18 Aug 2020

Structure of replicating SARS-CoV-2 polymerase (Nature)

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here P. Cremer et. al.  present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in active form, mimicking the replicating enzyme. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged ‘sliding poles’. These sliding poles can account for the known processivity of the RdRp that is required for replicating the long coronavirus genome3. Their results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19).

11 Aug 2020

Site-specific glycan analysis of the SARS-CoV-2 spike (Science)

The emergence of the betacoronavirus, SARS-CoV-2, the causative agent of COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, M. Crispin et. al.  reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan- processing states across the trimeric viral spike. They show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

11 Aug 2020

Controlling the SARS-CoV-2 spike glycoprotein conformation (Nat. Struct. Mol. Biol.)

The coronavirus (CoV) spike (S) protein, involved in viral–host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, R. Henderson, P. Acharya et. al. implemented a structure-based vector analysis of available β-CoV S-protein structures. Despite an overall similarity in domain organization, they found that S-proteins from different β-CoVs display distinct configurations. Based on this analysis, they developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs ‘down’ position or adopts ‘up’ state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.

14 May 2020

Fragment-based drug discovery using cryo-EM

Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Harren Jhoti et. al.  in Drug Discovery Today review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. They demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. In addition, they exemplify this using the test system β-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).

14 May 2020

Integrating cryo-EM and NMR data

Single-particle cryo-electron microscopy (cryo-EM) is increasingly used as a technique to determine the atomic structure of challenging biological systems. Recent advances in microscope engineering, electron detection, and image processing have allowed the structural determination of bigger and more flexible targets than possible with the complementary techniques X-ray crystallography and NMR spectroscopy. However, there exist many biological targets for which atomic resolution cannot be currently achieved with cryo-EM, making unambiguous determination of the protein structure impossible. Although determining the structure of large biological systems using solely NMR is often difficult, highly complementary experimental atomic-level data for each molecule can be derived from the spectra, and used in combination with cryo-EM data. Gunnar F. Schröder et.al. review in Current Opinion in Structural Biology strategies with which both techniques can be synergistically combined, in order to reach detail and understanding unattainable by each technique acting alone; and the types of biological systems for which such an approach would be desirable.

5 May 2020

Integrated multidisciplinarity in the natural sciences

The integration of multiple perspectives in both the arts and natural sciences is tremendously powerful and arguably necessary for capturing relevant features of complex phenomena. Individual methods and models comprise abstractions from and idealizations of nature, and only the integration of multiple models, methods, and representations provides a means to reach more accurate results than relying on any single approach. In the Mildred Cohn Award Lecture at the 2019 ASBMB meeting, and the subsequent Journal of Biological Chemistry award article, A. Gronenborn illustrates the power of such multidisciplinary work by highlighting the successful integration of data and multiple views afforded by NMR spectroscopy, cryo-electron micros- copy, cryo-electron tomography, X-ray crystallography, computation, and functional assays made possible through collaborative efforts by members of the Pittsburgh Center for HIV Protein Interactions. This approach permitted her to generate the first all-atom model of a native HIV-1 capsid core.

5 May 2020

Cryo-electron microscopy for the study of virus assembly

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In the Nature Chemical Biology review, D. Luque and J. R. Castón discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.

5 May 2020

Structural biology of multicomponent assemblies in DNA double-strand-break repair through non-homologous end joining

The mechanisms mediating the repair of DNA damage in human cells have been the focus of a multitude of studies since the middle of the previous century, and many of the proteins implicated in these processes have been identified as being part of large macromolecular assemblies. This review by A. Chaplin and T. L. Blundell, published in Current Opinion in Structural Biology, gives an overview of the current knowledge of protein structures specifically involved in the repair of DNA double strand breaks through Non-Homologous End Joining, with a focus on recent structures obtained via cryo-electron microscopy and prospects for how this rapidly evolving method will impact our understanding of DNA repair.

26 Mar 2020

Methyl TROSY spectroscopy: A versatile NMR approach to study challenging biological systems

A major goal in structural biology is to unravel how molecular machines function in detail. To that end, solution-state NMR spectroscopy is ideally suited as it is able to study biological assemblies in a near natural environment. Based on methyl TROSY methods, it is now possible to record high-quality data on complexes that are far over 100 kDa in molecular weight. In this review, Marco Sprangers and Stefan Schütz discuss the theoretical background of methyl TROSY spectroscopy, the information that can be extracted from methyl TROSY spectra and approaches that can be used to assign methyl resonances in large complexes. In addition, we touch upon insights that have been obtained for a number of challenging biological systems, including the 20S proteasome, the RNA exosome, molecular chaperones and G-protein-coupled receptors. We anticipate that methyl TROSY methods will be increasingly important in modern structural biology approaches, where information regarding static structures is complemented with insights into conformational changes and dynamic intermolecular interactions.

26 Mar 2020

The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membrane-less compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membrane-less organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution. Solution-state NMR spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. This review discusses practical aspects of studying LLPS by NMR, summarizes recent work on the molecular aspects of LLPS of various protein systems, and discusses future opportunities for characterizing the molecular details of LLPS to modulate phase separation.

26 Mar 2020

Cryo-electron microscopy for the study of virus assembly

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In this Review, recent viral assembly analyses by cryo-EM and cryo-ET are discussed, showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.