To determine the structure of the SNX5-IncE complex we generated a fusion protein encoding the human SNX5 PX domain (residues 22–170) and C. trachomatis IncE C-terminal sequence (residues 108–132) attached at the PX domain C-terminus Figure 4—figure supplement 1A). This construct readily crystallised in several crystal forms, and we were able to determine the structure of the complex in three different spacegroups (Figure 4; Table 3; Figure 4—figure supplement 1B). Confirming that the fusion does not alter complex formation, the short linker region is disordered, and the mode of IncE-binding to SNX5 is identical in all three structures (Figure 4—figure supplement 1C and D). Because of the higher resolution, we focus our discussions on the structure of the SNX5 PX-IncE complex observed in the P212121 crystal form. The first three IncE N-terminal residues (Pro107, Ala108, Asn109) and the last three IncE C-terminal residues (Val130, Thr131, Gln132) were not modeled due to lack of electron density, suggesting disorder and matching precisely with our mapping experiments showing these residues are not necessary for SNX5 association.10.7554/eLife.22311.011Figure 4.Structure of the SNX5 PX domain in complex with the IncE C-terminal domain.(A) Crystal structure of the SNX5 PX domain (yellow) in complex with IncE residues 107–132 (magenta) shown in cartoon representation. (B) Backbone atoms of the SNX5 and IncE proteins are shown to highlight the prominent β-sheet augmentation mediating the association between the two molecules. (C) Close up view of the SNX5-IncE interface highlighting specific contact areas at the N-terminus of the IncE peptide. (D) Close up of the SNX5-IncE interface highlighting specific contact areas at the hairpin loop of the IncE peptide shown at 90° to Figure 4C. (E). Close up of the SNX5-IncE interface highlighting contact areas at the C-terminus of the IncE peptide in approximately the same orientation as Figure 4C. Residues in SNX5 (Phe136) and IncE (Phe116) that are critical for binding based on mutagenesis are boxed.DOI: http://dx.doi.org/10.7554/eLife.22311.01110.7554/eLife.22311.012Figure 4—figure supplement 1.Supplementary images for SNX5-IncE crystal structures.(A) Sequence of the SNX5 PX domain fusion protein with the IncE C-terminal peptide. (B) Refined 2fo-fc electron density contoured at 1.5σ for the SNX5-IncE structure in spacegroup P212121. (C) Overlay of each independent SNX5-IncE complex observed in the three crystal forms. (D) Ribbon structures indicating the locations of the linker regions in each crystal form. The C-terminal SNX5 residues and the N-terminal IncE residues are shown by spheres with distances indicated.DOI: http://dx.doi.org/10.7554/eLife.22311.01210.7554/eLife.22311.013Table 3.Summary of crystallographic structure determination statistics*.DOI: http://dx.doi.org/10.7554/eLife.22311.013CrystalSNX5 PX-IncE Form 1SNX5 PX-IncE Form 2SNX5 PX-IncE Form 3PDB ID5TGI5TGJ5TGHData collectionWavelength (Å)0.953700.953700.95370Space groupP212121I2P32Cell dimensions a, b, c (Å)60.7, 67.5, 88.258.4, 80.3, 94.6100.6, 100.6, 71.7 α, β, γ (°)90, 90, 9090, 97.2, 9090, 90, 120Resolution (Å)60.7–1.98 (2.03–1.98)31.9–2.6 (2.72–2.60)50.3–2.80 (2.95–2.80)Rmerge0.104 (0.525)0.153 (0.659)0.101 (0.713)Rmeas0.112 (0.572)0.18 (0.777)0.124 (0.873)Rpim0.042 (0.225)0.096 (0.408)0.051 (0.363)<I> / σI12.4 (3.4)39.6 (3.2)11.7 (2.3)Total number reflections178868 (11000)46691 (5757)115149 (16861)Total unique reflections26075 (1805)13432 (1632)20001 (2923)Completeness (%)100 (100)99.9 (100.0)100 (100)Multiplicity6.9 (6.1)3.5 (3.5)5.8 (5.8)Half-set correlation (CC(1/2))0.997 (0.868)0.986 (0.55)0.997 (0.683)RefinementResolution (Å)45.1–1.98 (2.02–1.98)31.9–2.6 (2.69–2.60)41.2–2.8 (2.87–2.80)No. reflections/No. Rfree26021/200013421/1342 (1208/134)19975/1972 (1301/144)Rwork/Rfree0.192/0.214 (0.221/0.246)0.199/0.242 (0.276/0.332)0.236/0.254 (0.329/0.372)No. atoms Protein257926195189 Solvent281690Average B-factor (Å2)31.842.556.0R.m.s deviations Bond lengths (Å)0.0120.0110.015 Bond angles (°)1.271.151.27*Highest resolution shell is shown in parentheses.