About the practical course
This EMBO Practical Course aims to teach participants how to expedite structural biology projects involving macromolecular complexes by combining multiple experimental approaches. In particular, the course will cover the techniques used to produce, purify, reconstitute and characterize multi-subunit protein and protein/nucleic acid complexes for structural analysis.
Topics include: multi-subunit expression in bacteria, insect and mammalian cells; affinity, electrophoretic and centrifugation methods to isolate and purify sub-complexes and holo-complexes; 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:
(i) the latest advances in isolating complexes from endogenous sources, in recombinantly co-expressing binary and higher-order combinations of complex components, and in reconstituting complexes from individually purified components or sub-complexes;
(ii) techniques for assessing and improving sample quality (homogeneity, stoichiometry, monodispersity);
(iii) combining structure determination methods (crystallography, cryo-electron microscopy, small-angle scattering, NMR , native mass spectrometry, etc.) to obtain a multi- resolution view of a given complex.