Table S1.

Computational parameters used to generate designed variants and lists of mutations

Designed variant	Positions held fixed*,†	ΔΔGcalc cutoffs, R.e.u.‡	Mutations*
PfRH5ΔNLHS1 (strict MSA)	147, 149, 193, 196, 202, 205, 206, 209, 212, 213, 216, 327, 328, 331, 334, 335, 337, 338, 339, 340, 341, 342, 344, 452, 456, 194, 197, 200, 201, 202, 203, 204, 207, 222, 225, 226, 242, 243, 244, 245, 246, 247, 248, 249, 250, 346, 347, 349, 350, 352, 357, 358, 362, 447, 448, 449, 496, 198, 343, 345, 348, 353, 451	−0.45 (18)	I157L§, D183E, A233K, M304F, K312N, L314F, K316N, M330N, S370A, S381N, T384K, L392K, T395N, N398E, R458K, N464K, S467A, F505L
PfRH5ΔNLHS2 (permissive MSA)		−0.75 (25)	D183E, N191I, S192A, A233N, K236H, N308K, L314F, L369N, S370N, S381N, T384K, L392D, T395R, N398K, H414L, L444E, N445D, S463A, N464K, S467A, I470R, H474D, H495N, F505L, K511P
PfRH5ΔNLHS3 (permissive MSA)		−1.25 (15)	D183E, N191I, S192A, A233N, L369N, S381N, L392D, N398K, N445D, S463A, N464K, S467A, I470R, H474D, K511P

^*Numbering according to PDB ID code 4WAT.

^†Residues within 5 Å of either basigin, QA1, or 9AD4 were held fixed through all design simulations.

^‡PROSS energy threshold of designs that were selected for experimental validation. The cutoffs defined the subsets of individually stabilizing mutations. Each defined subset resulted in a single variant. R.e.u. refers to Rosetta energy units. Values in parentheses show the number of mutations relative to PfRH5ΔNL.

^§Two sequences contribute information at position 157, and both have ILE at this position. LEU was allowed and selected because the PSSM accounts also for BLOSUM62 statistics.