The advent of cryogenic-electron microscopy (cryo-EM) as a key tool for structural
biologists has brought forth a whole range of applications to gain structural information at
high-resolution. Cryo-EM single-particle analysis (SPA) approaches typically entail
overexpression and purification of the target protein. Larger and more complex molecular
assemblies often require extensive optimisation of expression, purification and
reconstitution procedures. Additionally, prior knowledge of the composition of the structure
of interest is required. In-situ approaches employing cryo-focused ion beam (FIB) milling
and cryo-electron tomography (cryo-ET) have proven incredibly useful in exploring protein
structures within cells, in some cases even at high-resolution. Such in-situ strategies do
not require purification of the target protein or protein complex yet are often still limited in
throughput and achievable resolution. During my talk, I will show how we expand the
range of samples attainable for SPA towards more native samples, specifically towards
complex macromolecular assemblies that cannot be reconstituted, and show how SPA is
used as a discovery tool for de-novo protein identification.