All parts together with the generated template-based model and secondary structure restraints were structurally concatenated by MODELLER.
Refinement was performed in Phenix [23] using the secondary structure restraints derived from the previously solved complex.
Secondary structure restraints and non-crystallographic symmetry restraints were applied for the refinements.
Secondary structure restraints for the Sec61 channel were generated in ProSMART (Nicholls et al., 2012).
Throughout refinement, reference and secondary structure restraints were applied to the ribosomal proteins using the Sc80S structure as a reference model (Nicholls et al., 2012).
A major point to note is that in each of these examples R osetta is used to complement structure restraints obtained early in the structure determination process.
Briefly, after applying the secondary structure restraints to the extended conformation, followed by energy minimization, the RMSD decreased from 7.1 Å to 4.4 Å.
Secondary structure restraints were generated in ProSMART (Nicholls et al., 2012 ), and nucleic acid base-pairing and stacking restraints were generated as before (Amunts et al., 2014 ) and were maintained throughout refinement to prevent overfitting.
Both secondary structure restraints (Trabuco et al., 2009) and symmetry restraints (Chan et al., 2011) were applied to prevent structural distortion under large force in the distance constrained simulation.
Secondary structure restraints were generated in ProSMART (Nicholls et al., 2012), and nucleic acid base-pairing and stacking restraints were generated as before (Amunts et al., 2014) and were maintained throughout refinement to prevent over-fitting.
The secondary structure restraints prevents some subtle changes in the structure, such as kinks observed previously in the upper part of TM1 in the open model of MscL (Deplazes et al., 2012).
