Adenosine triphosphate (ATP) is the molecular currency of intracellular energy transfer in living organisms. The enzyme ATP synthase is primarily responsible for ATP production in eukaryotes. In archaea and some bacteria, ATP is synthesized by V-ATPase that is related to ATP synthase both in structure and function. Both of these enzymes are reversible rotary motors capable of catalyzing ATP synthesis or hydrolysis. The rotation of the central rotor, which is powered by the flow of proton (or sometimes sodium ion) down the electrochemical gradient through the membrane-bound Fo/Vo region, leads to the chemical synthesis of ATP in F1/V1 region. The F1/V1 region, on the other hand, can catalyze ATP hydrolysis, which in turn leads to proton (or sodium) pumping across the membrane through rotation of the central rotor in the opposite direction. This thesis describes structure determination of both the intact F-type and V-type enzymes using single particle electron cryomicroscopy (cryo-EM), with the aim of better understanding their overall architecture, subunit organization and the mechanism of proton translocation. Our cryo-EM structural analysis on the F-type ATP synthase from Saccharomyces cerevisiae uncovered the arrangement of subunits a, b, c, and the two dimer-specific subunits e and g within the membrane-bound region of Fo. A model of oligomerization of the ATP synthase involving two distinct dimerization interfaces was proposed.The rotor-stator interaction within the membrane-bound region of both enzymes is responsible for proton translocation. Our cryo-EM structures of the V-ATPase from Thermus thermophilus reveal that the interaction between the rotary ring (rotor) and the I-subunit (stator) is surprisingly small, with only two subunits from the ring making contact with the I-subunit near the middle of the membrane. Furthermore, the spatial arrangement of transmembrane helices resolved in subunit I can form two passageways that could provide proton access through the membrane-bound region and is consistent with a two-channel model of proton translocation.

Adenosine triphosphate (ATP) is the molecular currency of intracellular energy transfer in living organisms. The enzyme ATP synthase is primarily responsible for ATP production in eukaryotes. In archaea and some bacteria, ATP is synthesized by V-ATPase that is related to ATP synthase both in structure and function. Both of these enzymes are reversible rotary motors capable of catalyzing ATP synthesis or hydrolysis. The rotation of the central rotor, which is powered by the flow of proton (or sometimes sodium ion) down the electrochemical gradient through the membrane-bound Fo/Vo region, leads to the chemical synthesis of ATP in F1/V1 region. The F1/V1 region, on the other hand, can catalyze ATP hydrolysis, which in turn leads to proton (or sodium) pumping across the membrane through rotation of the central rotor in the opposite direction. This thesis describes structure determination of both the intact F-type and V-type enzymes using single particle electron cryomicroscopy (cryo-EM), with the aim of better understanding their overall architecture, subunit organization and the mechanism of proton translocation. Our cryo-EM structural analysis on the F-type ATP synthase from Saccharomyces cerevisiae uncovered the arrangement of subunits a, b, c, and the two dimer-specific subunits e and g within the membrane-bound region of Fo. A model of oligomerization of the ATP synthase involving two distinct dimerization interfaces was proposed.The rotor-stator interaction within the membrane-bound region of both enzymes is responsible for proton translocation. Our cryo-EM structures of the V-ATPase from Thermus thermophilus reveal that the interaction between the rotary ring (rotor) and the I-subunit (stator) is surprisingly small, with only two subunits from the ring making contact with the I-subunit near the middle of the membrane. Furthermore, the spatial arrangement of transmembrane helices resolved in subunit I can form two passageways that could provide proton access through the membrane-bound region and is consistent with a two-channel model of proton translocation.

Translation is the process by which proteins are synthesized from the instructions in the genetic code. Translation is mediated by the ribosome, a large ribonucleoprotein complex, in concert with messenger RNA (mRNA), transfer RNA (tRNA), and a variety of proteins. The canonical mechanism of translation, introduced in Part I of my thesis, is divided into four distinct phases: initiation, elongation, termination, and recycling. Under unusual circumstances, each phase of translation can also proceed via a number of noncanonical mechanisms, many of which are vitally important for cellular growth or viral infectivity. My thesis describes structural insights into two such noncanonical mechanisms. The aim of the first project, described in Part II, was to structurally characterize a noncanonical mechanism of translational termination in bacteria. In the absence of a stop codon, ribosomes arrest at the 3′ end of an mRNA and are unable to terminate. In bacteria, the primary mechanism for rescuing such nonstop complexes is known as trans-translation. In the absence of a functional trans-translation system, however, the small protein ArfA recognizes the empty mRNA channel and recruits the release factor RF2 to the ribosome, enabling termination to occur. Using single-particle electron cryomicroscopy (cryo-EM), I obtained four high-resolution structures of nonstop complexes that reveal the mechanism of ArfA-mediated ribosome rescue and have wider implications for understanding canonical termination in bacteria. The aim of the second project, described in Part III, was to gain structural insights into a noncanonical mechanism of translational initiation in eukaryotes known as internal ribosome entry. Instead of a 5′ cap, many viruses contain intricately structured, cis-acting internal-ribosome-entry sites (IRESs) within their genomes that direct end-independent initiation. The IRES of hepatitis-C virus (HCV), for example, interacts directly with the mammalian ribosome and functionally replaces many of the canonical initiation factors. However, the mechanism by which the HCV IRES coordinates assembly of an initiation complex and progresses through the initiation phase remains poorly understood. I developed a method for purifying native ribosomal complexes from cell lysate that enabled me to obtain multiple cryo-EM maps of the HCV IRES in complex with the 80S ribosome, including a previously unseen conformation of the IRES induced by rotation of the ribosomal small subunit, and to make progress towards capturing earlier steps in the initiation pathway.

