David J. DeRosier - US grants

Each cell of Salmonella typhimurium possesses a chemosensory apparatus, which detects gradients in its environment, and a flagellum, which propels the cell up or down the gradient. A set of five different proteins comprise a sensor. Three of these form a stable complex. The sensor regulates the phosporylation of the response regulator, which carries the signal from the sensor to the flagellum. The flagellum is the cell's propulsion system. It contains an 18,000 rpm, reversible rotary motor and an external propeller. The motor is powered directly by the proton motive gradient across the cell membrane. About 40 different proteins are required for the regulation, assembly and operation of the flagellum. Nineteen of the 40 proteins are known to be part of the assembled structure. There are likely to be a few more found in the final tally but these will be involved in the export/assembly pathway. Our goal is to determine the structures and mechanisms of these two remarkable machines. We will use electron cryomicroscopy to produce 3D molecular- resolution maps of the machines into which we can dock atomic models for the components. This approach is a frontier of structural biology. We propose to produce an atomic model for the cytoplasmic portion of a sensor, which contains the signaling domain of the transmembrane receptor, a kinase, and an adapter molecule. The entire complex has a mass of 1.4 106 daltons, consisting of 28 copies of TarC (the cytoplasmic domain of the receptor), 6 copies of CheW (adapter protein), and 4 copies of CheA (histidine kinase). The atomic structures for TarC and most of CheA are known. The structure of CheW can be modeled because of its homology to CheA. We have good preliminary data on the whole complex, which augers well for completion of a 20Angstrom units map of the complex. We propose to obtain 3D maps at molecular resolution of the rotor of the flagellum. Our preparations retain three of the key torque-generating, direction-switching elements. They lack the transmembrane proton channel proteins. We plan to compare maps from clockwise-turning motors and from counterclockwise-turning motors. We plan to obtain maps with the response regulator bound. These maps will give insights into the motor's mechanism of torque generation and switching. Only one domain of one of the key proteins is available at atomic resolution, so an atomic model for the motor remains in the future. We propose to obtain 3D maps of the propeller apparatus (hook and filament) at about 4 Angstrom units resolution by electron cryomicroscopy. These maps should permit us to produce a chain tracing of the peptides.

The actin filament is a key element in the dynamic machinery of motile cells and in the cytoskeleton of non-mobile cells. Different assemblies of actin filaments fulfill different roles, even within the same cell. These assemblies differ both in their function and in their filament organization. We propose experiments to discover the properties of actin which make it a common element in the cytoskeletal structures. We also plan to study how these cytoskeletal structures are constructed. The approach, using combined techniques of electron microscopy and image analysis, is to study the structure of isolated actin filaments, free and in combination with other proteins, actin bundles, and a cytoplasmic actin gel. In particular, we plan: 1) a study of actin, myosin S-1 decorated actin, thin filament, and the actin-containing filaments from the acrosomal bundle in Limulus; 2) a comparative study of the structures of actin-fascin and actin-fimbrin bundles; 3) a study of the morphogenesis of the actin-scruin bundle found in the sperm of Limulus, the horseshoe crab; 4) a study of the structure, organization and morphogenesis of a naturally occurring actin gel, the cuticular plate, found in hair cells in the inner ear. The aim of these studies is to reconstruct the three-dimensional structure of actin; to determine the shapes of myosin S-1, scruin, fimbrin, and fascin; to locate their binding sites on actin; and to determine how their bonding modifies the structure of the actin filament. Next, we aim to determine the bonding rules for actin filaments in the cuticular plate, a naturally occurring actin gel. Finally, we aim to determine how bundles and gels are assembled in vivo by looking at the formation of these structures in development.

The proposed conference will be the third in a new series of Gordon Conferences on Three-dimensional Electron microscopy of Macromolecules. Electron microscopy has developed rapidly over the last few years as a tool for structure research at the molecular level. Important technical breakthroughs have been made which enable three-dimensional structure to be determined more accurately than before, and which open the way to a study of structure essentially under controlled physiological conditions. As a result, there are now exciting new opportunities for understanding biological mechanisms and principles of molecular design. The focus of this meeting will be on the novel electron microscopic methods being developed for preserving and imaging molecules in frozen solutions, for identifying specific sites on them by heavy atom labels, and for extracting near-atomic resolution three- dimensional information from images of molecules in isolation, in complex assemblies and in crystalline arrays. Important recent structural findings, relating especially to cell organelles, membrane and cytoskeletal proteins, will also be emphasized. This conference will help catalyze the further development of this field by bringing together many of the key investigators and allowing them to discuss their latest and perhaps even incomplete studies in a unique and informal setting. Speakers are being chosen on the basis of the importance of their recent research contributions and the potential of their particular approach in future structure research. Topics to be covered include: cryoelectron microscopy, new methods in averaging, three-dimensional reconstruction, use of site-specific labels, image correlation and classification (sorting), and recent structural results obtained for membranes, filaments, organelles, and viruses.

