CMS BIOCEV Newsletter
Heads of BIOCEV CIISB core facilities have summarised recent technological upgrades of their laboratories and presented them as CMS newsletter. The overview of recent updates is available here.
Research scientist for the Centre of Molecular Structure - Biophysical methods facility
Biophysical Methods facility of BIOCEV is looking for a research scientist specialised in biophysical techniques and/or spectroscopic techniques.
Instruct-ERIC is hiring a Director to join the Instruct Hub
The Integrated Structural Biology - European Research Infrastructure Consortium (Instruct-ERIC), a pan-European distributed infrastructure whose principal task is to support excellent science that integrates an understanding of biological structure with cellular function, seeks to recruit a director.
Two on-line Magnetic Resonance Courses
Ilja Kuprov and Marcel Utz from University of Southampton made entire Undergraduate Magnetic Resonance module available online: 20 hours of video and 100+ pages of handouts.
PhD Studentship Applications - Professor J. Lewandowski
University of Warwick together with GSK company offers a PhD studentship focused on "Solid/solution-state NMR spectroscopy and cryo-electron microscopy methodology for the characterisation of aggregation mechanisms in proteins".
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.
Highlights of Coronavirus Structural Studies
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.
Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2
Two nanobodies that bind SARS-CoV-2 spike RBD are shown to block interaction with receptor ACE2 and thus neutralize the virus, and have an additive effect with antibody CR3022. The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, Raymond Owens, James Naismith et al. have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K(D)of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Stabilizing the Closed SARS-CoV-2 Spike Trimer
The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, P.M. Langedijk et al. report the structure-based design of soluble S trimers, with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. They identify two regions in the S-protein critical for the protein’s stability: the heptad repeat region 1 of the S2 subunit and subunit domain 1 at the interface with S2. We combined a minimal selection of mostly inter-protomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.
Reader's Corner Archive
Solid-state NMR spectroscopy for characterization of RNA and RNP complexes
Ribonucleic acids are driving a multitude of biological processes where they act alone or in complex with proteins (ribonucleoproteins, RNP). To understand these processes both structural and mechanistic information about RNA is necessary. Due to their conformational plasticity RNA pose a challenge for mainstream structural biology methods. Solid- state NMR (ss-NMR) spectroscopy is an emerging technique that can be applied to biomolecular complexes of any size in close-to-native conditions. This review outlines recent methodological developments in ss-NMR for structural characterization of RNA and protein–RNA complexes and provides relevant examples.
Mass spectrometry-based methods for structural biology on a proteome-wide scale
Mass spectrometry (MS) has long been used to study proteins mainly via sequence identification and quantitation of expression abundance. In recent years, MS has emerged as a tool for structural biology. Intact protein structural analysis has been enabled by the development of methods such as native MS, top-down proteomics, and ion mobility MS. Other MS-based structural methods include affinity purification MS, chemical cross-linking, and protein footprinting. These methods have enabled the study of protein–protein and protein–ligand interactions and regions of conformational change. The coupling of MS with liquid chromatography has permitted the analysis of complex samples. This bottom-up proteomics workflow enables the study of protein structure in the native cellular environment and provides structural information across the proteome. It has been demonstrated that the crowded environment of the cell affects protein binding interactions and affinities. Performing studies in this complex environment is essential for understanding the functional roles of proteins. MS-based structural methods permit analysis of samples such as cell lysates, intact cells, and tissue to provide a more physiological view of protein structure. This mini-review discusses the various MS-based methods that can be used for proteome-wide structural studies and highlights some of their application.
The data universe of structural biology
The Protein Data Bank (PDB) has grown from a small data resource for crystallographers to a worldwide resource serving structural biology. The history of the growth of the PDB and the role that the community has played in developing standards and policies are described. This article also illustrates how other biophysics communities are collaborating with the worldwide PDB to create a network of interoperating data resources. This network will expand the capabilities of structural biology and enable the determination and archiving of increasingly complex structures.
Structural basis of the activation of a metabotropic GABA receptor
Metabotropic gamma-aminobutyric acid receptors (GABA(B)) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis. GABA(B)belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. In the Nature paper Gati and Cherezov et.al. present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA(B). Their results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.
Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol
The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, published in Science, Wang, Li, Besra and Rao et.al. determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, their work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.
Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system
Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR–Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase–nuclease Cas, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. In the recent Nature publication Sternberg and Fernandez et al. describe structures of a TniQ–Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3′ end of the CRISPR RNA (crRNA). The natural Cas8–Cas5 fusion protein binds the 5′ crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ–Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.
First Experiments in Structural Biology at the European X-ray Free-Electron Laser
Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.
In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer
Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharidevan. Kugelen et. al. report an electron cryo-microscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, they deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, they present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryo-tomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.