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Higher-order multi-protein complexes such as RNA poly-merase II (Pol II) complexes with transcription initiationfactors are often not amenable to X-ray structure determina-tion. Here, we show that protein cross-linking coupled tomass spectrometry (MS) has now sufficiently advanced as atool to extend the Pol II structure to a 15-subunit, 670kDacomplex of Pol II with the initiation factor TFIIF at peptideresolution. The N-terminal regions of TFIIF subunits Tfg1and Tfg2 form a dimerization domain that binds the Pol IIlobe on the Rpb2 side of the active centre cleft near down-stream DNA. The C-terminal winged helix (WH) domains ofTfg1 and Tfg2 are mobile, but the Tfg2 WH domain canreside at the Pol II protrusion near the predicted path ofupstream DNA in the initiation complex. The linkers be-tween the dimerization domain and the WH domains inTfg1 and Tfg2 are located to the jaws and protrusion,respectively. The results suggest how TFIIF suppressesnon-specific DNA binding and how it helps to recruitpromoter DNA and to set the transcription start site. Thiswork establishes cross-linking/MS as an integrated struc-ture analysis tool for large multi-protein complexes.The EMBO Journal (2010) 29, 717–726. doi:10.1038/emboj.2009.401; Published online 21 January 2010Subject Categories: chromatin & transcriptionKeywords: higher-order protein complex; integrated structureanalysis; mass spectrometry; multi-dimensional structureand dynamics of biological macromolecules; transcriptionand its regulation