image processing; computer graphics /printing; biomedical equipment purchase; chemical structure function; scanning electron microscopy; X ray crystallography; computer human interaction; chemical models;

This research is intended to elucidate the mechanism by which force is developed by the interaction of myosin and actin molecules in muscle (and in a number of other motile systems) and will focus on the dynamics of the changes taking place in the crossbridges in striated muscle during contraction. It has long been established that muscle contraction is brought about by a sliding filament mechanism, in which partially overlapping arrays of actin and myosin filaments are acted on by a relative sliding force which leads to shortening of the muscle. It is generally believed that this force is generated by a cyclic interaction of myosin crossbridges with sites on the actin filaments. However, the structural details of the process have proved to be particularly difficult to establish, partly because of the transient and unsynchronized nature of many of the changes taking place, and partly because of the technical difficulty of obtaining submicroscopic structural information about any rapidly changing biological system. The development of rapid freezing techniques by Heuser and others has now made it possible in principle to obtain electron microscope images of these transient structural states and suitable variations of these techniques will be developed to enable correlations to be made with evidence derived from time-resolved x-ray diffraction studies. These approaches should provide important new information about the structural behavior of the crossbridges as they interact with actin and develop force. When combined with new information from biochemical and physiological studies, the results of the present work should materially advance our understanding of this very fundamental biological mechanism.

The actin cytoskeleton is important in the machinery of motile and non-motile cells. Different cytoskeletal assemblies fulfill different roles, and their different functions are reflected in their different designs. Cytoskeletal design is mediated by the proportion, kind and organization of the actin-binding proteins present. We have been studying the organization and assembly of the cytoskeletons of specialized cells because their cytoskeletons are relatively simple in composition and structure. Several recent advances have opened a pathway to obtaining detailed models for in vivo cytoskeletal structures, in particular, actin bundles. The discovery that many of the proteins are modular in design permits us to dissect the cytoskeleton into parts. We propose in particular to study the fimbrin and fimbrin/villin bundles found respectively, in the specialized hair cells of the inner ear and in the brush border cells of the intestine. We propose to study rafts of actin crosslinked by EF1alpha, fascin, and aldolase. The results will tell us how actin is crossbridged by different bundling proteins and how the structure is deformed to accommodate them. We will also obtain maps of 2-D arrays of F-actin crosslinked by intact fimbrin or villin or both. We proposed to dock the atomic models for the components (all are known) into the maps obtained from the 2-D arrays and thereby derive a 3-D model for a row of crossbridged filaments. This model in turn will be used to construct a 3-D model of an in vivo 3-D bundle (e.g., the actin-villin bundle). In this way, we can begin the long task of constructing atomic models for the cytoskeleton. No one technique nor any one lab can accomplish all of this, but as evidenced in the recent atomic model for the acto-S1 I complex, it can be done by a combination of techniques and labs. We also propose to study the structural states of actomyosin. We plan to produce 3-D maps of actomyosin in the rigor, ADP and ATP states. We will use constructs of smooth muscle myosin and myosin V. We will also obtain maps of smooth muscle HMM in order to understand why double but not single headed myosin is regulated. We will also look at myosin V (actually a construct having 2 IQ domains) in the presence of calcium in order to visualize the changes associated with regulation.

