Single particle reconstruction (SPR) analysis of cryoelectron microscopy (cryoEM) data is a rapidly growing method for determining the near-atomic molecular structures of biological samples. This burgeoning interest stems from the benefits of the SPR technique, including [1] greatly reduced demand for sample (<100 g vs. > 10 mg for X-ray diffraction or NMR analysis); [2] the capacity to image large, multi-protein and/or dynamic complexes that are cryofixed in a submicron thick layer of vitreous ice; and [3] improved safety in instances where the sample is of pathogenic origin. Nonetheless, SPR requires large single particle populations, automated class-averaging methods, and advanced computational approaches to achieve near-atomic resolution. Increased sampling is partially addressed by microscope automation, particle picking, and direct detection; however, improvements in sample preparation and reliability are still lacking despite efforts to develop ?affinity grids?, modified graphene, SiN, Au, DNA origami, and engineered 2D protein arrays. This proposal seeks to accelerate the productivity of cryoEM SPR through the development of novel materials that will concentrate the biological sample onto a rod-like scaffold prior to deposition onto the TEM grid. We will achieve this objective by integrating molecular theory, molecular dynamics, and experimental studies in Aims 1 and 2 to design rod-like materials that actively discourage non-specific protein adsorption, while enhancing the capture and random presentation of target proteins on the rod-like scaffold. Materials conferring these properties will then be tested in Aim 3 for their capacity to capture only the target protein from complex mixtures such as cell lysates to elucidate the structures of His-GroEL as a standard and His-p97 in complex with a client protein via cryoEM SPR at near-atomic resolution.

Public Health Relevance

The proposed research will yield a family of molecularly-designed cryoEM sample preparation tools capable of accelerating near-atomic resolution protein structure elucidation by concentrating and depositing target proteins isolated from complex mixtures in multiple orientations onto EM grids for SPR analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21GM127958-02
Application #
9787549
Study Section
Biomaterials and Biointerfaces Study Section (BMBI)
Program Officer
Wu, Mary Ann
Project Start
2018-09-18
Project End
2020-08-31
Budget Start
2019-09-01
Budget End
2020-08-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Purdue University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907