Recombinant protein preparation and cloning were done using standard methods. In immunofluorescence experiments, we transfected cells with Fugene 200 or microinjected them with 10 nM protein, where indicated. We performed kinase assays with rabbit pEGFP–PAK2, which was immunoprecipitated from 293T cell lysates and incubated with the protein of interest in the presence of MBP, 10 µM ATP and 5 µCi γ32P[ATP]. Reactions were stopped by the addition of SDS buffer, separated by SDS–polyacrylamide gel electrophoresis and kinase activity was measured as 32P counts per minute. EspG–ARF6 and EspG–PAK2–Iα3 were purified by ion exchange and gel filtration chromatography and crystallized by the hanging-drop vapour diffusion method. We collected X-ray diffraction data at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory (USA). The structure of EspG–ARF6 was phased to a resolution of 2.5 Å by the multiwavelength anomalous dispersion method using selenomethionine-labelled EspG and ARF6 proteins. The EspG–PAK2 structure was solved to a resolution of 2.8 Å by the molecular replacement method using the EspG monomer of the EspG–ARF6 structure as the initial search model. Further details can be found in Supplementary Information.
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