The goal of this Training Program is to expose graduate students to fundamental issues of macromolecular function and to develop expertise in advanced aspects of this currently exploding subject. The theme of the program is a determined focus on the chemical and mechanistic principles governing the workings of biological macromolecules, and on how their functional behaviors can be understood in terms of their molecular structures. The Training Grant will provide support for graduate students in either the Graduate Program in Biochemistry or in the Graduate Program in Biophysics, two of the five life-science graduate programs at Brandeis University. There are currently 167 students in these five programs, and we propose to support 18 on this Training Grant. Twenty faculty, all carrying out grant-funded basic research on structure and function of macromolecules, participate in this Training Program. The Program provides specific training in the following areas: protein and nucleic acid structure determination by x-ray crystallography and multidimensional NMR, high-resolution electron microscopy, mechanistic enzymology, muscle and molecular motors, membrane protein structure, protein dynamics, ion channel structure and function, and catalytic RNA mechanisms. Most graduate students in the.Program come from undergraduate backgrounds in chemistry, biochemistry, physics, or mathematics. All graduate students take a program of rigorous, quantitative courses and advanced research seminars; moreover, in the first year all students engage in intense and demanding laboratory rotations in 3-7 different laboratories. Students choose Ph.D. thesis advisors at the beginning of the second academic year and commence working on their research projects.

In this application we describe research programs in structural biology at two different institutions, each of which requires the ability to analyze data obtained from our high resolution electron microscopes. In order to be able to fully utilize the capabilities of our EM Facilities and to overcome a major gap in the technological resources available to the life sciences communities at Brandeis University and at Boston University School of Medicine, we are proposing to purchase a high resolution precision microdensitometer scanning system. Considerable effort has been devoted to insure that the Rosenstiel Basic Medical Sciences Research Center (the Center) and BUSM, Department of Biophysics have the EM facilities necessary to pursue high-resolution structural studies. Over the past few years several projects have developed specimens extremely well suited for Cystallographic and single particle study using EM techniques. Optical diffraction of film recorded EM images and/or direct observation of film recorded electron diffraction patterns of a variety of these specimens have indicated that the information content of much of this data is great and that Programs for high-resolution structural determination are feasible. Central to working with any film recorded data is the ability to convert this information into digital form for computer-based processing and analysis. In neither institution is there currently a device able to digitize EM film recorded data in a form suitable for high-resolution structural studies. (The digitization equipment currently available is inadequate even for moderate resolution studies.) The few EM based high-resolution reconstructions performed on biological samples (bacteriorhodopsin, light harvesting complex and porin) in other laboratories were exclusively determined with data (images and diffraction patterns) digitized through precision microdensitometers. Both the Center's high resolution electron microscopes (Philips CM12 and Model 420) are routinely providing high resolution images for our structural biology research programs. However, in order to digitize these images for computer analysis it has been necessary for our investigators to travel to Albany, NY as there are no suitable scanners in New England. The instrument of choice for this application is currently being manufactured by Orbital Sciences Corporation (Pomona, CA). It is a flat-bed microdensitometer and is essentially identical to the Perkin-Elmer instrument which is no longer manufactured. It is widely regarded as the state-of-the-art instrument for image digitization and far exceeds the capabilities of our current scanner. The technique used in the Orbital microdensitometer system is almost completely free of optical aberrations and produces high fidelity data. The major advantage of this instrument is that an illuminated area of the specimen is optically projected onto a sample defining aperture as small as 3~m. Only that light which passes through the aperture is measured and digitized. Scanning is accomplished by laterally translating the specimen relative to the optical axis. The aperture maintains a fixed location on the optical axis, thereby minimizing distortions and the effects of optical aberrations. The Center's only available instrument is an Eikonix digitizer which operates by projecting a real image of the specimen to be digitized using a 35mm photographic lens. This real image is digitized via a linear photodiode array which is translated across the image plane. This approach, though relatively inexpensive, is subject to a multitude of optical and mechanical defects. The Eikonix scanner cannot support our high resolution structural studies reliably.

9513898 Lowey The goal of the research is to test the "rotating crossbridge model" for muscle contraction by two approaches: (1) cryo-electron microscopy and image analysis will be used to analyze the molecular structure of actin filaments decorated with recombinant myosin head fragments. By combining the X-ray crystal structures of actin and S1 with the computer generated three-dimensional reconstructions from electron micrographs of frozen-hydrated decorated F-actin, it may be possible to see whether conformational changes occur in myosin upon its binding to actin. (2) Multi-dimensional nuclear magnetic resonance (NMR) will be used to explore the structure of the regulatory light chain in the free and bound state, and its interactions upon phosphorylation. The light chain and its target peptide will be expressed in E. coli for isotope labeling. The structure of the skeletal muscle light chain will be compared to the smooth muscle light chain for a better understanding of the mechanism of regulation in these two major muscle types. %%% The most widely accepted theory of how muscles contract involves a reorientation of the myosin head relative to the actin filament as the filaments slide past one another. A fundamental problem with the "rotating crossbridge model" has been the failure to detect any large conformational changes within the myosin molecule that could account for the large distance over which an attached crossbridge can develop tension. The studies proposed here are designed to test models for muscle contraction based on the recently solved crystal structure of the myosin head. ***

