Cryo-EM Seminar @ ITQB NOVA - Julia Mahamid

Title: Enabling discovery by in-cell structural biology

Speaker: Julia Mahamid

Affiliation: European Molecular Biology Laboratory (DE)

Abstract: Most structural biology focuses on the structure and function of individual macromolecular complexes, but falls short of revealing how they come together to give rise to cellular functions. Here, cryo-electron tomography (cryo-ET) provides a unique opportunity for obtaining structural information across a wide range of spatial scales - from whole cells to individual macromolecules embedded in their native functional environment. We develop and employ advanced sample preparation techniques for in-cell cryo-ET, including cryo-focused ion beam thinning guided by three-dimensional correlative fluorescence microscopy. Preparations of site-specific ‘electron-transparent windows’ in cellular model systems enable assignment of molecular structures directly from three-dimensional stills of intact cells and reveal their molecular sociology. Using the genome-reduced human pathogen Mycoplasma pneumoniae as a minimal cell model, we further demonstrate the synergistic application of whole-cell crosslinking mass spectrometry and cellular cryo-ET to determine an in-cell integrative model of actively transcribing RNA polymerases coupled to a translating ribosomes. Recent computational breakthroughs now allow resolving these molecular machines to residue-level, reveal small molecule antibiotics bound to their active site within the intact pathogen, and provide snapshots of their structural dynamics along reaction cycles. These methodologies unlock an enormous potential for novel discovery enabled by label-free in-cell structural biology.

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