Readily deployable technology for reproducible cryogenic sample time-resolved electron microscopy (cryotrem)

The University of Colorado received a $2.11M Project Grant from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), awarded September 1, 2025, with a completion date of August 31, 2029. The project focuses on developing and deploying readily deployable technology for reproducible cryogenic sample time-resolved electron microscopy (CryoTREM). The primary deliverable is a patterned LED/LCD-based illumination system capable of capturing multiple temporal states on a single electron microscopy grid across a four-order-of-magnitude time range from 1 to 10,000 milliseconds. The resulting time-resolved datasets will be integrated with various data processing methods to determine ensembles of time-resolved structures and understand molecular mechanisms in multiple biological systems.

The technology will be applied to several biological systems including ligand-gated ion channels, lipid transferase, V-ATPase, and synaptic vesicle fusion, generating time-resolved structural data with intermediate conformational states. As a sub-awardee, the University of California, San Diego's Hibbs laboratory will test the CryoTREM uncaging instrument on GABAA and α7 nicotinic receptors, purify receptors for cryo-EM analysis, optimize the uncaging device using collected test datasets, and collaborate with the lead institution on data analysis and interpretation to support effective instrument optimization.

Abstract

This advanced technology grant focuses on enhancing cryogenic sample time-resolved electron microscopy (cryotrem) through the development of a patterned led/LCD-based illumination system capable of capturing multiple temporal states on a single EM grid with 4 orders of magnitude time range from 1 to 10,000 milliseconds. This system will be developed and deployed in producing time-resolved data sets for several different biological systems with intermediate states with varying degree of conformational magnitude. The resultant data will be integrated with a variety of data processing methods to determine ensembles of time-resolved structures, which can be leveraged to understand molecular mechanisms. These time-resolved EM experiments will be conducted on multiple biological systems and processes including ligand-gated ION channels, a lipid transferase, the v- atpase, and synaptic vesicle fusion.