Modeling Apo-Closed and Inward-Facing ABCG2 Conformations

In order to model the apo-closed conformation, the high-resolution structure of human ABCG2 bound to the 5D3 Fab and MZ29 inhibitor (PDBid 6ETI) was used as an initial template for model building and refinement. The 5D3 Fab and all ligands were removed from the structure, leaving just the ABCG2 TM and NBD domains. TM5 was manually shifted and rotated to match the proper helical character and sequence register observed in the closed conformation map. Similarly, the unraveled portion of TM2 was manually adjusted in COOT to fit the apo-closed conformation map. The model was then iteratively adjusted manually in COOT and refined in real-space with phenix.real_space_refine.
The structure of human ABCG2 bound to the 5D3 Fab and MZ29 inhibitor (PDBid 6ETI) was also used as an initial template for model building and refinement for inward facing ABCG2 structures. The 5D3 Fab and all ligands were removed from the structure, leaving just the ABCG2 TM and NBD domains. The resulting model was rigid body fit in UCSF Chimera^{48 (link)} into the inward facing conformation map of ABCG2 bound to imatinib, MXN, or SN38 and then refined by iterative rounds of manual adjustment in COOT^{49 (link)}, with manual placement and real-space refinement of ligands, followed by real-space refinement in phenix.real_space_refine^{50 (link)}. Chemical descriptions and refinement restraints for MXN and imatinib were obtained from the CCP4^{51 (link)} monomer library and phenix.ready_set. A molecule of SN38 was manually built in JLigand^{52 (link)}, and refinement restraints were generated in phenix.ready_set. Final calculation of map vs model FSC was performed by first simulating a map from the refined atomic models to Nyquist frequency using UCSF Chimera, and then using this simulated map to calculate FSC with the final map from Relion autorefine.