Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

qFit 3: Protein and ligand multiconformer modeling for X‐ray crystallographic and single‐particle cryo‐EM density maps

New X‐ray crystallography and cryo‐electron microscopy (cryo‐EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open‐source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0 .     

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV), a member of the membrane‐containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo‐microscopy (cryo‐EM), we determined the structure of an attenuated vaccine strain, TC‐83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.     

Cryo‐EM map interpretation and protein model‐building using iterative map segmentation

A procedure for building protein chains into maps produced by single‐particle electron cryo‐microscopy (cryo‐EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections between these elements. Since the high density in a map typically follows the main‐chain of the protein, the main‐chain connection between secondary structure elements can often be identified as the unbranched path between them with the highest minimum value along the path. This chain‐tracing procedure is then combined with finding side‐chain positions based on the presence of density extending away from the main path of the chain, allowing generation of a C_α model. The C_α model is converted to an all‐atom model and is refined against the map. We show that this procedure is as effective as other existing methods for interpretation of cryo‐EM maps and that it is considerably faster and produces models with fewer chain breaks than our previous methods that were based on approaches developed for crystallographic maps.     

Investigating the structural dynamics of the PIEZO1 channel activation and inactivation by coarse‐grained modeling

The PIEZO channels, a family of mechanosensitive channels in vertebrates, feature a fast activation by mechanical stimuli (eg, membrane tension) followed by a slower inactivation. Although a medium‐resolution structure of the trimeric form of PIEZO1 was solved by cryo‐electron microscopy (cryo‐EM), key structural changes responsible for the channel activation and inactivation are still unknown. Toward decrypting the structural mechanism of the PIEZO1 activation and inactivation, we performed systematic coarse‐grained modeling using an elastic network model and related modeling/analysis tools (ie, normal mode analysis, flexibility and hotspot analysis, correlation analysis, and cryo‐EM‐based hybrid modeling and flexible fitting). We identified four key motional modes that may drive the tension‐induced activation and inactivation, with fast and slow relaxation time, respectively. These modes allosterically couple the lateral and vertical motions of the peripheral domains to the opening and closing of the intra‐cellular vestibule, enabling external mechanical forces to trigger, and regulate the activation/inactivation transitions. We also calculated domain‐specific flexibility profiles, and predicted hotspot residues at key domain‐domain interfaces and hinges. Our results offer unprecedented structural and dynamic information, which is consistent with the literature on mutational and functional studies of the PIEZO channels, and will guide future studies of this important family of mechanosensitive channels.     

Apo-Hsp90 coexists in two open conformational states in solution

Background information. Hsp90 (90 kDa heat‐shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide‐binding‐dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue). Results and discussion. Here, we present the first nucleotide‐free structures of the entire eukaryotic Hsp90 (apo‐Hsp90) obtained by small‐angle X‐ray scattering and single‐particle cryo‐EM (cryo‐electron microscopy). We show that, in solution, apo‐Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo‐EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo‐forms require large movements of the NTD (N‐terminal domain) and MD (middle domain) around two flexible hinge regions. Conclusions. The present study shows, for the first time, the structure of the entire eukaryotic apo‐Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.     

Lipid nanotechnologies for structural studies of membrane‐associated proteins

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane‐associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo‐electron microscopy (cryo‐EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ～20 nm inner diameter and a few microns in length, that self‐assemble in aqueous solutions. The lipid nanodisks (NDs) are self‐assembled discoid lipid bilayers of ～10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane‐associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane‐bound coagulation factor VIII in vitro for structure determination by cryo‐EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three‐dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane‐associated proteins and complexes for structural studies by cryo‐EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane‐associated proteins, such as the coagulation factors, at a close to physiological environment. Proteins 2014; 82:2902–2909. © 2014 Wiley Periodicals, Inc.     

Integrative structure and function of the yeast exocyst complex

Exocyst is an evolutionarily conserved hetero‐octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative‐stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride cross‐links from chemical‐crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo‐EM structure, cross‐links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo‐EM structure, except for Sec5. While not observed in the cryo‐EM map, the integrative model localizes the N‐terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low‐resolution structural data can inform biologically relevant hypotheses, even in the absence of high‐resolution data.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Structure of a eukaryotic cytoplasmic pre‐40S ribosomal subunit

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre‐RNA. Using cryo‐electron microscopy (cryo‐EM), we have determined the structure of a yeast cytoplasmic pre‐40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre‐rRNA adopts a highly distorted conformation of its 3′ major and 3′ minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre‐40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre‐40S particle toward the mature form capable of engaging in translation.     

