Protein complexes can be modeled with data arising from crosslinking mass spectrometry, but it is difficult to collect such data from cellular sources, particularly for proteins that are part of extensive interaction networks. Here, we present a tandem-affinity purification crosslinking mass spectrometry (xTAP‑MS) method that enables structural analysis of low-stoichiometry complexes directly from cellular lysate. Applying this approach to the DNA-PK/protein phosphatase 6 (PP6) interaction, we selectively enriched endogenous DNA-PK/PP6 assemblies and generated inter- and intra-protein crosslinks suitable for the generation of a working model of the structure. Integrating crosslinking data with AlphaFold3 modeling positions the PP6 holoenzyme at the base of the DNA-PKcs HEAT-repeat cradle, proximal to a major autophosphorylation loop yet spatially compatible with kinase activation. Structure-guided truncations and mutagenesis of PPP6R3 are consistent with the model. Together, these results establish xTAP‑MS as an effective bridge between interactome discovery and mechanistic structural analysis, generating testable hypotheses for subsequent investigations.
