Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement

Cryo-EM has become one of the prime methods for protein
structure
elucidation, frequently yielding density maps with near-atomic or
medium resolution. If protein structures cannot be deduced unambiguously
from the density maps, computational structure refinement tools are
needed to generate protein structural models. We have previously developed
an iterative Rosetta-MDFF protocol that used cryo-EM densities to
refine protein structures. Here we show that, in addition to cryo-EM
densities, incorporation of other experimental restraints into the
Rosetta-MDFF protocol further improved refined structures. We used
NMR chemical shift (CS) data integrated with cryo-EM densities in
our hybrid protocol in both the Rosetta step and the molecular dynamics
(MD) simulations step. In 15 out of 18 cases for all MD rounds, the
refinement results obtained when density maps and NMR chemical shift
data were used in combination outperformed those of density map-only
refinement. Notably, the improvement in refinement was highest when
medium and low-resolution density maps were used. With our hybrid
method, the RMSDs of final models obtained were always better than
the RMSDs obtained by our previous protocol with just density refinement
for both medium (6.9 Å) and low (9 Å) resolution maps. For
all the six test proteins with medium resolution density maps (6.9
Å), the final refined structure RMSDs were lower for the hybrid
method than for the cryo-EM only refinement. The final refined RMSDs
were less than 1.5 Å when our hybrid protocol was used with 4
Å density maps. For four out of the six proteins the final RMSDs
were even less than 1 Å. This study demonstrates that by using
a combination of cryo-EM and NMR restraints, it is possible to refine
structures to atomic resolution, outperforming single restraint refinement.
This hybrid protocol will be a valuable tool when only low-resolution
cryo-EM density data and NMR chemical shift data are available to
refine structures.