Multi-Pole Approach to Structural Science

May 10 - 13, 2015

Multi-resolution refinement and fitting of atomic models into cryo-EM reconstructions
Piotr Neumann

Recent progress in high-resolution structure determination of large macromolecular complexes by single particle electron cryo-microscopy (cryo-EM) made it possible to obtain cryo-EM reconstructions with the overall quality comparable to the crystal structure derived electron density maps of aforementioned complexes, like ribosomes. Although local resolution of the cryo-EM reconstructions can be non-uniform and structures available to date are usually reported to an overall resolution lower than 3 Å, the level of visible structural details legitimates building of atomic models, which can not be modeled by simple rigid body fit of known 3D structures or their fragments/domains. In order to be able to build a model observed in cryo-EM map conformational state of the macromolecular complex, the initial atomic model needs to be fitted and refined against the target cryo-EM density. This raises the need to develop new tools for cryo-EM based 3D modeling or customize the usage of existing powerful programs already available for X-ray crystallography. Here we present an atomic model of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65 - 2.9 Å resolution obtained by cryo-EM reconstruction and a pseudo-crystallographic refinement approach. The superior quality of the cryo-EM reconstruction allowed for the first time to build all 35 RNA modifications in the bacterial ribosome explaining their roles in fine-tuning ribosome structure and function. The starting model of the crystal structure of Escherichia coli ribosome has been refined against reciprocal structure factors obtained from EM maps using a combination of efficient conformational sampling with “Deformable Elastic Network” restraints, manual modeling and standard crystallography-like refinement. Application of available crystallographic refinement programs (CNS, PHENIX) and newly established protocols to yield a reliable atomic model based on cryo-EM maps will be discussed.