posted on 2020-03-20, 18:36authored byCarlos
F. Rodríguez, Mohinder Pal, Hugo Muñoz-Hernandez, Laurence H. Pearl, Oscar Llorca
The number of high-resolution
structures of protein complexes obtained
using cryo-electron microscopy (cryo-EM) is increasing rapidly. Cryo-EM
maps of large macromolecular complexes frequently contain regions
resolved at different resolution levels, and modeling atomic structures de novo can be difficult for domains determined at worse
than 5 Å in the absence of atomic information from other structures.
Here we describe the details and step-by-step decisions in the strategy
we followed to model the RUVBL2-binding domain (RBD), a 14 kDa domain
at the C-terminus of RNA Polymerase II associated protein 3 (RPAP3)
for which atomic information was not available. Modeling was performed
on a cryo-EM map at 4.0–5.5 Å resolution, integrating
information from secondary structure predictions, homology modeling,
restraints from cross-linked mass spectrometry, and molecular dynamics
(MD) in AMBER. Here, we compare our model with the structure of RBD
determined by NMR to evaluate our strategy. We also perform new MD
simulations to describe important residues mediating the interaction
of RBD with RUVBL2 and analyze their conservation in RBD homologous
domains. Our approach and its evaluation can serve as an example to
address the analysis of medium resolution regions in cryo-EM maps.