The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
- MRC Laboratory of Molecular Biology, United Kingdom
Abstract
The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.
Data availability
The atomic coordinates for the Rea1 AAA+ ring and the Rea1 linker in the ADP state have been deposited with PDB IDs 6HYP and 6HYD, respectively. The accession codes for the Rea1 and Rea1_ΔAAA2L-H2α models in the AMPPNP state are 6I26 and 6I27, respectively. The accession codes for the cryoEM maps of the Rea1 AAA+ ring and the Rea1 linker in the ADP state are EMD-0309 and EMD-0308, respectively. The accession code for the unsharpened cryoEM map of the Rea1 AAA+ ring in the ADP state is EMD-0330. The cryoEM maps of the Rea1 and Rea1_ΔAAA2L-H2α AMPPNP states have the EMD accession codes EMD-0328 and EMD-0329, respectively.
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Sharpened map of the Rea1 AAA+ ring ADP stateElectron Microscopy Data Bank, EMD-0309.
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Unsharpened map of the Rea1 linker ADP stateElectron Microscopy Data Bank, EMD-0308.
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Unsharpened map of the Rea1 AAA+ ring ADP stateElectron Microscopy Data Bank, EMD-0330.
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Sharpened map of the Rea1 AMPPNP stateElectron Microscopy Data Bank, EMD-0328.
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Sharpened map of the Rea1_ΔAAA2L-H2α AMPPNP stateElectron Microscopy Data Bank, EMD-0329.
Article and author information
Author details
Funding
HS-ATIP-avenir grant (CDP 0B1INSB-HS-9ADO1051)
- Helgo Schmidt
LabEX startup grant (GIE I-LABEX-HS)
- Helgo Schmidt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Sosnowski et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1
eLife 7:e39163.
https://doi.org/10.7554/eLife.39163
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We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