Single particle electron cryomicroscopy (cryo-EM) is a method of structure determination that uses many randomly oriented images of the specimen to construct a three-dimensional density map. In this thesis, single particle cryo-EM has been used to determine the structure of intact adenosine triphosphate (ATP) synthase from bovine heart mitochondria, an approximately 550 kDa membrane protein complex. In respiring organisms, ATP synthase is responsible for synthesizing the majority of ATP, a molecule that serves as an energy source for many cellular reactions. In order to understand the mechanism of ATP synthase, knowledge of the arrangement of subunits in the intact complex is necessary. To obtain maps of intact ATP synthase showing internal density distributions by single particle cryo-EM, methodological improvements to image acquisition, map refinement, and data selection were developed. Further, a novel segmentation algorithm was developed to aid in interpretation of maps. The use of these tools allowed for construction and interpretation of two maps of ATP synthase, solubilized in different membrane mimetics, in which the arrangement of subunits could be identified. These maps revealed interactions within the complex important for its function. In addition, evidence was obtained for curvature of membrane mimetics around ATP synthase, suggesting a role for the complex in maintenance of mitochondrial membrane morphology.

Single particle electron cryomicroscopy (cryo-EM) is a method of structure determination that uses many randomly oriented images of the specimen to construct a three-dimensional density map. In this thesis, single particle cryo-EM has been used to determine the structure of intact adenosine triphosphate (ATP) synthase from bovine heart mitochondria, an approximately 550 kDa membrane protein complex. In respiring organisms, ATP synthase is responsible for synthesizing the majority of ATP, a molecule that serves as an energy source for many cellular reactions. In order to understand the mechanism of ATP synthase, knowledge of the arrangement of subunits in the intact complex is necessary. To obtain maps of intact ATP synthase showing internal density distributions by single particle cryo-EM, methodological improvements to image acquisition, map refinement, and data selection were developed. Further, a novel segmentation algorithm was developed to aid in interpretation of maps. The use of these tools allowed for construction and interpretation of two maps of ATP synthase, solubilized in different membrane mimetics, in which the arrangement of subunits could be identified. These maps revealed interactions within the complex important for its function. In addition, evidence was obtained for curvature of membrane mimetics around ATP synthase, suggesting a role for the complex in maintenance of mitochondrial membrane morphology.

Emerging arthropod-borne viruses, such as alphaviruses and bunyaviruses, represent a serious threat to human and animal health worldwide, and for most of them, vaccines and specific treatments are unavailable. Viral host cell entry can be divided into several entry checkpoints, and the most important checkpoints for low pH-dependent enveloped viruses, such as bunyaviruses and alphaviruses, include receptor binding at the cell surface and, followed by endocytosis, low pH dependent membrane fusion from within intracellular compartments. A more thorough understanding of the detailed mechanisms allowing the viruses to pass these checkpoints is a pre-requisite for the design of viral entry inhibitors. This thesis reports the in vitro analysis of native alphavirus-receptor interactions, with the help of electron cryo-microscopy and icosahedral reconstruction of virus-recaptor complexes, using the prototypic alphavirus Semliki Forest virus (SFV) and the C-type lectin DC-SIGN. Together with results from collaborative work on SFV glycosylation, this study provides progress in defining the binding sites of DC-SIGN at the surface of SFV. Second, an in vitro system for phlebovirus fusion was developed using standard fluorometry, and has been characterized with the help of electron cryo-microscopy. It was discovered that negatively charged phospholipids with a conical shape, including the late endosomal phospholipid BMP, allow efficient phlebovirus fusion in vitro, thereby providing a possible rationale for phlebovirus fusion in late endosomes. Furthermore, electron cryo-microscopy of phlebovirus-liposome complexes allowed the capture of early stage fusion intermediates and laid the basis for possible future higher resolution studies of these fusion intermediates.