Fig. 1: Mapping the proximal environment of kinetochore kinases KKT2, KKT3, CLK2 with XL-BioID.

a Schematic of XL-BioID. The addition of exogenous biotin initiates in vivo biotinylation of proteins proximal to the BirA* tagged bait protein. A membrane permeable cross-linker, DSP, is then used in a second in vivo proximity capturing step to covalently link any proximal proteins that escaped biotinylation. Biotin tagging and covalent linking survive harsh lysis enabling the complex to be solubilised for affinity purification. b Comparison of kinetochore protein amounts purified in conventional BioID and XL-BioID. Label-free intensities of kinetochore proteins (KKTs) were summed and normalised to the amount of purified bait (BirA*::KKT3). Median and standard deviation are shown for three independent replicates. c Fragmentation spectra matching KKTs in BioID and XL-BioID. A median number of spectra matching to KKTs from three independent replicates. d KKT2, KKT3, CLK2-proximal proteins identified with XL-BioID. Enrichment against a BirA*-BDF5 spatial reference is plotted. Red points are proximal proteins, grey points are non-proximal. The dashed line indicates a 1% false discovery rate (FDR) threshold. e Selected KKT2, KKT3 and CLK2-proximal proteins. Ninety-eight proteins were significantly enriched and are classed as proximal at 1% (FDR). Only those proteins with annotated functions are shown. Edges indicate bait-prey associations.