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- EMDB-25474: CryoEM structure of the N-Terminal deleted Rix7 AAA-ATPase -

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Basic information

Entry
Database: EMDB / ID: EMD-25474
TitleCryoEM structure of the N-Terminal deleted Rix7 AAA-ATPase
Map data
Sample
  • Complex: CryoEM structure of the N-terminal-deleted Rix7 AAA-ATPase
    • Protein or peptide: Rix7
    • Protein or peptide: polyleucine
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA-ATPase / ribosome biogenesis / substrate translocation / RIBOSOMAL PROTEIN
Function / homology
Function and homology information


peroxisome organization / preribosome binding / ribosome biogenesis / ATP hydrolysis activity / RNA binding / ATP binding / nucleus
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
Peroxisomal ATPase PEX1
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsKocaman S / Stanley RE
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
DepositionNov 20, 2021-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7swl
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25474.png

Downloads & links

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Map

FileDownload / File: emd_25474.map.gz / Format: CCP4 / Size: 10.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 154 pix.
= 162.393 Å		1.05 Å/pix.
x 141 pix.
= 148.684 Å		1.05 Å/pix.
x 126 pix.
= 132.867 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.0545 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.5 / Movie #1: 0.6
Minimum - Maximum-3.1339645 - 5.779644
Average (Standard dev.)0.048774414 (±0.3361542)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin879287
Dimensions141126154
Spacing126141154
CellA: 132.867 Å / B: 148.6845 Å / C: 162.39299 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.05451.05449645390071.0545
M x/y/z126141154
origin x/y/z0.0000.0000.000
length x/y/z132.867148.684162.393
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS928787
NC/NR/NS126141154
D min/max/mean-3.1345.7800.049

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Supplemental data

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Sample components

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Entire : CryoEM structure of the N-terminal-deleted Rix7 AAA-ATPase

EntireName: CryoEM structure of the N-terminal-deleted Rix7 AAA-ATPase
Components
  • Complex: CryoEM structure of the N-terminal-deleted Rix7 AAA-ATPase
    • Protein or peptide: Rix7
    • Protein or peptide: polyleucine
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: CryoEM structure of the N-terminal-deleted Rix7 AAA-ATPase

SupramoleculeName: CryoEM structure of the N-terminal-deleted Rix7 AAA-ATPase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Chaetomium thermophilum (fungus)
Molecular weightTheoretical: 100 KDa

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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: 69.008492 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASMTGGQQM GRGSMSILDI AGVDDTLQRL LKEVWFPLRG GEACEKMGYR YDNGVLLHGP SGCGKTTLAH AIAGSIGVAF IPVSAPSVI GGTSGESEKN IRDVFDEAIR LAPCLIFLDQ IDAIAGRRES ANKGMESRIV AEIMNGMDRI RQNTPLGKNV V VLAATNRP ...String:
MASMTGGQQM GRGSMSILDI AGVDDTLQRL LKEVWFPLRG GEACEKMGYR YDNGVLLHGP SGCGKTTLAH AIAGSIGVAF IPVSAPSVI GGTSGESEKN IRDVFDEAIR LAPCLIFLDQ IDAIAGRRES ANKGMESRIV AEIMNGMDRI RQNTPLGKNV V VLAATNRP EFLDPAIRRR FSVEIDMGMP SERAREQILR SLTRDLSLAD DINFKELAKM TPGYVGSDLQ YVVKAAVSES FQ ANIDSLL AQARAKHPAD HLANVSQPQR DWLLLEAHRD EEVSWPSTKI TMEQFRKAVS LVQPASKREG FSTIPDTTWS HVG ALEDVR KKLEMSIIGP IKNPELFTRV GIKPAAGILL WGPPGCGKTL VAKAVANESK ANFISIKGPE LLNKYVGESE RAVR QLFSR AKSSAPCILF FDQMDALVPR RDDSLSDASA RVVNTLLTEL DGVGDRSGIY VIGATNRPDM IDEAIRRPGR LGTSI YVGL PSAEDRVKIL KTLYRNTVKA PKKREGTNGE DVDMTDAAAE QQHQGTTDAD LEKVALDLRC TGFSGADLGN LMQAAA QAC LERVYTQRQQ KRKEGGSVAE EEEIEPVITM EDWEKALNEV KPSVKDPEKY MHSGFAAALE HHHHHH

UniProtKB: Peroxisomal ATPase PEX1

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Macromolecule #2: polyleucine

MacromoleculeName: polyleucine / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.733803 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
LLLLLLLLLL LLLLLLLLLL LLLL

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 9 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

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

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 9 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1200000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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