Figure 1:

Schematic diagram illustrating the two structure refinement protocols used in this study. The initial models that were inputted to the protocols were obtained using Rosetta ab initio model building without using any experimental restraints. In the first protocol which is the same protocol used in our previous publication, three rounds of Rosetta-MDFF were run iteratively without using any chemical shift information. Only cryo-EM density data was used in this refinement. In the new hybrid protocol three rounds of Rosetta-MDFF were performed using both chemical shift data and cryo-EM density data. The final models were obtained after the last round of Rosetta and validated by their chemical shift agreement using the ShiftX2 program. The chemical shift data was used in molecular dynamics simulations with the PLUMED program which was integrated into the MD flexible fitting algorithm. In the Rosetta steps of the protocol, chemical shifts were used in picking the fragments in the model building step.