Validation of AlphaFold modeling of the PIKfyve complex. (A) Flow path for processing the AlphaFold3 search of PIKfyve with Fig4 and two copies of Vac14. The model is colored by chain to identify each protein, then colored by pLDDT to indicate regions of high and low confidence. The final panel shows the structure used in Fig. 1 A, where residues with a pLDDT score <50 and domains that do not form high-confidence contacts with the complex have been deleted. (B) Flow path for processing the AlphaFold3 search of Fig4 and five copies of Vac14. The model is colored by chain to identify each protein, then colored by pLDDT to indicate regions of high and low confidence. The final panel shows the structure used in Fig. 1 A, where residues with a pLDDT score <50 and domains that do not form high-confidence contacts with the complex have been deleted. (C) PAE for AlphaFold3 search of PIKfyve with Fig4 and two copies of Vac14. The colors indicate the PAE and are colored according to the legend. Note that the PAE plot is not an inter-residue distance map or a contact map. Instead, the red-blue color indicates the expected distance error. The color at (x, y) corresponds to the expected distance error in residue x’s position (Angstroms), when the predictions are aligned on residue y (more information can be found at https://alphafold.ebi.ac.uk/). The regions highlighted in gray indicate domains that have been deleted because they do not form high-confidence interfaces with the complex. (D) PAE for AlphaFold3 search of Fig4 and five copies of Vac14. The colors indicate the PAE and are colored according to the legend. Note that the PAE plot is not an inter-residue distance map or a contact map. Instead, the red-blue color indicates the expected distance error. The color at (x, y) corresponds to the expected distance error in residue x’s position (Angstroms), when the predictions are aligned on residue y (more information can be found at https://alphafold.ebi.ac.uk/). The regions highlighted in gray indicate domains that have been deleted because they do not form high-confidence interfaces with the complex. (E) Flow path for how the full model of the PIKfyve complex was generated for Fig. 1 A. The searches described in A and B were aligned at Fig4, and the two overlapping Vac14 arms were deleted from the Vac14 pentamer. PAE, predicted aligned error.