Cryo-EM in molecular and cellular biology.

This review summarizes the current state of methods and results achievable by cryo-electron microscopy (cryo-EM) imaging for molecular, cell, and structural biologists who wish to understand what is required and how it might help to address their research questions. It covers some of the main issues in sample preparation, microscopes and data collection, image processing, three-dimensional (3D) reconstruction, and validation and interpretation of the resulting EM density maps and atomic models.

Structural basis of ubiquitin-independent PP1 complex disassembly by p97.

The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.

ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin.

The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.

Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase.

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.

A Liquid to Solid Phase Transition Underlying Pathological Huntingtin Exon1 Aggregation.

Huntington's disease is caused by an abnormally long polyglutamine tract in the huntingtin protein. This leads to the generation and deposition of N-terminal exon1 fragments of the protein in intracellular aggregates. We combined electron tomography and quantitative fluorescence microscopy to analyze the structural and material properties of huntingtin exon1 assemblies in mammalian cells, in yeast, and in vitro. We found that huntingtin exon1 proteins can form reversible liquid-like assemblies, a process driven by huntingtin's polyQ tract and proline-rich region. In cells and in vitro, the liquid-like assemblies converted to solid-like assemblies with a fibrillar structure. Intracellular phase transitions of polyglutamine proteins could play a role in initiating irreversible pathological aggregation.

Structural basis of substrate progression through the bacterial chaperonin cycle.

The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP·BeF3, and GroEL-ADP·AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, suggesting how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.

A structural basis for prion strain diversity.

Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular ß-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations.

Chaperone machines for protein folding, unfolding and disaggregation.

Molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. Their increased expression in response to stress is a key factor in the health of the cell and longevity of an organism. Unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecular machines that operate on a wide range of substrates. The structural basis of their mechanism of action is being unravelled (in particular for the heat shock proteins HSP60, HSP70, HSP90 and HSP100) and typically involves massive displacements of 20-30 kDa domains over distances of 20-50 Å and rotations of up to 100°.

Human Hsp70 Disaggregase Reverses Parkinson's-Linked α-Synuclein Amyloid Fibrils.

Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.

The PDB and protein homeostasis: From chaperones to degradation and disaggregase machines.

This review contains a personal account of the role played by the PDB in the development of the field of molecular chaperones and protein homeostasis, from the viewpoint of someone who experienced the concurrent advances in the structural biology, electron microscopy, and chaperone fields. The emphasis is on some key structures, including those of Hsp70, GroEL, Hsp90, and small heat shock proteins, that were determined as the molecular chaperone concept and systems for protein quality control were emerging. These structures were pivotal in demonstrating how seemingly nonspecific chaperones could assist the specific folding pathways of a variety of substrates. Moreover, they have provided mechanistic insights into the ATPase machinery of complexes such as GroEL/GroES that promote unfolding and folding and the disaggregases that extract polypeptides from large aggregates and disassemble amyloid fibers. The PDB has provided a framework for the current success in curating, evaluating, and distributing structural biology data, through both the PDB and the EMDB.

Spiers Memorial Lecture: Challenges in cryo electron microscopy.

This article provides an introductory background and overview of the discussion meeting. It begins with an account of a few key milestones in the development of the cryo EM field, followed by an overview of the presentations that will form the basis of the discussion.

Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress.

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.

The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins.

The membrane attack complex and perforin proteins (MACPFs) and bacterial cholesterol-dependent cytolysins (CDCs) are two branches of a large and diverse superfamily of pore-forming proteins that function in immunity and pathogenesis. During pore formation, soluble monomers assemble into large transmembrane pores through conformational transitions that involve extrusion and refolding of two α-helical regions into transmembrane ß-hairpins. These transitions entail a dramatic refolding of the protein structure, and the resulting assemblies create large holes in cellular membranes, but they do not use any external source of energy. Structures of the membrane-bound assemblies are required to mechanistically understand and modulate these processes. In this Commentary, we discuss recent advances in the understanding of assembly mechanisms and molecular details of the conformational changes that occur during MACPF and CDC pore formation.

A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum-infected erythrocytes.

Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.

The molecular basis for perforin oligomerization and transmembrane pore assembly.

Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.

Conformational changes during pore formation by the perforin-related protein pleurotolysin.

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 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.

Blob-ology and biology of cryo-EM: an interview with Helen Saibil.

Helen Saibil is Bernal Professor of Structural Biology at Birkbeck, University of London. After undergraduate work at McGill University, Canada, Helen moved to London for her PhD at Kings College. After stints at CEA Grenoble and the University of Oxford, she moved to Birkbeck where her lab studies the operation of macromolecular machinery-including molecular chaperones, protein folding/misfolding, and host cell interactions with pathogens. Helen is a Fellow of the Royal Society, Fellow of the Academy of Medical Sciences, and an Honorary Member of both the British Biophysical Society and the Royal Microscopical Society. She talked to us about the background, recent developments, and future prospects in cryo-electron microscopy.