9977556

Abstract

This project involves the acquisition and installation of a 300 kEV field emission gun (FEG) transmission electron microscope (TEM) to determine the structures of cellular machinery. Cellular machines such as the actin cytoskeleton are often large structures difficult to study by methods other than electron cryomicroscopy. For example, it has been possible to dock atomic models for the cytoskeletal components obtained by x-ray crystallography into the molecular maps of the cytoskeletal complexes obtained by electron cryomicroscope. Analysis of filamentous structures has been extended to 10 A resolution in many cases. A state of the art microscope is essential for these studies. The microscope to be obtained and installed during this project will be utilized for the study of the splicesome, voltage-gated ion channels, the actin bundle of the intestinal microvillus, the bacterial flagellar motor and filament, the bacterial gas vesicle, the receptor-kinase signaling complex and the complex of actin with myosin. The aim of these studies are atomic models of the structures. In some instances the structure will be obtained to atomic resolution using electron cryomicroscopy alone. In other cases, atomic models obtained by x-ray crystallography will be docked into molecular maps obtained by cryomicroscopy. The 300 kEV FEG TEM is essential to these projects because the quality of the micrographs limits the resolution of the map in the same way that the quality of the objective lens limits the resolution of the light microscope. No matter how sophisticated, computer processing cannot extract what is not present in the images. The two key features of this microscope in this regard are the higher voltage and the FEG. With these, it is possible to take highly defocused images which have strong contrast at low resolution but which preserve high resolution detail. The strong contrast at low resolution is essential for accurate alignment of images prior to averaging. If the alignment (or correction for distortion) is not accurate, one will average non-equivalent features and resolution will be lost. This microscope will extend the resolution of current maps and therefore, the ability to obtain accurate atomic models whether it be directly or by the docking of atomic models into molecular maps.

The cryomicroscope is essential to the training of young scientists. Since the kind of microscopy being used represents a frontier in structural studies of cellular machinery, students and post doctoral fellows must be trained in these techniques. The need for such scientists is increasing rapidly now that the power of cryoelectron microscopy has been demonstrated. There are currently few places equipped for this kind of training.

Description: (Applicant?s abstract) This is a joint Harvard-Brandeis program project, with goals that include both methodological developments and cell-biological applications. The four major goals are: (1) advance methods for obtaining atomic models of large structure by docking and refining atomic models of the component parts in molecular maps from electron cryomicroscopy (cryoEM); (2) improve the accuracy and resolution of the molecular maps we can obtain from cryoEM of single particles, helical arrays, and imperfectly ordered 2D crystals; (3) enhance use of electron diffraction data to accelerate 2D crystal structure determination, through development of molecular replacement and phase extension methods; (4) improve use of molecular maps from cryoEM to initiate phase determination in studies of large assemblies by x-ray crystallography. There are four major projects, three cores, and a set of associated projects. The major and associated projects focus on the following biological systems: (1) the actin cytoskeleton; (2) viruses and viral entry into cells; (3) membrane proteins, especially members of the MIP and MAPEG families; (4) antibiotic peptide synthetases; (5) spliceosome assemblies; (6) the phage T7 replication complex ("replisome"). The cores, which are a central component of the collaborative effort, support shared state-of-the-art electron microscope facilities, software development, and administration of the consortium.

The overall goal is to establish a high performance connection to the Abilene advanced Internet facility to support science research. To achieve this connection Brandeis will establish the advanced connection via Northern CrossRoads (NoX) to the Abilene network. Brandeis will link to the NoX gigapop directly through a leased fiber connection provided by RCN, which will also provide the end switches and the circuit monitoring equipment as well as manage the circuit.

The proposers have identified four areas of research that require the use of a high performance network connection. The investigators in each of these areas of research have coped with inadequate bandwidth for a number of years. They have been remarkably adaptive in working out ways to deal with this situation, but the time has come to sustain their research with a high performance Internet connection.