substantial contributions to free energies, consequential enough that they must be considered in any discussion of fundamental nanocrystal properties or applications.
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Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. We developed the SIMPLE open-source image-processing suite for analysing cryo-EM images of single-particles. A core component of SIMPLE is the probabilistic PRIME algorithm for identifying clusters of images in 2D and determine relative orientations of single-particle projections in 3D. Here, we extend our previous work on PRIME and introduce new stochastic optimization algorithms that improve the robustness of the approach. Our refined method for identification of homogeneous subsets of images in accurate register substantially improves the resolution of the cluster centers and of the ab initio 3D reconstructions derived from them. We now obtain maps with a resolution better than 10 Å by exclusively processing cluster centers. Excellent parallel code performance on over-the-counter laptops and CPU workstations is demonstrated.
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Algoritmos , Microscopía por Crioelectrón , Imagenología Tridimensional , Programas InformáticosRESUMEN
Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.