Joint Proteomics/IQB/SEBS Recruiting Seminars

Seminar 1 | Download Flyer

Hui-Ting Chou, Ph.D.
Harvard Medical School
Molecular Architecture of Membrane Trafficking Machineries Revealed by Single Particle Electron Microscopy

Date: Monday, February 27, 2017
Time: 3:00 pm
Location: Proteomics, Room 120

In membrane trafficking pathways, multisubunit tethering complexes (MTCs) are recruited to the organelles through Rab GTPasees and orchestrate the events from the vesicle recognition to membrane fusion with a number of proteins. Here we characterized the molecular architecture of three tethering complexes, Golgi-associated retrograde protein (GARP) complex, conserved oligomeric Golgi (COG) complex, and homotypic fusion and vacuole protein sorting (HOPS) complex using single particle electron microscopy. GARP promotes fusion of endosome-derived vesicles to the trans-Golgi network and COG is required for vesicle docking to and within the Golgi apparatus. Our analysis showed that GARP and COG subcomplex, Cog1-4, share the same subunit organization, even though the subunits have very low sequence similarity. We also found the overall structures of COG complex, Cog1-8, and subcomplex, Cog5-8, and identified the subunit arrangement of Cog5-8.  HOPS is associated to the lysosome acting in the endolysosomal pathway and belongs to different MTC subfamily. Our electron microscopy class averages revealed that the HOPS particles containing flexible rod-like extensions were markedly different from the more globular and rigid particles in previous report. The class averages of full and sub-complexes showed HOPS forms a core composed of Vps16, Vps33 and Vps41, and three flexible legs including Vps11, Vps18 and Vps39. Our data suggested that the HOPS, COG and GARP complexes are similarly multilegged and share a common building plan, reflecting their analogous functions.

Seminar 2 | Download Flyer

Jiansen Jiang, Ph.D.
The University of California, Los Angeles
CryoEM structure of  Tetrahymena telomerase: insights into functions and interactions of the subunits

Date: Thursday, March 2, 2017
Time: 3:00 pm
Location: Proteomics, Room 120

Telomerase is a large ribonucleoprotein complex that adds telomeric DNA repeats to chromosome ends to maintain genome integrity in eukaryotic cells. Structural study on telomerase has been highly challenging. We solved the first structure of telomerase holoenzyme from Tetrahymena thermophila by cryoelectron microscopy (cryoEM). Using a combination of biophysical and biochemical approaches, we unambiguously localized the known subunits in the cryoEM structure of Tetrahymena telomerase and more surprisingly discovered two new protein subunits that are important to telomerase function. This study provides the first view of the interactions among nine protein subunits and the RNA within the telomerase holoenzyme and shed light on their roles in telomerase activity as well as interactions with other proteins associated with telomere maintenance.

Seminar 3 | Download Flyer

Priyanka Abeyrathne, Ph.D.
HHMI |  HHMI Janelia Farms
Cryo-EM studies of the translocation of a viral IRES through the ribosome and a small bacterial chloride channel

Date: Monday, March 6, 2017
Time: 3:00 pm
Location: Proteomics, Room 120

Cryogenic electron microscopy (cryo-EM) is uniquely suited to studying the structure of proteins, in particular membrane proteins, and macromolecular complexes, which conduct a wide range of essential cellular tasks. Understanding the details of their structures is critical to unraveling their roles in cellular functions in health and disease.

My current work combines biochemistry and cryo-EM to understand the molecular mechanisms that underlie the regulation of eukaryotic ribosomes. In my seminar, I will discuss how the internal ribosome entry site (IRES) RNA of the Taura syndrome virus can hijack host ribosomes to make them translate the viral proteins necessary for virus propagation. Using cryo-EM of a single sample, I was able to determine several high-resolution structures (3.4 - 4.2 Å) that capture different states of the ribosome in complex with the viral IRES RNA and sordarin-bound elongation factor eEF2.GTP. These structures describe the near-complete trajectory of IRES RNA translocation through the ribosome and provide an unprecedented view of eEF2 dynamics. The structures suggest missing links in our understanding of tRNA translocation.

Cryo-EM has also become a popular method for obtaining high-resolution structural information of membrane proteins. However, even once a sufficient amount of protein has been expressed and purified, small membrane proteins (MW <200 kDa) are still difficult to study by cryo-EM, because of the low contrast they generate in images. Furthermore, the density arising from the detergent micelle around the hydrophobic belt of the protein complicates the assessment of structural information, which is particularly challenging for membrane proteins that are almost entirely embedded in the lipid bilayer. I will present approaches to overcome these challenges using as example ClC-rm1, a small membrane protein (~116 kDa) from Ralstonia metalidurance that belongs to the prokaryotic chloride channel (ClC) family.

Seminar 4 | Download Flyer

Jesper Pallesen, Ph.D., MBA
The Scripps Research Institute
Heterogeneity in Viral Glycoprotein Complexes

Date: Thursday, March 9, 2017
Time: 3:00 PM
Location: Proteomics, Room 120

Viral fusion glycoproteins (GPs) are the principal mediators of enveloped virus fusion with host cell membranes during infection. Consequently, prefusion viral GPs are targets of neutralizing antibodies elicited during host humoral immune response. GPs are inherently metastable in their prefusion conformations; to mediate fusion, GPs undergo substantial rearrangements to arrive at a stable lower-energy postfusion conformation.

Through a series of cryo-EM studies of GPs complexed with various ligands (e.g., neutralizing antibodies, receptor analogues or receptors), we seek to understand the underlying mechanisms of viral fusion and viral interplay with host humoral immune response. As a result, we have characterized several GP cryo-EM structures that describe various antibody epitopes as well as various conformations probed in the prefusion state and during the fusion process.

Seminar 5 | Download Flyer

Arek Kulczyk, Ph.D.
Harvard University
Single-molecule studies of the replisome structure and dynamics

Date: Monday, March 27, 2017
Time: 3:00 PM
Location: Proteomics, Room 120

The antiparallel nature of the two strands in duplex DNA pose a topological problem for their simultaneous synthesis.  The “trombone” model of the replication fork postulates that the lagging-strand forms a loop such that the leading and lagging-strand replication proteins contact one another.  The replisome now can move in one direction along the DNA while synthesizing both strands.  Physical interactions between the replication proteins and DNA coordinate processive synthesis of leading and lagging strands.

I will present a structure of the ~650 kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork.  A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy.  The two molecules of DNA polymerase adopt different spatial arrangement at the replication fork reflecting their roles in leading- and lagging-strand synthesis.  The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis.

The replisome is highly dynamic, with components exchanging and entire replisomes collapsing and reassembling.  We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork.  By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange.  Since mechanisms of DNA replication are highly conserved the observations are relevant to other replication systems.