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- EMDB-23392: Cryo-EM structure of the Mpa hexamer in the presence of ATP and t... -

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Entry
Database: EMDB / ID: EMD-23392
TitleCryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
Map dataCryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
Sample
  • Complex: Cryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
    • Protein or peptide: AAA ATPase forming ring-shaped complexes
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsMycobacterial proteasomal ATPase / Mycobacterium tuberculosis / structural biology / cryo-EM / ATP-Bound / STRUCTURAL PROTEIN
Function / homology
Function and homology information


proteasome complex / proteasomal protein catabolic process / modification-dependent protein catabolic process / ATP hydrolysis activity / ATP binding
Similarity search - Function
Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal 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 ...Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal 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 / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
AAA ATPase forming ring-shaped complexes
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsYin Y / Li H
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Complementary and Integrative Health (NIH/NCCIH)AI070285 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)AI088075 United States
CitationJournal: J Biol Chem / Year: 2021
Title: The mycobacterial proteasomal ATPase Mpa forms a gapped ring to engage the 20S proteasome.
Authors: Yanting Yin / Amanda Kovach / Hao-Chi Hsu / K Heran Darwin / Huilin Li /
Abstract: Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. ...Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal proteasome-activating nucleotidase and eukaryotic proteasomal Rpt1-6 ATPases, such as the N-terminal coiled-coil domain, oligosaccharide-/oligonucleotide-binding domain, and ATPase domain. However, Mpa has a unique β-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle (CP). Thus, it is unclear how Mpa binds to proteasome CPs. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide-binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP hydrolysis-driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its β-grasp domains and activating C termini to facilitate engagement with proteasome CPs. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.
History
DepositionJan 29, 2021-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ljf
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23392.png

Downloads & links

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Map

FileDownload / File: emd_23392.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.025549376 - 0.05442437
Average (Standard dev.)0.0001871242 (±0.002086138)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 214.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8260.8260.826
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z214.760214.760214.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0260.0540.000

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of the Mpa hexamer in the presence of ATP and t...

EntireName: Cryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
Components
  • Complex: Cryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
    • Protein or peptide: AAA ATPase forming ring-shaped complexes
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Cryo-EM structure of the Mpa hexamer in the presence of ATP and t...

SupramoleculeName: Cryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 390 KDa

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Macromolecule #1: AAA ATPase forming ring-shaped complexes

MacromoleculeName: AAA ATPase forming ring-shaped complexes / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 66.582969 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MGESERSEAF GIPRDSPLSS GDAAELEQLR REAAVLREQL ENAVGSHAPT RSARDIHQLE ARIDSLAARN SKLMETLKEA RQQLLALRE EVDRLGQPPS GYGVLLATHD DDTVDVFTSG RKMRLTCSPN IDAASLKKGQ TVRLNEALTV VEAGTFEAVG E ISTLREIL ...String:
MGESERSEAF GIPRDSPLSS GDAAELEQLR REAAVLREQL ENAVGSHAPT RSARDIHQLE ARIDSLAARN SKLMETLKEA RQQLLALRE EVDRLGQPPS GYGVLLATHD DDTVDVFTSG RKMRLTCSPN IDAASLKKGQ TVRLNEALTV VEAGTFEAVG E ISTLREIL ADGHRALVVG HADEERVVWL ADPLIAEDLP DGLPEALNDD TRPRKLRPGD SLLVDTKAGY AFERIPKAEV ED LVLEEVP DVSYADIGGL SRQIEQIRDA VELPFLHKEL YREYSLRPPK GVLLYGPPGC GKTLIAKAVA NSLAKKMAEV RGD DAHEAK SYFLNIKGPE LLNKFVGETE RHIRLIFQRA REKASEGTPV IVFFDEMDSI FRTRGTGVSS DVETTVVPQL LSEI DGVEG LENVIVIGAS NREDMIDPAI LRPGRLDVKI KIERPDAEAA QDIYSKYLTE FLPVHADDLA EFDGDRSACI KAMIE KVVD RMYAEIDDNR FLEVTYANGD KEVMYFKDFN SGAMIQNVVD RAKKNAIKSV LETGQPGLRI QHLLDSIVDE FAENED LPN TTNPDDWARI SGKKGERIVY IRTLVTGKSS SASRAIDT

UniProtKB: AAA ATPase forming ring-shaped complexes

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / 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: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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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: NITROGEN

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 345023

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