Figure 1: A deep mutational scan individually characterizes all single amino-acid mutations in rubisco.

A) A summary of the metabolism of Δrpi the rubisco-dependent strain. B) Δrpi grows with a rate that is proportional to the flux through rubisco. C) Schematic of the library selection. A library of rubisco single amino-acid mutants was transformed into Δrpi then selected in minimal media with supplemented glycerol at elevated CO₂. Samples were sequenced before and after selections and barcode counts were used to determine the relative fitness of each mutant. D) Correspondence between 2 example biological replicates, each point represents the median fitness among all barcodes for a given mutant. E) Fitness of 77 mutants with measurements in previous studies compared to the catalytic rates measured in those studies (${\mathrm{k}}_{\text{cat}}$). The outlier is I190T, see supplemental text for discussion. F) Histogram of all variant fitnesses (grey) were normalized between values of 0 and 1 with 0 representing the average of fitnesses of mutations at a panel of known active-site positions (red distribution, average is plotted as a red dashed line) and 1 representing the average of WT barcodes (white dashed line). G) A heatmap of variant fitnesses. Conservation by position and the sequence logo were determined from a multiple sequence alignment of all rubiscos. Black triangle indicates G186, an example of a position with high conservation that is mutationally tolerant.