+Open data
-Basic information
Entry | Database: PDB / ID: 7t0v | ||||||
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Title | CryoEM structure of the crosslinked Rix7 AAA-ATPase | ||||||
Components |
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Keywords | RIBOSOMAL PROTEIN / CryoEM / AAA-ATPase / ribosome biogenesis / substrate translocation | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Chaetomium thermophilum (fungus) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å | ||||||
Authors | Kocaman, S. / Stanley, R.E. / Lo, Y.H. / Krahn, J. / Dandey, V.P. / Sobhany, M. / Petrovich, M. / Williams, J.G. / Deterding, L.J. / Borgnia, M.J. / Etigunta, S. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7t0v.cif.gz | 589.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7t0v.ent.gz | 482.5 KB | Display | PDB format |
PDBx/mmJSON format | 7t0v.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/t0/7t0v ftp://data.pdbj.org/pub/pdb/validation_reports/t0/7t0v | HTTPS FTP |
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-Related structure data
Related structure data | 25582MC 7swlC 7t3iC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 89418.266 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 | | Mass: 2298.024 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #3: Chemical | ChemComp-ATP / #4: Chemical | ChemComp-MG / #5: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cryoem structure of the crosslinked Rix7 AAA-ATPase / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.1 MDa / Experimental value: YES |
Source (natural) | Organism: Chaetomium thermophilum (fungus) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 201000 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 94.89 Å2 | ||||||||||||||||||||||||
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