Project G

Associated PIs: Francisco Asturias, Wah Chiu, Joachim Frank, Nikolaus Grigorieff, Eva Nogales, Phoebe L. Stewart

The program project will include the work of a total of 6 principal investigators as associated projects. The research of each associated project is, in all cases, work that is currently funded from other sources, and no new funding is requested for this work as a part of the program project.

Inclusion of the associated projects will benefit the program project by providing examples of the research context that motivated our development of high-throughput computational technology. In addition, the PIs and staff of the associated projects will provide a base of beta-site testers who will give feedback on how well the computational technology works, what bugs exist that need to be fixed, and what features a user would like to have that are not yet available in the software.

Although the program project will not provide new (additional) funding for the associated projects, their affiliation with the program should result in very substantial improvement in the ability that each has to speed up and more effectively accomplish their respective research goals. Each affiliated project will have early access (as a beta-site user) to the software technology that is needed to take advantage of highly parallel computers. Each will also have access to the 80-node cluster at LBNL, on which they can run this software. Extremely valuable improvements (speed-ups) in computational turnaround will therefore become available to each of these associated project, when the number of single particles in each of their data sets rises significantly about 10⁴ to 2X10⁴ particles.

Project G₁-Yeast Mediator Complex and Pol II Holoenzyme (Francisco Asturias)

The long-term goal of Project G₁ (funded by NIH) is to elucidate the mechanism of regulation of DNA transcription by RNA polymerase II. Our experimental approach concentrates on 3-D electron microscopy structural studies of the multi-protein co-activator complex termed Mediator and of the "holoenzyme" that it forms with RNA polymerase II. The holoenzyme complex is the molecular centerpiece of the transcription machinery as it represents the minimal entity that is responsive to DNA regulatory sequences. Specific aims of the project include (1) the determination of the 3-D structure of th yeast Saccharomyces cerevisiae Mediator complex from unstained specimens preserved in amorphous ice (using cryo-EM), and (2) the determination of the 2-D structure of the Mediator/RNA polymerase II holoenzyme complex, also using unstained specimens. Docking the atomic resolution structure of RNA polymerase II to the EM envelope for the holoenzyme complex will reveal the location and possible role of Mediator/polymerase contacts.

Project G₂-Various (Wah Chiu)

Dr. Wah Chiu directs the National Center for Macromolecular Imaging at Baylor College of Medicine where he has engaged with many collaborators in a variety of research projects ranging from viruses, cytoskeletal protein complexes, and oligometric proteins. Many of these projects can be benefited from the the software development as described in this program project. The two projects described in the program proposal involve (1) skeletal Ca²⁺ release channel, and (2) human fatty acid synthase. The ultimate goal in wither project is to determine their structures towards atomic resolution, whereas the immediate goal is to resolve their structures at ~8-9Å resolution. Both projects are supported by independent grants of the collaborators and the NCRR Center grant of Dr. Wah Chiu.

Project G₃-Ribosome Structure and Function (Joachim Frank)

This subproject is spurred by our recent discovery of evidence for a rotational movement of the 30S subunit with respect to the 50S subunit in response to EF-G binding in its GTP state (a 6 degree rotation) and GDP state (a 3 degree reverse rotation from the GTP state). This relative movement is accompanied by large-scale conformational change in both subunits. For example, the entrance and exit channels in the 30S subunit that conduct the mRNA are seen to open and close, and the central protuberance of the 50S subunit changes its architecture dramatically as the bridges connecting it to the 30S subunit head have to follow the 20-Å movement at the ribosome's periphery. While mechanisms involving relative movements of the subunits of the ribosome have been proposed by a number of authors over the past 30 years, our study provides the first direct experimental evidence and shows that the movement is ratchet-like.

We will develop an approach of large-scale modeling to explain the observed changes in terms of the rearrangement of domains, relative movements of connected, relatively rigid RNA elements, and strains on these elements that might result in bending or torsion. This information in turn can be used to analyze the dynamics of local conformational rearrangements in RNA components.

Project G₄-Structural Analysis of the Shaker Potassium Channel (Nikolaus Grigorieff)

Voltage-gated ion channels carry out the essential task of signal propagation in electrically excitable tissue in all living organisms. These channels combine a number of fascinating functions: their voltage sensitivity is higher than that of a transistor; their turnover rates, which approach diffusion limits, are several orders of magnitude larger than those of any know transporter or pump. Yet they are highly selective with a 10,000-fold variation in conductance of ions differing in their radius by only a few tenths of an Angstrom. While some of these elements are well understood in terms of the recently determined atomic structure of the simpler voltage-insensitive potassium channel KcsA, other elements remain a mystery. The Shaker potassium channel from Drosophila is the most thoroughly studied voltage-gated ion channel and extensive mutagenesis has been carried out to gain insight into its structure and function.

The aims of this project are (1) to determine the 3-D structure on the Shaker potassium channel at 15Å resolution or better, and (2) to determine the physical location of the voltage sensor by site-specific labeling. These aims target current research topics with important biological implications and are well within range of current electron microscopical methods. A 15Å 3-D structure will show how the mass of the additional four transmembrane segments in Shaker which are not present in KcsA is arranged and what their functional roles may be. It will add a solid framework to the numerous speculations about how the voltage sensor might do its work.

Project G₅-Eukaryotic Transcription Initiation Machinery (Eva Nogales)

The main research objective for this project is the structural characterization of the multi-protein complexes involved in gene transcription initiation and regulation in eukaryotes. The first step of transcription involves the binding of the general transcription factor TFIID to the core promoter to facilitate the assembly of the whole transcriptional machinery. TFIID is also involved int he interaction with gene-specific activators. Our initial objective is to characterize the structure of TFIID and its interaction with other general factors and with activators using cryo-electron microscopy and single-particle image reconstruction. Our ultimate goal is to understand how the eukaryotic transcription initiation machinery assembles and functions, and the structural mechanisms for gen transcription regulation that are essential to maintain the health of the organism.

The initial step (which is most pertinent to the program project) will be creating a 3-D reconstruction of frozen-hydrated complexes of human TFIID. This, of course, will be done by cryo-EM. The immediate priority is to obtain a 3-D reconstruction of frozen-hydrated human TFIID complexes at a resolution of 25Å. The long-tern objective is to obtain a reconstruction at 10Å where the secondary structure of the subunits will start to be discernible. This goal will require a large number of high quality images and the automation of data analysis, and will benefit from the technology developed by the program project.

Project G₆-Reconstruction of Ribonucleoprotein Vault Particles (Phoebe L. Stewart)

The research focus of the Stewart laboratory is cryo-EM and single particle reconstruction of a variety of biological assemblies including adenovirus, the ribonucleoprotein vault, DNA-dependent protein kinase, and members of the small heat-shock protein family.

The overall goal of this project is to determine the molecular architecture of ribonucleoprotein vault particles and use the structural information to help understand their biological function. The original specific aims of this federally funded vault project were to (1) determine a reconstruction of the vault, (2) discover the location of the RNA within the vault, and (3) localize the major vault protein (MVP) within the vault. An additional aim (aim #4) is to determine a reconstruction of the vault isolated from TEP1 knock out mice (lacking one of the three vault proteins) and compare it to a reconstruction of the appropriate wild-type control mouse vault.

The specific aim proposed for this program project is to dramatically increase the number of vault particles included in a vault reconstruction (from a current maximum of 4,317 particle images to 20,000+ particle images). We anticipate that processing of such a large dataset will result in a significant improvement in the resolution attainable by cryo-EM single particle reconstruction of the vault.

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