About the Practical Course
This EMBO Practical Course aims to teach participants how to integrate structural biology approaches to expedite characterization of large macromolecular complexes from the atomic to cellular scale. Lectures and practical sessions will explain the techniques used to produce, purify, reconstitute and characterize multi-subunit protein and protein/nucleic acid complexes for structural analysis.
Specifically, the course will cover multi-subunit protein expression in bacteria, insect and mammalian cells; production, folding, and purification of non-coding RNAs; affinity, electrophoretic and centrifugation methods to isolate and purify sub-complexes and holocomplexes; biochemical and biophysical experiments that inform on the stoichiometric and conformational homogeneity of complexes; and strategies for determining the overall 3D architecture of large macromolecular assemblies and ultimately their structure at atomic or pseudo-atomic resolution.
Special emphasis will be placed on:
- the latest advances in producing targets directly from endogenous or recombinant sources or from cell free approaches (including production of RNA by in vitro transcription), and in reconstituting complexes from individually purified components or sub-complexes;
- techniques for assessing and improving sample quality (homogeneity, stoichiometry, monodispersity); and
- integration of structure determination methods (crystallography, cryo-electron microscopy, small-angle scattering, NMR , native mass spectrometry, FRET, super-resolution light microscopy) to obtain a multi-resolution view of a given complex.