Virus structures revealed by advanced cryoelectron microscopy methods.

Before the resolution revolution, cryoelectron microscopy (cryo-EM) single-particle analysis (SPA) already achieved resolutions beyond 4 Å for certain icosahedral viruses, enabling ab initio atomic model building of these viruses. As the only samples that achieved such high resolution at that time, cryo-EM method development was closely intertwined with the improvement of reconstructions of symmetrical viruses. Viral morphology exhibits significant diversity, ranging from small to large, uniform to non-uniform, and from containing single symmetry to multiple symmetries. Furthermore, viruses undergo conformational changes during their life cycle. Several methods, such as asymmetric reconstruction, Ewald sphere correction, cryoelectron tomography (cryo-ET), and sub-tomogram averaging (STA), have been developed and applied to determine virus structures in vivo and in vitro. This review outlines current advanced cryo-EM methods for high-resolution structure determination of viruses and summarizes accomplishments obtained with these approaches. Moreover, persisting challenges in comprehending virus structures are discussed and we propose potential solutions.

Structure of Semliki Forest virus in complex with its receptor VLDLR.

Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV.

Supramolecular assembly of chloroplast NADH dehydrogenase-like complex with photosystem I from Arabidopsis thaliana.

Cyclic electron transport/flow (CET/CEF) in chloroplasts is a regulatory process essential for the optimization of plant photosynthetic efficiency. A crucial CEF pathway is catalyzed by a membrane-embedded NADH dehydrogenase-like (NDH) complex that contains at least 29 protein subunits and associates with photosystem I (PSI) to form the NDH-PSI supercomplex. Here, we report the 3.9 Å resolution structure of the Arabidopsis thaliana NDH-PSI (AtNDH-PSI) supercomplex. We constructed structural models for 26 AtNDH subunits, among which 11 are unique to chloroplasts and stabilize the core part of the NDH complex. In the supercomplex, one NDH can bind up to two PSI-light-harvesting complex I (PSI-LHCI) complexes at both sides of its membrane arm. Two minor LHCIs, Lhca5 and Lhca6, each present in one PSI-LHCI, interact with NDH and contribute to supercomplex formation and stabilization. Collectively, our study reveals the structural details of the AtNDH-PSI supercomplex assembly and provides a molecular basis for further investigation of the regulatory mechanism of CEF in plants.

Structural basis for nucleosome-mediated inhibition of cGAS activity.

Activation of cyclic GMP-AMP synthase (cGAS) through sensing cytosolic double stranded DNA (dsDNA) plays a pivotal role in innate immunity against exogenous infection as well as cellular regulation under stress. Aberrant activation of cGAS induced by self-DNA is related to autoimmune diseases. cGAS accumulates at chromosomes during mitosis or spontaneously in the nucleus. Binding of cGAS to the nucleosome competitively attenuates the dsDNA-mediated cGAS activation, but the molecular mechanism of the attenuation is still poorly understood. Here, we report two cryo-electron microscopy structures of cGAS-nucleosome complexes. The structures reveal that cGAS interacts with the nucleosome as a monomer, forming 1:1 and 2:2 complexes, respectively. cGAS contacts the nucleosomal acidic patch formed by the H2A-H2B heterodimer through the dsDNA-binding site B in both complexes, and could interact with the DNA from the other symmetrically placed nucleosome via the dsDNA-binding site C in the 2:2 complex. The bound nucleosome inhibits the activation of cGAS through blocking the interaction of cGAS with ligand dsDNA and disrupting cGAS dimerization. R236A or R255A mutation of cGAS impairs the binding between cGAS and the nucleosome, and largely relieves the nucleosome-mediated inhibition of cGAS activity. Our study provides structural insights into the inhibition of cGAS activity by the nucleosome, and advances the understanding of the mechanism by which hosts avoid the autoimmune attack caused by cGAS.

Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein.

Global emergencies caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 significantly endanger human health. The spike (S) glycoprotein is the key antigen and its conserved S2 subunit contributes to viral entry by mediating host-viral membrane fusion. However, structural information of the post-fusion S2 from these highly pathogenic human-infecting coronaviruses is still lacking. We used single-particle cryo-electron microscopy to show that the post-fusion SARS-CoV S2 forms a further rotated HR1-HR2 six-helix bundle and a tightly bound linker region upstream of the HR2 motif. The structures of pre- and post-fusion SARS-CoV S glycoprotein dramatically differ, resembling that of the Mouse hepatitis virus (MHV) and other class I viral fusion proteins. This structure suggests potential targets for the development of vaccines and therapies against a wide range of SARS-like coronaviruses.

Structural insights into assembly, operation and inhibition of a type I restriction-modification system.

Type I restriction-modification (R-M) systems are widespread in prokaryotic genomes and provide robust protection against foreign DNA. They are multisubunit enzymes with methyltransferase, endonuclease and translocase activities. Despite extensive studies over the past five decades, little is known about the molecular mechanisms of these sophisticated machines. Here, we report the cryo-electron microscopy structures of the representative EcoR124I R-M system in different assemblies (R2M2S1, R1M2S1 and M2S1) bound to target DNA and the phage and mobile genetic element-encoded anti-restriction proteins Ocr and ArdA. EcoR124I can precisely regulate different enzymatic activities by adopting distinct conformations. The marked conformational transitions of EcoR124I are dependent on the intrinsic flexibility at both the individual-subunit and assembled-complex levels. Moreover, Ocr and ArdA use a DNA-mimicry strategy to inhibit multiple activities, but do not block the conformational transitions of the complexes. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into assembly, operation and inhibition mechanisms of type I R-M systems.

Structural basis for energy and electron transfer of the photosystem I-IsiA-flavodoxin supercomplex.

Under iron-deficiency stress, which occurs frequently in natural aquatic environments, cyanobacteria reduce the amount of iron-enriched proteins, including photosystem I (PSI) and ferredoxin (Fd), and upregulate the expression of iron-stress-induced proteins A and B (IsiA and flavodoxin (Fld)). Multiple IsiAs function as the peripheral antennae that encircle the PSI core, whereas Fld replaces Fd as the electron receptor of PSI. Here, we report the structures of the PSI3-IsiA18-Fld3 and PSI3-IsiA18 supercomplexes from Synechococcus sp. PCC 7942, revealing features that are different from the previously reported PSI structures, and a sophisticated pigment network that involves previously unobserved pigment molecules. Spectroscopic results demonstrated that IsiAs are efficient light harvesters for PSI. Three Flds bind symmetrically to the trimeric PSI core-we reveal the detailed interaction and the electron transport path between PSI and Fld. Our results provide a structural basis for understanding the mechanisms of light harvesting, energy transfer and electron transport of cyanobacterial PSI under stressed conditions.

Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase.

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains.

The envelope spike (S) proteins of MERS-CoV and SARS-CoV determine the virus host tropism and entry into host cells, and constitute a promising target for the development of prophylactics and therapeutics. Here, we present high-resolution structures of the trimeric MERS-CoV and SARS-CoV S proteins in its pre-fusion conformation by single particle cryo-electron microscopy. The overall structures resemble that from other coronaviruses including HKU1, MHV and NL63 reported recently, with the exception of the receptor binding domain (RBD). We captured two states of the RBD with receptor binding region either buried (lying state) or exposed (standing state), demonstrating an inherently flexible RBD readily recognized by the receptor. Further sequence conservation analysis of six human-infecting coronaviruses revealed that the fusion peptide, HR1 region and the central helix are potential targets for eliciting broadly neutralizing antibodies.

Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol.

Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.