Mark Yeager - US grants

Overview The EM Crystallography Core will have access to the facilities and equipment within the National Resource for Automated Molecular Microscopy (NRAMM). Housed at TSRI, this resource has four electron cryomicroscopes and the necessary expertise and auxiliary equipment for high-throughput automated electron cryomicroscopy.

DESCRIPTION (provided by applicant): Gap junctions are specialized regions of cell-to-cell contact in which hexameric oligomers, called connexons, dock end-to-end across a narrow extracellular gap, allowing the intercellular exchange of nutrients, metabolites, ions and small molecules. Previously, this research program focused on the 43 kDa connexin (Cx43) channel, which mediates ionic conduction between cardiac myocytes, thereby regulating the normal heartbeat, but also mediating potentially fatal cardiac arrhythmias. We have now expanded our research to include Cx26, mutations in which are the predominant cause of inherited, nonsyndromic deafness, and Cx40, which forms high conductance channels in the specialized conducting tissue of the heart. For the next cycle, we will pursue 3 specific aims: (1) Our major aim, to which we will devote 70% effort, is to use X-ray crystallography to determine an atomic resolution structure of one or more hexameric connexons, which includes improving upon the resolution of a recent 3.5 E structure of Cx26 (Maeda et al., 2009), as well as testing our cryoEM-based C1 model for the assignment and packing of the transmembrane ?-helices. (2) Our second aim, to which we will devote 20% effort, is to use electron cryocrystallography of 2D crystalline gap junction plaques isolated from cells to examine the higher resolution structure of the extracellular loops, in order to understand the molecular basis for isoform selectivity and how the packing of the loops forms a tight molecular seal to exclude the extracellular environment. (3) A third minor aim, to which we will devote 10% effort, is to use cryoEM and single particle image analysis of connexons to explore gating and regulation of a number of Cx26 truncation mutants. Of particular interest is the recent discovery that the N-tail of Cx26 forms a central gating plug (Oshima et al., 2007). We will perform a structure/function analysis by correlating the structure of truncation mutants with functional studies of channels incorporated into liposomes. Our research is enriched by several key collaborations: Dr. Andrew Harris uses a liposome assay to test the function of our Cx mutants;Dr. Ray Stevens is an expert membrane protein X-ray crystallographer;Dr. Qinghai Zhang has synthesized custom detergents for channel stabilization to enable 3D crystallization trials;and Dr. Anchi Chang is an expert in electron cryocrystallography. By this integrated approach, we will continue our quest to visualize an atomic structure of a gap junction channel. PUBLIC HEALTH RELEVANCE: The cells within all tissues of our body have to communicate with one another in order to coordinate their metabolic activities. This is accomplished by a unique set of molecular pores called gap junction channels, which are assembled from protein subunits called connexins. Gap junction channels are formed by the coupling of two half channels called connexons, which are themselves formed by a ring of 6 connexin subunits. A cylindrical connexon in the surface membrane of one cell is coupled to a corresponding connexon in the adjacent cell. This molecular conduit mediates the passage of ions and small molecules and thereby coordinates the metabolic activity of the tissue. Much of our effort is now focused on a particular connexin, Cx26, mutations in which are a major cause of hereditary deafness. We have generated substantial quantities of the protein so that we can use biophysical techniques such as electron and X-ray crystallography to determine an atomic structure of the channel. Such a detailed molecular picture will provide insight into how these channels are involved in such diverse processes as regulating the heartbeat and hereditary deafness.

The EM Crystallography Core will have access to the facilities and equipment within the National Resource for Automated Molecular Microscopy (NRAMM). Housed at TSRI, this resource has four electron cryomicroscopes and the necessary expertise and auxiliary equipment for high-throughput automated electron cryomicroscopy.

Summary Funds are requested to purchase a direct electron detector and advanced operating software for a Titan Krios transmission electron microscope. The new generation of direct electron detectors replace scintillators and CCDs for recording EM images, and offer a highly dramatic improvement in both sensitivity and resolution. The Falcon II camera requested will also offer a three-fold increase in the data collection rate over our existing CCD camera, but the main advantage comes from the huge increase in the signal-to-noise ratio and the greatly improved point spread function. While the three-fold increase in the rate of acquiring images could be met with our existing CCD camera by simply acquiring images over a three-fold longer period, no amount of images acquired with the CCD would ever recover the high resolution information that is either completely absent in the CCD images or buried in the noise. These advances with the direct electron detector will allow us to image a large number of specimens at an unprecedented resolution, greatly aiding a number of NIH-supported projects. Four Major Users (Egelman, Yeager, Stewart and Zhang) and a number of Minor Users will use the Titan Krios as a shared facility. The incredible throughput of this microscope with the proposed camera (~ 2,000 images per day), combined with the robotic operation of the microscope on a 24/7 schedule, means that data analysis will become the rate-limiting step and not image acquisition. This high resolution, high throughput mode of imaging will largely transform EM. Thus, our Krios can truly function as a facility much like synchrotrons do in providing a unique service to the structural biology community. The specimens that will be examined at high resolution on the Krios with the proposed detector range from viruses and bacterial pili to complexes involved in the innate immune recognition of foreign RNA, allowing for conceptual advances in understanding many systems that are important to human health.

