Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION

Nakane, Takanori; Kimanius, Dari; Lindahl, Erik; Scheres, Sjors H.W.

doi:10.7554/elife.36861

Cited by 473 publications

(405 citation statements)

References 34 publications

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“…S1D and S2), we obtained a reasonably isotropic reconstruction using cisTEM. After per-particle, projection-based CTF and motion correction (Bayesian polishing) in RELION 3.0 (Nakane et al, 2018;Zivanov et al, 2018), refinement in cisTEM yielded a map with an average resolution of 3.3 Å (FSC=0.143 criterion; Figs.…”

Section: Cryo-em Structure Determinationmentioning

confidence: 99%

Structure of a rabies virus polymerase complex from electron cryo-microscopy

Horwitz

Jenni

Whelan

2019

Preprint

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Non-segmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS-RNA viruses carry all the enzymatic functions required for viral mRNA transcription and replication: RNA polymerization, mRNA capping, cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a pre-initiation conformation. Comparison with the likely elongation state, seen in two partial structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.

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Section: Cryo-em Structure Determinationmentioning

confidence: 99%

Structure of a rabies virus polymerase complex from electron cryo-microscopy

Horwitz

Jenni

Whelan

2019

Preprint

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“…One last iteration of CTF refinement, particle polishing and refinement was performed and led to a reconstructions at 3.07 Å resolution. The core was then subjected to multibody refinement by masking each of the three lobes separately 45 . The arm, head and body refined to 3.23, 3.14, and 2.96 Å respectively.…”

Section: Cryo-em Data Processingmentioning

confidence: 99%

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement

Patel

Moore

Greber

et al. 2019

Preprint

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Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility 1,2 . The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair 2-4 . Here we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored through the interactions between the N-terminus of Sth1 and the core. Our cryo-EM analysis also shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of Rsc8 SWRIM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes. MainEukaryotes have four major families of chromatin remodelers: SWI/SNF, ISWI, CHD, and INO80 2 . Each of these remodelers plays distinct roles based on how they select and affect target nucleosomes. Together, these remodelers give rise to the distinct chromatin landscapes observed in eukaryotic cells and determine how genetic information is organized, replicated, transcribed, and repaired 5 . In S. cerevisiae there are two members of the SWI/SNF family of chromatin remodelers: RSC and SWI/SNF 3,4 . RSC is essential for viability and is ten times more abundant than SWI/SNF 4 . While both complexes play a role in remodeling nucleosomes during transcription initiation, RSC is also involved in transcription-independent processes such as mitotic division, double stranded break repair, and telomere maintenance 6-12 .RSC is a ~1-MDa complex composed of 17 proteins, with two copies of Rsc8 and one copy of either Rsc1 or Rsc2 (Fig. S1) 4,13 . In order to determine the structure of RSC, we purified the complex from S. cerevisiae and performed cryo-EM analysis (Fig. S2). We find that RSC is composed of five main lobes, three that form a rigid core (head, body and arm) and two that are flexibly attached (leg and tail) ( Fig. 1a, b; Fig. S3, S4). We determined the structure of the core to ~3.1 Å and mapped 14 proteins within this region: Rsc1/2, Rsc3, Rsc4, Rsc6, Rsc8 (2 copies), Rsc9, Rsc30, Rsc58, Ldb7, Npl6, Htl1, Sfh1, and Sth1 (Fig. 1c, d, e). Our negative stain analysis of the yeast SWI/SNF complex shows that, like RSC, it has head, body, and arm regions that define a rigid core, and a flexible leg that occupies similar overall positions (Fig. S5). However, SWI/SNF lacks a tail lobe, indicating that while RSC and SWI/SNF share a conserved core architecture, ...

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“…Multibody refinement, additional SSU head domain movement amplitude a Views of the two extreme states of the head and head extension, relative to the body of the SSU, calculated using the multibody refinement implemented in RELION312 , showing the movement in two different planes. b All ten states reveals a movement amplitude of 30°.…”

mentioning

confidence: 99%

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome

Waltz

Soufari

Bochler

et al. 2019

Preprint

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The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6–10. In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.

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Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION

Cited by 473 publications

References 34 publications

Structure of a rabies virus polymerase complex from electron cryo-microscopy

Structure of a rabies virus polymerase complex from electron cryo-microscopy

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome

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