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- PDB-7t3i: CryoEM structure of the Rix7 D2 Walker B mutant -

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Basic information

Entry
Database: PDB / ID: 7t3i
TitleCryoEM structure of the Rix7 D2 Walker B mutant
Components
  • Rix7
  • substrate peptide
KeywordsRIBOSOMAL PROTEIN / AAA-ATPase / Rix7 / substrate translocation
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding
Similarity search - Function
AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / AAA+ ATPase domain-containing protein
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsKocaman, S. / 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: PNAS Nexus / Year: 2022
Title: Communication network within the essential AAA-ATPase Rix7 drives ribosome assembly.
Authors: Seda Kocaman / Yu-Hua Lo / Juno M Krahn / Mack Sobhany / Venkata P Dandey / Matthew L Petrovich / Suhas K Etigunta / Jason G Williams / Leesa J Deterding / Mario J Borgnia / Robin E Stanley /
Abstract: Rix7 is an essential AAA+ ATPase that functions during the early stages of ribosome biogenesis. Rix7 is composed of three domains including an N-terminal domain (NTD) and two AAA+ domains (D1 and ...Rix7 is an essential AAA+ ATPase that functions during the early stages of ribosome biogenesis. Rix7 is composed of three domains including an N-terminal domain (NTD) and two AAA+ domains (D1 and D2) that assemble into an asymmetric stacked hexamer. It was recently established that Rix7 is a presumed protein translocase that removes substrates from preribosomes by translocating them through its central pore. However, how the different domains of Rix7 coordinate their activities within the overall hexameric structure was unknown. We captured cryo-electron microscopy (EM) structures of single and double Walker B variants of full length Rix7. The disordered NTD was not visible in the cryo-EM reconstructions, but cross-linking mass spectrometry revealed that the NTD can associate with the central channel in vitro. Deletion of the disordered NTD enabled us to obtain a structure of the Rix7 hexamer to 2.9 Å resolution, providing high resolution details of critical motifs involved in substrate translocation and interdomain communication. This structure coupled with cell-based assays established that the linker connecting the D1 and D2 domains as well as the pore loops lining the central channel are essential for formation of the large ribosomal subunit. Together, our work shows that Rix7 utilizes a complex communication network to drive ribosome biogenesis.
History
DepositionDec 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Rix7
B: Rix7
C: Rix7
D: Rix7
E: Rix7
F: Rix7
G: substrate peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)542,98416
Polymers538,8317
Non-polymers4,1529
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Rix7


Mass: 89419.250 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0002840 / Production host: Escherichia coli (E. coli) / References: UniProt: G0RZG1
#2: Protein/peptide substrate peptide


Mass: 2315.846 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: CryoEM structure of Rix7 D2 Walker B mutant / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Chaetomium thermophilum (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 defocus max: 2700 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 584000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 90.92 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002327246
ELECTRON MICROSCOPYf_angle_d0.544936908
ELECTRON MICROSCOPYf_chiral_restr0.04084196
ELECTRON MICROSCOPYf_plane_restr0.00434794
ELECTRON MICROSCOPYf_dihedral_angle_d4.52023780

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