Upgrade of the workflow for cryo-electron tomography and microscopy
The CIISB Cryo-electron microscopy core facility at CEITEC Masaryk University is expanding its services in the sample preparation for electron microscopy. The facility has recently acquired high-pressure freezer Leica EM ICE for vitrification of bulky biological specimen (up to 200 mm thickness). In addition, the freeze-substitution unit Leica EM AFS2 for resin embedding of the high-pressure frozen samples and the ultramicrotom Leica EM UC7 with the adapter for cryo-ultramicrotomy were purchased in order to provide the facility users with the complete workflow for preparation of thin section samples for both room-temperature electron microscopy and cryo-electron microscopy.
Emerging Topics in Biomolecular Magnetic Resonance – Nick Cox & Kendra Frederick
The series Emerging topics in Biomolecular Magnetic Resonance will continue on July 9th at 16:00 CEST with the following lecturers and topics:
Nick Cox (Australian National University): Spin state evolution during the biological water splitting reaction
Kendra Frederick (UT Southwestern): In cell structural biology enabled by DNP MAS NMR
Emerging Topics in Biomolecular Magnetic Resonance – 4th edition
The series Emerging topics in Biomolecular Magnetic Resonance will continue on June 25th at 16:00 CEST with the following lecturers and topics:
Melinda J. Duer (University of Cambridge): Understanding extracellular matrix disease states with solid-state NMR?
Claudio Luchinat (University of Florence): NMR for metabolomics: again the ‘second best’ technique?
Emerging Topics in Biomolecular Magnetic Resonance – 3rd edition
The series Emerging topics in Biomolecular Magnetic Resonance will continue on June 18th at 16:00 CEST with the following lecturers and topics:
Tuo Wang (Louisiana State University): Elucidation of carbohydrate structure in plant biomass and fungal pathogens using solid-state NMR and DNP methods
Rina Rosenzweig (Weizmann Institute of Science): Molecular Chaperones in Protein Disaggregation - What we can learn from NMR
Emerging Topics in Biomolecular Magnetic Resonance – On-line lecture series
The series will continue on Thursday, June 11, 2020 at 16:00 CEST with two cutting-edge 30 minutes presentations:
Putative Amyloids in Human Health— Insights from NMR (Ann E. McDermott, Columbia University)
An RNA dynamic ensemble at atomic resolution (Hashim Al-Hashimi, Duke University)
New timsTOF Pro Mass Spectrometer installed at CEITEC.
New timsTOF Pro Mass Spectrometer (Bruker) has been installed at the Proteomics Core Facility, CEITEC MU. The new instrument brings another separation dimension (according collisional cross sections) in qualitative and quantitative characterization of complex protein samples as it is equipped by trapped ion mobility spectrometry (TIMS) module.
Highlights of Coronavirus Structural Studies
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.
Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase (Cell)
Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. This study published in Cell examines the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, authors investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.
SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects
SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. Antoni Wrobel, Donald Benton, Steven Gamblin et al. determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; they compared these with recently reported structures for uncleaved SARS-CoV-2 S. They also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.
Reader's Corner Archive
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.
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.
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.
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.
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.
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.
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.
Integrative Structural Biology of Protein-RNA Complexes
Ribonucleoprotein complexes (RNPs) are central to all processes in the cell. One of the prerequisites to understand how RNPs work is to determine their high-resolution structures. With the recent revolution in cryo-electron microscopy this task has become easier for large RNP machines, such as ribosomes, spliceosomes, and polymerases. However, the transient and highly dynamic nature of many RNPs makes structure determination a challenging task. Thus, an integrative structural and molecular biology approach is required, tackling three key challenges: (1) identification of cognate RNA sequences; (2) collection of structural data by conducting X-ray crystallography, NMR, electron microscopy, small-angle scattering (SAS), and other experiments; and (3) the creation of structural models that integrates all experimental restraints. Given the breadth of expertise required, Janosch Henning et. al. in Structure present an overview of available methods and successful examples with the goal to provide readers with a selection of promising options for structure determination of RNPs.