High-resolution cryo-electron microscopy and its application to challenging targets

In order to enable biological discovery, we need to address challenges that arise during structure determination of biomolecular complexes by cryo-electron microscopy (cryo-EM). We are particularly interested in three challenges:

Structural flexibility: The molecular assemblies that we are working with are flexible. Some exhibit intrinsic continuous flexibility, others require the ability to undergo structural transitions to perform their biological function. We aim to exploit the ability of cryo-EM to visualise flexible assemblies and combine these results with biophysical methods to create "molecular movies" of the processes we study.

​Fragile complexes: Interaction of molecular complexes with the air-water interface in the extremely thin film of sample that is generated during cryo-EM specimen preparation can lead their denaturation and destruction. We aim to exploit available techniques to circumvent these issues and obtain structures of intact and fully functional fragile molecular assemblies.

Cryo-EM map of the human CDK-activating kinase, with CDK7 shown in grey, cyclin H in brown, and MAT1 in orange. The map encompasses approximately 85 kDa of protein mass. At the time of publication, this was one of the smallest asymmetric protein complexes resolved to better than 3 Å by cryo-EM.

Greber BJ#, Perez-Bertoldi J M, Lim K, Iavarone AT, Nogales E# (2020). The Cryoelectron Microscopy Structure of The Human CDK-Activating Kinase. Proc. Natl. Acad. Sci. U.S.A. 549 (7672): 414-417. doi: 10.1073/pnas.2009627117. #corresponding authors PubMed

High resolution structure determination of small complexes: Cryo-EM has the proven potential to achieve true atomic resolution for relatively large and rigid biomolecular complexes. However, many therapeutically relevant proteins or protein complexes are comparably small and more difficult to visualise at high resolution. At the same time, applications such as structure-based drug design benefit from high-resolution structural data. We are therefore interested in high-resolution structure determination of small complexes. Our work on the CDK-activating kinase is the first example of these efforts.

Cryo-EM for structure-based drug design

Due to its involvement in the regulation of transcription and the cell cycle, the human CDK-activating kinase is a promising target for cancer therapeutics, and several compounds have entered clinical trials. To enable structure-based discovery of next-generation therapeutics, we are working to improve the resolutions of our cryo-EM maps and determine structures in complex with clinical inhibitors.

Cryo-EM density of human CDK-activating kinase at approx. 2.5 Å resolution. At this resolution, side chain rotamers and carbonyl positions become discernible. The map shown here was computed from approx. 200,000 particles selected from a dataset of more than 10 million initial particle picks.

We are currently collaborating with the laboratories of Prof. Simak Ali and Prof. Matthew Fuchter at Imperial College on this topic.