Figure 1.
Cryo-EM structures of IrtAB from Mtb. (A) Representative 2D classification averages showing full-length IrtAB in different orientations. The SID of IrtA is observed in some views indicated by the red boxes. (B) Cryo-EM map of full-length IrtAB. IrtA is in slate, IrtB is in salmon, the detergent micelle is in grey. (C) Cartoon representation of inward-facing IrtAB viewed from the plane of the membrane. The membrane is shown in grey. IrtA and IrtB are in slate and salmon, respectively. The cavity of IrtAB is shown as a grey surface. (D) Superposition of ATP-free (yellow) and one AMP-PNP-bound (IrtA, slate; IrtB, salmon) structures. The ATP analogue AMP-PNP is drawn as spheres and binds to the NBD of IrtA. (E) Superimposition of IrtA (slate) and IrtB (salmon) structures. The transmembrane helices are numbered. (F) The asymmetric opening of the NBD interface. The distances between equivalent residues in the two ATPase sites are indicated. The Walker A and the ABC signature motifs in NBDs are coloured red and orange, respectively. (G) View of the NBDs of IrtAB without nucleotide bound from the cytoplasm. The NBDs are shown as transparent surface and in cartoon representation. The density map of C-terminal extension of the NBD of IrtB (red cartoon) is shown as grey mesh and contoured at 10 σ. (H) View of the TM region interface between IrtA and IrtB. Non-protein cryo-EM map density contoured at 7 σ is shown as green mesh. (I) Cartoon representation of NBD of IrtA binding AMP-PNP. The Walker A and A-loop are coloured red and green, respectively. The density map of AMP-PNP is shown as grey mesh and contoured at 9 σ. (J) The ATPase activities of five IrtAB variants and SID-truncated IrtAB (IrtABΔSID) were normalized relative to that of the wild-type (WT) IrtAB. Error bars represent mean ± SD based on three independent measurements.
