R35GM139453

Structural Studies of Macromolecular Assemblies Using Cryo-EM - Project Summary/Abstract

The main objective of the proposed studies is the elucidation of fundamental processes of translation, translational regulation, and translational quality control. To this end, single-particle cryo-electron microscopy, the technique pioneered in the PI's lab, is used in collaborations with world specialists on bacterial and eukaryotic translation.

We make use of two techniques of sample preparation, standard and time-resolved cryo-EM. In the standard application of cryo-EM, samples are pipetted onto the grid, excess liquid is removed by blotting, and the grid is then plunged into the cryogen. Since this procedure requires several seconds, it is not possible to capture short-lived (less than 1000 millisecond) states of a molecule following a reaction.

The alternative technique developed in this lab is time-resolved cryo-EM, whereby a reaction is started by mixing two components in a microfluidic chip, allowing them to react in a channel of defined, variable length (10 to 1000 ms), and then spraying the reaction products onto the grid as the latter is plunged into the cryogen. In this way, the kinetics of a reaction can be followed, and at the same time, intermediate states can be captured and visualized at high resolution.

These two techniques are used to study the following processes: translation initiation in E. coli and yeast, translation termination, recycling and quality control in mammalian, EMCV virus takeover of the host's ribosome.

Another objective of the proposed studies is the exploration of a novel method of data analysis that seeks to generate a low-dimensional map of states existing in a continuum from a large dataset of single-particle cryo-EM images of a biological macromolecule. Such a mapping can be used to determine the free-energy landscape of the molecule, containing information on the function-related conformational trajectories. This method will be applied in collaborations with leading experts to two membrane-associated molecules with eminent biological and public health significance: rotary ATPase and cystic fibrosis transmembrane conductance regulator (CFTR).

The Trustees Of Columbia University In The City Of New York

THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES (NIGMS) SUPPORTS BASIC RESEARCH THAT INCREASES OUR UNDERSTANDING OF BIOLOGICAL PROCESSES AND LAYS THE FOUNDATION FOR ADVANCES IN DISEASE DIAGNOSIS, TREATMENT, AND PREVENTION. NIGMS ALSO SUPPORTS RESEARCH IN SPECIFIC CLINICAL AREAS THAT AFFECT MULTIPLE ORGAN SYSTEMS: ANESTHESIOLOGY AND PERI-OPERATIVE PAIN, CLINICAL PHARMACOLOGY ?COMMON TO MULTIPLE DRUGS AND TREATMENTS, AND INJURY, CRITICAL ILLNESS, SEPSIS, AND WOUND HEALING.? NIGMS-FUNDED SCIENTISTS INVESTIGATE HOW LIVING SYSTEMS WORK AT A RANGE OF LEVELSFROM MOLECULES AND CELLS TO TISSUES AND ORGANSIN RESEARCH ORGANISMS, HUMANS, AND POPULATIONS. ADDITIONALLY, TO ENSURE THE VITALITY AND CONTINUED PRODUCTIVITY OF THE RESEARCH ENTERPRISE, NIGMS PROVIDES LEADERSHIP IN SUPPORTING THE TRAINING OF THE NEXT GENERATION OF SCIENTISTS, ENHANCING THE DIVERSITY OF THE SCIENTIFIC WORKFORCE, AND DEVELOPING RESEARCH CAPACITY THROUGHOUT THE COUNTRY.

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

New York, New York 10032 United States

Single Zip Code

Maximizing Investigators' Research Award (R35 - Clinical Trial Optional) (PAR-19-367)

Amendment Since initial award the total obligations have increased 506% from $640,351 to $3,880,563.