Structural dynamics of human ribosomes in situ reconstructed by exhaustive high-resolution template matching
John Peter Rickgauer, Heejun Choi, Andrew S. Moore, Winfried Denk, Jennifer Lippincott‐Schwartz
Abstract
Protein synthesis is central to life and requires the ribosome, which catalyzes the stepwise addition of amino acids to a polypeptide chain by undergoing a sequence of structural transformations. Here, we employed high-resolution template matching (HRTM) on cryoelectron microscopy (cryo-EM) images of directly cryofixed living cells to obtain a set of ribosomal configurations covering the entire elongation cycle, with each configuration occurring at its native abundance. HRTM’s position and orientation precision and ability to detect small targets (∼300 kDa) made it possible to order these configurations along the reaction coordinate and to reconstruct molecular features of any configuration along the elongation cycle. Visualizing the cycle’s structural dynamics by combining a sequence of >40 reconstructions into a 3D movie readily revealed component and ligand movements, some of them surprising, such as spring-like intramolecular motion, providing clues about the molecular mechanisms involved in some still mysterious steps during chain elongation. • HRTM localizes and aligns macromolecules down to ∼300 kDa in cryo-EM images in situ • Small-target detection reveals conformations within and between ribosomes • Stitching together >40 3D conformations reveals dynamics of the elongation cycle • Molecular dynamics during elongation include bending and “spring-like” motion Rickgauer et al. dissect molecular motion of human ribosomes in situ using high-resolution template matching. Detecting individual ribosome subunits provides maps of native conformations, revealing new conformations within and between translating ribosomes. Conformation-specific 3D reconstruction captures structural snapshots of the ribosome’s transient binding partners and internal molecular motion during elongation.