Abstract The Molecular Electron Microscopy Core (MEMC) facility at the University of Virginia (UVa) is comprised of three transmission electron microscopes. An FEI 120 kV Spirit is housed on the second floor of the Snyder Translational Research Building at Fontaine Research Park. An FEI 200 kV F20 and a 300 kV Titan Krios are located in a specially designed suite in the underground Life Sciences Annex (LiSA) adjacent and connected to the Snyder Building. Anticipating future growth in high-resolution cryoEM research, the suite was designed to accommodate two Titan Krios electron microscopes. The Titan Krios has an autoloader that robotically manipulates up to 12 frozen grids inserted at one time, uses constant current lenses for greater stability, is equipped with an XFEG for improved illumination and a Falcon II direct electron detector. In 2009, the abstract of our G20 grant that supported the build-out of the LiSA suite stated, ?The MEMC facility will (1) enhance the quality of research at UVa, (2) increase scientific productivity, (3) foster communication, interactions and collaboration amongst scientists, (4) boost the ability of UVa to attract and maintain premier faculty and students, and (5) strengthen the competitiveness of our faculty for funding. The MEMC facility will not only be available to School of Medicine faculty members but also to other scientists in other schools and departments at UVa. This will be a unique Core laboratory at UVa, and we envision that scientists throughout Virginia and the surrounding states will visit UVa to take advantage of such a cutting-edge laboratory for performing molecular resolution electron microscopy.? We have a strong spirit of collegiality and collaboration at UVa and welcome the opportunity to share the MEMC facility within UVa and beyond. We believe that our vision of the MEMC facility as a state-of-the-art cryoEM center for scientists in the mid-Atlantic region has already been realized. Over the last two years, 23 labs at UVa and 12 external labs have collected data using the MEMC. 14 labs have collected data using the Titan Krios. Half of the available time will be devoted to Consortium PIs, who have substantial experience in cryoEM and image analysis: Drs. Jenny Hinshaw (NIH), Deborah Kelly (Virginia Tech), Melanie Ohi (Vanderbilt), Montserrat Samso (VCU), and Peijun Zhang (Pitt). As the primary internal UVa users of the MEMC, Drs. Yeager and Egelman have more than 5 decades of experience. As Director of the Core, Dr. Kelly Dryden interacts closely with all users. The U24 grant, combined with $300K/year provided by the UVa School of Medicine, would make the MEMC financially sustainable.

Abstract Connexin (Cx) proteins form hexameric hemichannels (HCs) that dock end-to-end to form gap junction channels (GJCs) across the extracellular gap, allowing intercellular exchange of nutrients, metabolites, ions and signaling molecules. Over the last 3 decades our research program has explored the structure and regulation of two Cx isoforms, Cx43 and Cx26. Each is found in many tissues. The former most notably mediates electrical conduction between cardiac myocytes, enabling the normal heartbeat, but also mediating potentially fatal cardiac arrhythmias. The latter, is most well known for its role in the inner ear; mutations of Cx26 are the predominant cause of inherited deafness. Over the last dozen years we have focused on the regulation of Cx26 channels during tissue injury, associated with Ca2+ overload and acidic pH. We determined X-ray structures of the human Cx26 GJC with and without bound Ca2+. To our surprise, the two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Computational analysis revealed that the binding of Ca2+ ions creates a positive electrostatic barrier that blocks K+ permeation. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion. To examine pH-mediated gating of Cx26 GJCs we used cryoEM and single-particle image analysis coupled with H/D exchange and crosslinking mass spectrometry. The results support a steric ?ball-and-chain? mechanism in which association of the acetylated N-termini form a pore-occluding, gating particle. Building on this rigorous structural and biophysical analysis of WT channels, we are now in a position to explore (1) the effects of deafness-causing mutations of Cx26 that involve residues that participate in Ca2+ coordination, (2) the effects of mutations of residues implicated in pH regulation, (3) the structure of undocked hemichannels and (4) structures of other connexins, particularly Cx32, mutations of which cause peripheral neuropathy, and also the cardiac connexins Cx43, in the working myocardium, and Cx40, in the specialized conducting tissue. Our structural studies utilize X-ray crystallography, cryoEM, crosslinking, H/D exchange mass spectrometry (HDX) and EPR spectroscopy in a synergistic manner. Functional studies include electrophysiology and proteoliposome-based transport assays. Our research program is fortified by fruitful collaborations with 3 experts: Drs. Andrew Harris (electrophysiology and functional assays), Patrick Griffin (HDX mass spectrometry) and David Cafiso (EPR spectroscopy). Our proposed research provides an opportunity to understand aspects of GJC and HC channel function that have been long-desired, and to initiate exploration of how those structure-function properties operate in several members of the Cx family. Given the importance of proper Cx channel function in development, pathophysiology and response to disease and trauma, this understanding will have substantial biomedical impact. !