A critical step in the analysis of novel cryogenic electron microscopy (cryo-EM) single-particle datasets is the identification of homogeneous subsets of images. Methods for solving this problem are ...important for data quality assessment, ab initio 3D reconstruction, and analysis of population diversity due to the heterogeneous nature of macromolecules. Here we formulate a stochastic algorithm for identification of homogeneous subsets of images. The purpose of the method is to generate improved 2D class averages that can be used to produce a reliable 3D starting model in a rapid and unbiased fashion. We show that our method overcomes inherent limitations of widely used clustering approaches and proceed to test the approach on six publicly available experimental cryo-EM datasets. We conclude that, in each instance, ab initio 3D reconstructions of quality suitable for initialization of high-resolution refinement are produced from the cluster centers.
•Generation of improved 2D class averages from large single-particle cryo-EM datasets•Production of a reliable 3D starting model in a rapid and unbiased fashion•Overcoming inherent limitations in widely used clustering approaches•The method is many times faster than other widely used approaches
Reboul et al. describe a new algorithm for simultaneous 2D alignment and clustering of cryo-EM images that generates improved 2D class averages that can be used to produce a reliable 3D starting model in a rapid and unbiased fashion.
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.
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.
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 ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-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 β-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 β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-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.
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 β-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.
•The MACPF/CDC family of pore forming toxins (PFTs) form transmembrane pores distinct in size and structure from other PFTs.•MACPF/CDC PFTs achieve membrane insertion via a common pore forming mechanism.•Members have also evolved variations of the general mechanism including method of binding to the membrane surface.
The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In ...contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.
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 β-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.
Abstract
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.
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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