Computational Advances in Structural Biology

Recent developments in single-particle cryo-EM have led to an explosion of structural insights into the molecular machines that replicate, repair, and transcribe eukaryotic genomes; however, static structures alone cannot reveal how these complex machines function in their biochemical context. Inspired by progress in cryo-tomography and single molecule imaging, we set out to develop single-particle EM-based ‘visual biochemistry’ tools to determine the molecular mechanisms underpinning the first step in eukaryotic DNA replication – loading of the MCM helicase onto DNA. To this end, we reconstituted MCM loading in vitro using chromatinised origins of replication and performed time-resolved EM experiments to identify new MCM loading intermediates and quantify their assembly and disassembly over time. Next, we performed ‘in silico reconstitution’ to generate images of hundreds of complete origins of replication at distinct stages of the MCM helicase loading process. Combined, these experiments showed both when (temporal order) and where (relative to specific origin features) each intermediate in the pathway forms, which enabled us to define a sequential and coordinated mechanism for MCM loading. In my talk, I will describe the development and use these EM-based visual biochemistry tools to reveal the mechanisms of MCM loading and will touch on future perspectives for investigating other multicomponent biochemical reactions that occur on flexible substrates.