A strategy for dissecting the architectures of native macromolecular assemblies

The molecular architecture of protein complexes can be determined using an optimal approach for isolating GFP-tagged complexes at native levels, combined with cross-linking, mass spectrometry analysis, and structure modeling from mass spectrometry-derived distance restraints. It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.