Extended Data Fig. 3: PfPTRAMP and PfCSS form a complex and the PfCSS C30 residue appears to be essential.

a. Volcano plot illustrating the log₂ protein ratios of immunoprecipitated PfCSS-HA proteins versus 3D7 control as analysed by mass spectrometry analysis. Differential protein expression analysis was performed using Limma which involves a moderated t-test. Benjamini and Hochberg’s method was used to adjust the p-values for multiple testing. Proteins were deemed differentially regulated in the log2 fold change in protein expression was 1-fold and exhibited an adjusted p-value ≤ 0.05. N = 3 biologically independent samples used. b. Scheme to construct P. falciparum lines that express PfCSS with the amino acid Cys30 mutated to Ser30 using CRISPR. Shown is the Cas9 cleavage site (red) near the protospacer adjacent motif and the resulting recombination event that replaces the endogenous pfcss gene with one mutated to encode Ser30. Both constructs included a HA-tag near the N-terminus of the PfCSS protein. In the grey box is the expected amino acid sequence expected after each insertion event. The HA-tagged pfcss gene that retained expression of C30 but inserted a HA-tag was successfully obtained and confirmed by sequencing (bottom panel). Parasites from multiple transfections with the construct that was identical to the former but would result in mutation of C30 to S30 was not successfully obtained. Therefore, we conclude that the C30 amino acid and the disulfide bond with PfPTRAMP was essential.