Structure determination

All datasets were collected at 100 K at the Australian Synchrotron MX2 beamline using an ADSC quantum 315R CCD detector (BCL-2 WT:venetoclax and BCL-2 F104L:venetoclax only) or an Eiger X 16M direct detector (all other datasets)³². Diffraction experiments were processed in XDS³³ and scaled in either XDS or Aimless³⁴. The phase problem was solved by molecular replacement using Phaser³⁵ and the BCL-2 WT:navitoclax structure chain A with all waters and ligands removed (PDBid 4LVT) as a search model¹⁰. The model was refined by iterative reciprocal and real space refinement using PHENIX refine³⁶ and Coot³⁷, respectively with at least one round of cartesian simulated annealing refinement in PHENIX prior modelling ligands to avoid phase bias. Ligand initial models and restraints for venetoclax and {"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 were generated using the Grade web server (version 1.2.9, Global Phasing Ltd.). For atoms with multiple conformations initial occupancies were set to 0.5 modelling each conformer into appropriate density prior to multiple rounds of refinement in phenix using the refine occupancy function in addition to standard refinement procedures. X-ray data to geometry weights or atomic displacement factors were determined automatically using the optimize X-ray/stereochemistry weight and optimise X-ray/ADP weight respectively. Model validation was performed in Coot and MolProbity³⁸. Data statistics were calculated in PHENIX using the generate Table 1 for journal function. Stereo images with electron density are displayed in Supplementary Fig. ⁷.

The BCL-2 G101V:venetoclax data processed and solved in the orthorhombic spacegroup P2₁2₁2₁ with a single protein chain in the assymetric unit. However, during refinement R_fact and R_free increased in successive refinement cycles, giving final values that were unreasonable when compared to similar resolution structures from the protein data bank. This did not occur when the structure was modelled in the monoclinic spacegroup P2₁ with two protein chains in the assymetric unit. As a consequence, the monoclinic model and data were used.

The BCL-2 G101V:{"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 structure processed in the monoclinic spacegroup P2₁ with two protein chains in the assymetric unit. The data were anisotropic with data extending to 2.7 Å in the a* and 2.0 Å in the b* and c* directions, and were ellipsoidally truncated and scaled by the diffraction anisotropy server³⁹ without applying B-factor sharpening. After applying ellipsoidal truncation the data completeness in high resolution shells dropped, with completeness dropping below 90% from 2.5–2.0 Å. Furthermore, the electron density for {"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 in one of the protein chains was more poorly resolved. This copy of {"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 was modelled into the electron density according to the {"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 orientation from the BCL-2 WT:{"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 structure (PDB id 6GL8)¹². To avoid phase bias no coordinates from the original WT:{"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"2205"}}S55746 structure were used in refinement and orientations were matched by protein alignments and visual inspection only. The analysis presented here relates to the protein chain without this problem.