Rosetta Relaxation and Ensemble Generation

Antibody structural ensembles with 10 members were generated using either the Rosetta FastRelax (48, 49) or Rosetta KIC protocol (50), and Rosetta version 2017.26-dev59567 was used for all simulations (corresponding to weekly release version 2017.26). The Rosetta FastRelax protocol consists of five cycles of side-chain repacking and gradient-based energy minimization in the REF2015 version of the Rosetta energy function (51). Thus, FastRelax ensembles explore the local energy minimum of the crystal structure. KIC ensembles are more diverse and representative of RosettaAntibody homology models: each ensemble member was generated by running the CDR-H3 refinement step of the RosettaAntibody protocol, consisting of V_H–V_L docking, CDR-H3 loop remodeling, and all-CDR loop minimization (38, 39). Sample command lines are given in Supplementary Material. The structural ensembles produced by both FastRelax and KIC were used for rigidity analysis. For technical reasons, 6 targets could not be analyzed from the FastRelax ensemble, and 177 targets from the KIC ensemble were omitted due to non-trivial incompatibilities between the input structure numbering and Rosetta’s internal antibody numbering scheme and a computing cluster time limitation. The excluded targets were randomly distributed and likely would not affect the conclusions.