Postdoc positions at CEITEC – Cryo-ET and Cryo-EM of Viral Transcription-Translation Coupling
Two postdoc positions are available in newly established Gabriel Demo lab (CEITEC, Masaryk University, Brno, Czech Republic) to participate in research focused on structural mechanisms of transcription-translation coupling in bacteria and potentially in virus-infected mammalian cells.
New CIISB newsletter has been published
Newsletter summarising new developments at CIISB in the period February - July 2020 and planned events is now available.
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.
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".
Highlights of Coronavirus Structural Studies
Human neutralizing antibodies elicited by SARS-CoV-2 infection (Nature)
The emerging coronavirus SARS-CoV-2 pandemic presents a global health emergency in urgent need of interventions. SARS-CoV-2 entry into the target cells depends on binding between the receptor-binding domain (RBD) of the viral Spike protein and the ACE2 cell receptor. Here, Wang, Z. Zhang, L. Zhang et. al. report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells of eight SARS-CoV-2 infected individuals. They identified antibodies with potent anti-SARS-CoV-2 neutralization activity that correlates with their competitive capacity with ACE2 for RBD binding. Surprisingly, neither the anti-SARS-CoV-2 antibodies nor the infected plasma cross-reacted with SARS-CoV or MERS-CoV RBDs, although substantial plasma cross-reactivity to their trimeric Spike proteins was found. Crystal structure analysis of RBD-bound antibody revealed steric hindrance that inhibits viral engagement with ACE2 and thereby blocks viral entry. These findings suggest that anti-RBD antibodies are viral species-specific inhibitors. The antibodies identified here may be candidates for the development of SARS-CoV-2 clinical interventions.
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).
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.
Reader's Corner Archive
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.
Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS
Technical developments are unifying molecular and cellular biology. A recent electron cryo-tomography study by von Kugelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.
Mapping Structural Dynamics of Proteins with Femtosecond Stimulated Raman Spectroscopy
The structure–function relationships of biomolecules have captured the interest and imagination of the scientific community and general public since the field of structural biology emerged to enable the molecular understanding of life processes. Proteins that play numerous functional roles in cellular processes have remained in the forefront of research, inspiring new characterization techniques. In this review in Annual Review of Physical Chemistry, Chong Fang and Longteng Tang present key theoretical concepts and recent experimental strategies using femtosecond stimulated Raman spectroscopy (FSRS) to map the structural dynamics of proteins, highlighting the flexible chromophores on ultrafast timescales. In particular, wavelength-tunable FSRS exploits dynamic resonance conditions to track transient-species-dependent vibrational motions, enabling rational design to alter functions. Various ways of capturing excited-state chromophore structural snapshots in the time and/or frequency domains are discussed. Continuous development of experimental methodologies, synergistic correlation with theoretical modeling, and the expansion to other nonequilibrium, photo-switchable, and controllable protein systems will greatly advance the chemical, physical, and biological sciences.
The regulation and functions of DNA and RNA G-quadruplexes
DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In the paper published in Nature Reviews Molecular Cell Biology, Balasubramanian, S. et al. first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. They then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, they consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, they discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.
The architecture of the Gram-positive bacterial cell wall
The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor. In Gram-positive bacteria, peptidoglycan is tens of nanometers thick, generally portrayed as a homogeneous structure that provides mechanical strength. S. J. Foster & J. K. Hobbs et.al. applied atomic force microscopy to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The paper published in Nature shows that the mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface, providing information complementary to traditional structural biology approaches.
Emerging solution NMR methods to illuminate the structural and dynamic properties of proteins
The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-resolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Hari Arthanari, Gerhard Wager et. al. in the Current Opinion in Structural Biology review summarize recent technical landmarks in solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic resolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.
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).