Methods for time-resolved cryo-EM using pulsed infrared radiation and vapour phase deposition

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Grigoras, Ioana

Advisor dc:contributor.advisor

• Russo, Christopher

• time-resolved cryo-EM
• pulsed IR melting and revitrification
• GABAA receptors

Licence

• https://www.repository.cam.ac.uk/bitstreams/71c7d03e-5016-488f-81b0-4bf962833144/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.125300

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/395971