Higher order structures of Adalimumab,Infliximab and their complexes with TNFα revealed by electron microscopy

Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface‐exposed sites for Adalimumab, Infliximab, and TNF receptor binding. Here, we report the structures of Adalimumab‐TNFα and Infliximab‐TNFα complexes modeled from negative stain EM and cryo‐EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab‐TNFα and Infliximab‐TNFα. The 2:2 complex structures of Adalimumab‐TNFα and Infliximab‐TNFα show diamond‐shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab‐TNFα or Infliximab‐TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo‐EM analysis of 3:2 Adalimumab‐TNFα complex generated a low‐resolution density consistent with a TNFα trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNFα trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNFα.     

Covid‐19.bioreproducibility.org: A web resource for SARS‐CoV‐2‐related structural models

The COVID‐19 pandemic has triggered numerous scientific activities aimed at understanding the SARS‐CoV‐2 virus and ultimately developing treatments. Structural biologists have already determined hundreds of experimental X‐ray, cryo‐EM, and NMR structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. To help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid19.bioreproducibility.org, that aggregates expert‐verified information about SARS‐CoV‐2‐related macromolecular models. In this article, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein.     

Structural basis for HIV‐1 DNA integration in the human genome,role of the LEDGF/P75 cofactor

Integration of the human immunodeficiency virus (HIV‐1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild‐type full‐length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA‐binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral‐induced pathologies.     

Structural basis for assembly of the CBF3 kinetochore complex

Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP‐A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP‐A^Cse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single‐particle cryo‐EM reconstruction of the core CBF3 complex, incorporating the sequence‐specific DNA‐binding protein Cep3 together with regulatory subunits Ctf13 and Skp1. This provides the first structural data on Ctf13, defining it as an F‐box protein of the leucine‐rich‐repeat family, and demonstrates how a novel F‐box‐mediated interaction between Ctf13 and Skp1 is responsible for initial assembly of the CBF3 complex.     

Cryo‐EM model validation using independent map reconstructions

An increasing number of cryo‐electron microscopy (cryo‐EM) density maps are being generated with suitable resolution to trace the protein backbone and guide sidechain placement. Generating and evaluating atomic models based on such maps would be greatly facilitated by independent validation metrics for assessing the fit of the models to the data. We describe such a metric based on the fit of atomic models with independent test maps from single particle reconstructions not used in model refinement. The metric provides a means to determine the proper balance between the fit to the density and model energy and stereochemistry during refinement, and is likely to be useful in determining values of model building and refinement metaparameters quite generally.     

Probing the structural dynamics of the SNARE recycling machine based on coarse‐grained modeling

Membrane fusion in eukaryotes is driven by the formation of a four‐helix bundle by three SNARE proteins. To recycle the SNARE proteins, they must be disassembled by the ATPase NSF and four SNAP proteins which together form a 20S supercomplex. Recently, the first high‐resolution structures of the NSF (in both ATP and ADP state) and 20S (in four distinct states termed I, II, IIIa, and IIIb) were solved by cryo‐electron microscopy (cryo‐EM), which have paved the way for structure‐driven studies of the SNARE recycling mechanism. To probe the structural dynamics of SNARE disassembly at amino‐acid level of details, a systematic coarse‐grained modeling based on an elastic network model and related analyses were performed. Our normal mode analysis of NSF, SNARE, and 20S predicted key modes of collective motions that partially account for the observed structural changes, and illuminated how the SNARE complex can be effectively destabilized by untwisting and bending motions of the SNARE complex driven by the amino‐terminal domains of NSF in state II. Our flexibility analysis identified regions with high/low flexibility that coincide with key functional sites (such as the NSF‐SNAPs‐SNARE binding sites). A subset of hotspot residues that control the above collective motions, which will make promising targets for future mutagenesis studies were also identified. Finally, the conformational changes in 20S as induced by the transition of NSF from ATP to ADP state were modeled, and a concerted untwisting motion of SNARE/SNAPs and a sideway flip of two amino‐terminal domains were observed. In sum, the findings have offered new structural and dynamic details relevant to the SNARE disassembly mechanism, and will guide future functional studies of the SNARE recycling machinery. Proteins 2016; 84:1055–1066. © 2016 Wiley Periodicals, Inc.