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- EMDB-25659: CryoEM structure of the Rix7 D2 Walker B mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-25659
TitleCryoEM structure of the Rix7 D2 Walker B mutant
Map data
Sample
  • Complex: CryoEM structure of Rix7 D2 Walker B mutant
    • Protein or peptide: Rix7
    • Protein or peptide: substrate peptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: PHOSPHATE IONPhosphate
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
AAA+ ATPase domain-containing protein
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsLo YH / Krahn J
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0133
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0133
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7t3i
  • Surface level: 0.0133
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25659.png

Downloads & links

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Map

FileDownload / File: emd_25659.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04967 Å
Density
Contour LevelBy AUTHOR: 0.0133 / Movie #1: 0.0133
Minimum - Maximum-0.037107896 - 0.07339655
Average (Standard dev.)1.6170701e-05 (±0.003256747)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.7155 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0496718751.0496718751.049671875
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z268.716268.716268.716
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0370.0730.000

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Supplemental data

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Sample components

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Entire : CryoEM structure of Rix7 D2 Walker B mutant

EntireName: CryoEM structure of Rix7 D2 Walker B mutant
Components
  • Complex: CryoEM structure of Rix7 D2 Walker B mutant
    • Protein or peptide: Rix7
    • Protein or peptide: substrate peptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: PHOSPHATE IONPhosphate

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Supramolecule #1: CryoEM structure of Rix7 D2 Walker B mutant

SupramoleculeName: CryoEM structure of Rix7 D2 Walker B mutant / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Chaetomium thermophilum (fungus)

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Macromolecule #1: Rix7

MacromoleculeName: Rix7 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Molecular weightTheoretical: 89.41925 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSRRPTLRLG LDRDVYNIVL NLEQQGTDEN GKRPRLTVDY VYDTIKRSNS SLARQKKRML EDSIERVLAV RKEQAKAEEE TDSDDLIEA QERERERQKA AQAQRDANLL NRQIAKSWGF ASSPGAKAAD GEKGTDTGSI ATPAPATPAV AENMAADTPT T STGPVLPA ...String:
MSRRPTLRLG LDRDVYNIVL NLEQQGTDEN GKRPRLTVDY VYDTIKRSNS SLARQKKRML EDSIERVLAV RKEQAKAEEE TDSDDLIEA QERERERQKA AQAQRDANLL NRQIAKSWGF ASSPGAKAAD GEKGTDTGSI ATPAPATPAV AENMAADTPT T STGPVLPA SSTDRQPNGE PRPKKRKAAP KEIDRTPPTK VSILDIAGVD DTLQRLLKEV WFPLRGGEAC EKMGYRYDNG VL LHGPSGC GKTTLAHAIA GSIGVAFIPV SAPSVIGGTS GESEKNIRDV FDEAIRLAPC LIFLDEIDAI AGRRESANKG MES RIVAEI MNGMDRIRQN TPLGKNVVVL AATNRPEFLD PAIRRRFSVE IDMGMPSERA REQILRSLTR DLSLADDINF KELA KMTPG YVGSDLQYVV KAAVSESFQA NIDSLLAQAR AKHPADHLAN VSQPQRDWLL LEAHRDEEVS WPSTKITMEQ FRKAV SLVQ PASKREGFST IPDTTWSHVG ALEDVRKKLE MSIIGPIKNP ELFTRVGIKP AAGILLWGPP GCGKTLVAKA VANESK ANF ISIKGPELLN KYVGESERAV RQLFSRAKSS APCILFFDQM DALVPRRDDS LSDASARVVN TLLTELDGVG DRSGIYV IG ATNRPDMIDE AIRRPGRLGT SIYVGLPSAE DRVKILKTLY RNTVKAPKKR EGTNGEDVDM TDAAAEQQHQ GTTDADLE K VALDLRCTGF SGADLGNLMQ AAAQACLERV YTQRQQKRKE GGSVAEEEEI EPVITMEDWE KALNEVKPSV KDPEKYMHS GFAAALEHHH HHH

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Macromolecule #2: substrate peptide

MacromoleculeName: substrate peptide / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.315846 KDa
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #4: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6mat

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 584000

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