We are investigating bacterial and eukaryotic ribosomes and their functional complexes to obtain insights into the process of protein synthesis. Building on our studies aimed at revealing the structures of eukaryotic cytosolic and mitochondrial ribosomes, we are now investigating eukaryotic translation initiation, targeting of proteins to membranes, regulation of protein synthesis, and how viruses reprogram host translation. Previously, we studied how Hepatitis C virus genomic RNA can bind mammalian ribosomes to achieve translation of viral mRNAs in the absence of some canonical cellular translation initiation factors. With our recent research activities we contributed to the understanding of how SARS-CoV-2, the virus that is responsible for the COVID-19 pandemic, shuts off host translation to prevent cellular defence mechanisms against the virus (Schubert et al. 2020). Furthermore, using a combination of cryo-electron microsocpy and biochemical assays we also investigated the mechanism of programmed ribosomal frameshifting, one of the key events during translation of the SARS-CoV-2 RNA genome that leads to synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins (Bhat et al. 2021).

Schubert K, Karousis ED, Jomaa A, Scaiola A, Echeverria B, Gurzeler LA, Leibundgut M, Thiel V, Mühlemann O, Ban N. (2020) SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nat Struct Mol Biol. (10):959-966

Bhatt PR, Scaiola A, Loughran G, Leibundgut M, Kratzel A, McMillan A, O’ Connor KM, Bode JW, Thiel V, Atkins JF and Ban N, 2021, Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome, Science, doi: 10.1126/science.abf3546.

Single Particle Analysis (SPA) application of cryo-electron microscopy (cryo-EM) has become one of the dominating methods for 3D structure determination of a wide variety of biological macromolecules to understand their function, mechanism of action^[1] and protein ligand/drug interactions. However, as the popularity of this technique increases, so does the need for accessibility and improved efficiency. In this abstract, we describe two cryo-Transmission Electron Microscopes (cryo-TEMs), that are equivalent to home source X-ray diffractometers, but for cryo-EM.

The first is the Thermo Scientific Tundra cryo-TEM operating at 100kV with a semi-automated grid loading system and automated data collection for SPA. Tundra allows users to load the sample in an effortless and robust way. Using this new microscope, we solved structures of several soluble and membrane protein samples. Standard sample such as apoferritin protein (equivalent to lysozyme crystals for X-ray crystallography) was solved to 2.6 Å resolution. More challenging samples such as homo-pentameric human GABA_A (gamma-aminobutyric acid type A) receptor was resolved to 3.4 Å reconstruction. The GABA_A receptor is a small membrane protein and ligand-gated chloride-ion channel that mediates inhibitory neurotransmission. GABA_A receptors are important therapeutic drug targets and hence it is vital to understand the molecular mechanism by which these receptors mediate neurotransmission. After decades of efforts, in 2014, this same sample of GABA_A receptor was crystallized and structure resolved to 3.0 Å^[2]. With cryo-EM on Tundra, we obtained similar resolution without the need of crystallization and in near native conditions.

To further push for more automation and high-throughput, we used the Thermo Scientific Glacios^TM cryo-TEM. Glacios has an Autoloader^TM, with a robotic arm which can load 12 grids simultaneously and switch the grids automatically. To push for higher resolution, Glacios is also equipped with direct electron detector (DED) and can be combined with Selectris energy filter. Using this system, we achieved a 2.4 Å resolution cryo-EM map for the same GABA_A receptor. Both these microscopes are not only good for sample screening and optimization but are also capable for generating high resolution structures comparable to those obtained from X-ray crystallography experiments.

Flaviviruses pose a complex threat to human health including a few global pathogens and numerous viruses with an epidemic potential. In the context of the co-circulation of closely-related viruses, non-neutralising immune responses may aggravate subsequent heterologous infections. Sub-optimal responses to vaccination entails a similar risk. To address these challenges, a detailed structural understanding of flavivirus infectious particles is essential to characterise quaternary epitopes responsible for broadly protective responses or, on the contrary, deleterious immune responses. Immature-like features and conformational “breathing” in circulating virions have been linked to the latter prompting for a better understanding of structural transitions underpinning viral maturation.

Taking advantage of an insect-specific flavivirus (ISF), we have determined high-resolution structures of immature and mature particles revealing key features in the maturation process. First, we produced chimeric viruses between the ISF and medically-relevant flaviviruses. We show that the outer shell of the chimeric viruses is native, which allowed cryo-EM structure determination at high-resolution for West Nile virus, Murray Valley Encephalitis virus and dengue virus. The structure of the dengue virus chimera at a resolution of 2.5Å reveals lipid-like ligands with a structural role likely to be conserved across all pathogenic flaviviruses. The structure of the immature ISF particle at a resolution of 3.9Å shows how the stem region of the E protein, where these ligands bind, is remodelled during maturation. Unexpectedly, the immature spike adopts a topology where prM forms a central pillar rather than the peripheral drawstring proposed earlier (Fig. 1A). This topology implies a revised organisation of the immature virion, which supports a collapse model for viral maturation (Fig. 1B). In this model, folding down of prM onto the membrane guides the collapse of the trimeric spikes.

Together, these structures provide new avenues to target the stem regions of E and prM for the development of improved vaccines and new therapeutics. More generally, we propose that the chimeric platform could be a largely applicable tool to investigate flavivirus biology.

Most of the rhinoviruses, which are the leading cause of common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. Before genome release, rhinoviruses convert to activated particles that contain pores in the capsid, lack capsid proteins VP4, and have altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here we present the structures of the native rhinovirus 14 and rhinovirus14-ICAM-1 complex at a resolution of 2.6 and 2.4 Å. The structures revealed a mechanism by which binding of rhinovirus 14 to ICAM-1 primes the virus for activation and subsequent genome release. The attachment of rhinovirus 14 to ICAM-1 induces conformational changes in the virion, which include translocation of the C-termini of VP4 subunits towards twofold symmetry axes of the capsid. Thus, VP4 subunits become poised for release through pores that open in the capsid upon particle activation. The cryo-EM reconstruction of rhinovirus 14 virion contains the resolved density of octa-nucleotides from the RNA genome, which interact with VP2 subunits near two-fold symmetry axes of the capsid. VP4 subunits with altered conformation, induced by the binding of rhinovirus 14 to ICAM-1, block the RNA-VP2 interactions and expose patches of positively charged residues around threefold symmetry axes of the capsid. The conformational changes of the capsid induce reorganization of the virus genome. The rearrangements of the capsid and genome probably lower the energy barrier of conversion of rhinovirus 14 virions to activated particles. The structure of rhinovirus 14 in complex with ICAM-1 represents an essential intermediate in the pathway of enterovirus genome release.

Enolase, a conserved glycolytic enzyme, that catalyzes the conversion of 2-phosphoglycerate (2PG) to phosphoenol pyruvate, important for energy production is an essential enzyme for mycobacterial growth. However, additionally enolase is a known moonlighting protein with additional functions in the cytoplasm as well as on the cell surface. It plays an important role in Mtb virulence by acting as cell surface receptor of human plasminogen. To derive a mechanistic insight into the function of this enzyme, we have deciphered the atomic level details of Mtb enolase structure in native as well as 2PG/PEP bound forms by both XRD and CryoEM microscopy. Notably, through X-ray structure superimposition of the enolase/2PG bound structures shows two binding confirmations of the 2PG in the active site. The cryoEM structure reveals the octameric conformation of Mtb enolase. P-P docking and simulation studies of enolase and plasminogen helps us to understand the molecular interaction of the complex.