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Methods for time-resolved cryo-EM using pulsed infrared radiation and vapour phase deposition https://doi.org/10.17863/CAM.125300 Grigoras, Ioana Russo, Christopher Electron cryo-microscopy (cryo-EM) is an imaging technique for determining high spatial resolution structures of proteins. Technological advances have made it possible to attain atomic resolution and attention may now be directed towards the challenge of extending the time resolution of cryo-EM from milliseconds to microseconds. If cryo-EM had the additional capability of high temporal resolution, transient conformational states of proteins could be imaged. Here, I present a laser melting and revitrification apparatus for controlling the phase of the water in cryo-EM specimens using pulsed infrared radiation. I demonstrate rapid melting and revitrification, and I show that it is possible to determine high spatial resolution structures of proteins from cryo-EM specimens that have been melted and revitrified using 3 μm laser pulses. I also present a cryogenic deposition instrument for preparing an initial, out-of-equilibrium state using vapour phase deposition. I demonstrate that it is possible to coat a cryo-EM specimen with a thin film of small molecules. GABA_A receptors, ion channels with a rich pharmacology, were chosen as a biological system for testing the two first-generation time-resolved cryo-EM instruments. I describe experiments conducted to explore the interactions of GABA_A receptors with various ligands using vapour deposition followed by laser pulsing. This work represents a step in the direction of realising a toolkit for time-resolved cryo-EM for studying microsecond dynamics associated with protein function. University of Cambridge 2025-09-30 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/395971 embargo 2027-01-29 https://www.repository.cam.ac.uk/bitstreams/d80fd0b1-87d8-402a-b872-b6a9991d061e/download 868786178bb39a74d6a5c50013bf374f https://www.repository.cam.ac.uk/bitstreams/71c7d03e-5016-488f-81b0-4bf962833144/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved time-resolved cryo-EM pulsed IR melting and revitrification GABAA receptors