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- EMDB-13694: Substrate-engaged mycobacterial Proteasome-associated ATPase - fo... -

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Basic information

Entry
Database: EMDB / ID: EMD-13694
TitleSubstrate-engaged mycobacterial Proteasome-associated ATPase - focused 3D refinement (state A)
Map dataSubstrate-engaged mycobacterial proteasome activator bound to 20S CP in state A (focused 3D refinement)
Sample
  • Complex: Mycobacterial Proteasome-associated ATPase in complex with substrate and open-gate 20SCP
    • Protein or peptide: AAA ATPase forming ring-shaped complexes
    • Protein or peptide: Prokaryotic ubiquitin-like protein Pup
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


protein pupylation / proteasome binding / proteasome complex / proteasomal protein catabolic process / modification-dependent protein catabolic process / protein tag activity / ATP hydrolysis activity / ATP binding
Similarity search - Function
Prokaryotic ubiquitin-like protein Pup / Pup-like protein / 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) ...Prokaryotic ubiquitin-like protein Pup / Pup-like protein / 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
Prokaryotic ubiquitin-like protein Pup / AAA ATPase forming ring-shaped complexes
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsJomaa A / Kavalchuk M / Weber-Ban E
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex.
Authors: Mikhail Kavalchuk / Ahmad Jomaa / Andreas U Müller / Eilika Weber-Ban /
Abstract: Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In ...Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.
History
DepositionOct 8, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 26, 2022-
Current statusJan 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0195
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0195
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7px9
  • Surface level: 0.0195
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13694.png

Downloads & links

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Map

FileDownload / File: emd_13694.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubstrate-engaged mycobacterial proteasome activator bound to 20S CP in state A (focused 3D refinement)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 352 pix.
= 430.144 Å		1.22 Å/pix.
x 352 pix.
= 430.144 Å		1.22 Å/pix.
x 352 pix.
= 430.144 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.222 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0195 / Movie #1: 0.0195
Minimum - Maximum-0.038947657 - 0.09120464
Average (Standard dev.)-0.00038488954 (±0.0026848782)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 430.144 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2221.2221.222
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z430.144430.144430.144
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ440440440
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0390.091-0.000

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Supplemental data

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Sample components

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Entire : Mycobacterial Proteasome-associated ATPase in complex with substr...

EntireName: Mycobacterial Proteasome-associated ATPase in complex with substrate and open-gate 20SCP
Components
  • Complex: Mycobacterial Proteasome-associated ATPase in complex with substrate and open-gate 20SCP
    • Protein or peptide: AAA ATPase forming ring-shaped complexes
    • Protein or peptide: Prokaryotic ubiquitin-like protein Pup
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Mycobacterial Proteasome-associated ATPase in complex with substr...

SupramoleculeName: Mycobacterial Proteasome-associated ATPase in complex with substrate and open-gate 20SCP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.1 MDa

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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: 67.48793 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 SASRAIDTES NLGQYL

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Macromolecule #2: Prokaryotic ubiquitin-like protein Pup

MacromoleculeName: Prokaryotic ubiquitin-like protein Pup / type: protein_or_peptide / ID: 2 / Details: GS residues are left after tag cleavage / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 7.095416 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMAQEQTKR GGGGGDDDDI AGSTAAGQER REKLTEETDD LLDEIDDVLE ENAEDFVRAY VQKGGQ

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / 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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 5 / 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 / 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: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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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: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 860718
CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 5 / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 48054

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Atomic model buiding 1

Initial modelPDB ID:

5kwa

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7px9:
Substrate-engaged mycobacterial Proteasome-associated ATPase - focused 3D refinement (state A)

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