Yeast in vivo growth complementation assay for human CBS function. Yeast in vivo growth complementation assay for human CBS function. (A) Scatter plot showing the predicted change in free energy (Predicted ΔΔG, x-axis) for 52 human single-amino-acid CBS substitutions vs. their conferred growth rates expressed relative to that of the major human CBS allele grown with 400 ng/mL pyridoxine supplementation in HEM1 yeast strains (y-axis). The alleles were categorized by growth phenotype as benign, sensitive, or nonfunctional (see Results). (B) Assessment of the calculated changes in free energy to predict the phenotypic outcome for 52 human CBS substitutions. The alleles were binned based on the predicted change in free energy (ΔΔG) in 0.5 kcal/mol increments. For each incremental threshold a percentage “correct” was calculated by summing the number of alleles growing below the threshold added to the number of alleles not growing above the threshold, divided by the total number of alleles (i.e., 52). The dotted line indicates a predicted ΔΔG of 3.5 kcal/mol Rosetta units. Dago Dimster-Denk et al. G3 2013;3:1619-1628 ©2013 by Genetics Society of America