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- PDB-6mat: Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7 -

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Basic information

Entry
Database: PDB / ID: 6mat
TitleCryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7
Components
  • Rix7 mutant
  • unknown protein
KeywordsRIBOSOMAL PROTEIN / AAA-ATPase
Function / homologyAAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / P-loop containing nucleoside triphosphate hydrolase / AAA ATPase, AAA+ lid domain / ATPase family associated with various cellular activities (AAA) / AAA+ lid domain / ATP binding / Uncharacterized protein
Function and homology information
Biological speciesChaetomium thermophilum (fungus)
Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLo, Y.H. / Sobhany, M. / Hsu, A.L. / Ford, B.L. / Krahn, J.M. / Borgnia, M.J. / Stanley, R.E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIA ES103247 United States
CitationJournal: Nat Commun / Year: 2019
Title: Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7.
Authors: Yu-Hua Lo / Mack Sobhany / Allen L Hsu / Brittany L Ford / Juno M Krahn / Mario J Borgnia / Robin E Stanley /
Abstract: Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly ...Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-α7 insertions within both AAA domains important for Rix7 function.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 28, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 6, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Rix7 mutant
B: Rix7 mutant
C: Rix7 mutant
D: Rix7 mutant
E: Rix7 mutant
F: Rix7 mutant
G: unknown protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)544,40418
Polymers538,8257
Non-polymers5,57911
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53540 Å2
ΔGint-187 kcal/mol
Surface area162790 Å2

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Components

#1: Protein
Rix7 mutant


Mass: 89418.266 Da / Num. of mol.: 6 / Mutation: E303Q/E602Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0002840 / Production host: Escherichia coli (E. coli) / References: UniProt: G0RZG1
#2: Protein/peptide unknown protein


Mass: 2315.846 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: AAA-ATPase Rix7 / Type: RIBOSOME / Entity ID: 1, 2 / Source: RECOMBINANT
Source (natural)Organism: Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3 4C
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: -2700 nm / Nominal defocus min: -1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4CTFFIND4.1.10CTF correction
7Coot0.8.9.1model fitting
11RELION2.1.0classification
12RELION2.1.03D reconstruction
13CNS1.3model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64386 / Symmetry type: POINT
Atomic model buildingDetails: 5C18 was used for reference restraints in refinement
Atomic model buildingPDB-ID: 5FTK
RefinementHighest resolution: 4.5 Å

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