Investigating conformational heterogeneity and transition states using hybrid methods and experimental data as cryo-EM maps

The importance of characterizing transient intermediate states is apparent when considering, for example, the search of enzyme inhibitors that target the transition state of the enzymatic reaction. Powerful inhibitors suggest transition state features. Characterizing intermediates is also essential for understanding the molecular mechanisms associated with the functional conformational changes.  There are numerous examples where data generated by experimental techniques (such as cryo-EM and X-ray crystallography) show proteins with distinct states, such as open and closed. In the last years, we have investigated that conformational changes, dynamics and transient conformations are critically important for protein function, yet methods to map out pathways and identify transient structures lag behind the methods used for structure determination of well-populated stable states.  Several hybrid methods combining Normal Modes Analysis, Molecular Dynamics and Experimental data have been developed. We provide examples showing how these methodologies can be efficient to study the heterogeneity and the transition states for molecular complexes of proteins and improve the study of large macromolecular complexes  using Cryo-EM maps.