X-ray crystallography
Initial crystallization conditions for single effector proteins and all protein complexes described here were determined with the JSG Core I-IV, Pact and Protein Complex suites from Qiagen. The sitting-drop vapor diffusion method was used, with a reservoir volume of 70 μl and a drop volume of 0.1 μl protein (15–25 mg ml−1) and 0.1 μl reservoir solution at 20°C. The best conditions were then optimized using the hanging-drop vapor diffusion method in order to obtain well diffracting crystals. The seleno-L-methionine labelled Mical-31841–1990 was finally crystallized in 0.1 M Tris pH 7.0 und 45 – 50% PEG 200 (protein concentration 5.5 – 11 mg/ml). Mical-cL534–683 in a 1 to 1 complex ratio with all tested Rab proteins crystallized in similar conditions. The complex with Rab1bFl was crystallized in 0.1 M bis-tris-propane pH 8.4–8.6, 0.2 M tri-sodium citrate and 20–22% (w/v) PEG 3350, with Rab8aFl in 0.1 M bis-tris-propane pH 8.3–8.7, 0.2 M tri-sodium citrate and 18–20% (w/v) PEG 3350 and finally with Rab10Fl in 0.2–0.3 M sodium acetate and 18–22% (w/v) PEG 3,350. The hybrid Rab1bR8N (chimera) in complex with Mical-cL534–683 was crystallized in 0.1 M bis-tris pH 7.5, 0.2 M sodium malonate and 20% (w/v) PEG3350. Mical-1918–1067 crystallized with Rab101-175 in a 1 to 2 ratio under the following conditions: 0.1 M imidazole pH 7.6–8.0 and 6–10% (w/v) PEG 8,000.
Best diffracting crystals were flash-cooled in liquid nitrogen and diffraction data were collected on beamline X10SA at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) and processed with XDS (Kabsch, 2010). The structure of Mical-31841–1992 was solved by the single anomalous diffraction method using data collected at the selenium absorption edge. Initial phases and an initial model were obtained with PHENIX AutoSol (Adams et al., 2010). All protein complex structures were solved by the maximum likelihood molecular replacement method using the structures of Mical-31841–1992, Mical-cL534–683, Rab1b (pdb id 3nkv) and Rab8a (pdb id 4lhw) as search models. The initial structure models were completed by hand in Coot (Emsley et al., 2010) and refined with phenix.refine (Adams et al., 2010) or Refmac5 (Murshudov et al., 1997) of the CCP4 package (Winn et al., 2011) using the TLS option. Data collection and refinement statistics, as well the Protein Data Bank accession numbers of each presented structure are summarized in Table 1.