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- EMDB-13695: Open-gate mycobacterium 20S CP proteasome in complex MPA - global... -

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Basic information

Entry
Database: EMDB / ID: EMD-13695
TitleOpen-gate mycobacterium 20S CP proteasome in complex MPA - global 3D refinement
Map datamycobacterium 20S CP open-gate
Sample
  • Complex: Mycobacterial Proteasome-associated ATPase in complex with substrate and open-gate 20SCP
    • Protein or peptide: Proteasome subunit alpha
    • Protein or peptide: AAA ATPase forming ring-shaped complexes
    • Protein or peptide: Proteasome subunit beta
KeywordsAAA motor / ATPAse / mycobacterium / proteasome activator / 20S CP / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / proteasome complex / modification-dependent protein catabolic process / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / : / : / Proteasome alpha-type subunit ...Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / : / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / ATPase, AAA-type, conserved site / AAA-protein family signature. / Nucleophile aminohydrolases, N-terminal / 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
Proteasome subunit beta / AAA ATPase forming ring-shaped complexes / Proteasome subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJomaa A / Kavalchuk M
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-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0236
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0236
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pxa
  • Surface level: 0.0236
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7pxa
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13695.png

Downloads & links

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Map

FileDownload / File: emd_13695.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmycobacterium 20S CP open-gate
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: 0.0236 / Movie #1: 0.0236
Minimum - Maximum-0.06599888 - 0.15186606
Average (Standard dev.)0.00026931084 (±0.005247084)
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-0.0660.1520.000

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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: Proteasome subunit alpha
    • Protein or peptide: AAA ATPase forming ring-shaped complexes
    • Protein or peptide: Proteasome subunit beta

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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: all
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 1.1 MDa

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Macromolecule #1: Proteasome subunit alpha

MacromoleculeName: Proteasome subunit alpha / type: protein_or_peptide / ID: 1 / Details: first 7 residues were removed (open-gate mutant) / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 26.911039 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSFPYFISPE QAMRERSELA RKGIARAKSV VALAYAGGVL FVAENPSRSL QKISELYDRV GFAAAGKFNE FDNLRRGGIQ FADTRGYAY DRRDVTGRQL ANVYAQTLGT IFTEQAKPYE VELCVAEVAH YGETKRPELY RITYDGSIAD EPHFVVMGGT T EPIANALK ...String:
MSFPYFISPE QAMRERSELA RKGIARAKSV VALAYAGGVL FVAENPSRSL QKISELYDRV GFAAAGKFNE FDNLRRGGIQ FADTRGYAY DRRDVTGRQL ANVYAQTLGT IFTEQAKPYE VELCVAEVAH YGETKRPELY RITYDGSIAD EPHFVVMGGT T EPIANALK ESYAENASLT DALRIAVAAL RAGSADTSGG DQPTLGVASL EVAVLDANRP RRAFRRITGS ALQALLVDQE SP QSDGESS G

UniProtKB: Proteasome subunit alpha

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Macromolecule #2: AAA ATPase forming ring-shaped complexes

MacromoleculeName: AAA ATPase forming ring-shaped complexes / type: protein_or_peptide / ID: 2 / Number of copies: 7 / 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

UniProtKB: AAA ATPase forming ring-shaped complexes

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Macromolecule #3: Proteasome subunit beta

MacromoleculeName: Proteasome subunit beta / type: protein_or_peptide / ID: 3
Details: propeptide (first N-terminal 57 residues) was removed
Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 30.332006 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTWPLPDRLS INSLSGTPAV DLSSFTDFLR RQAPELLPAS ISGGAPLAGG DAQLPHGTTI VALKYPGGVV MAGDRRSTQG NMISGRDVR KVYITDDYTA TGIAGTAAVA VEFARLYAVE LEHYEKLEGV PLTFAGKINR LAIMVRGNLA AAMQGLLALP L LAGYDIHA ...String:
MTWPLPDRLS INSLSGTPAV DLSSFTDFLR RQAPELLPAS ISGGAPLAGG DAQLPHGTTI VALKYPGGVV MAGDRRSTQG NMISGRDVR KVYITDDYTA TGIAGTAAVA VEFARLYAVE LEHYEKLEGV PLTFAGKINR LAIMVRGNLA AAMQGLLALP L LAGYDIHA SDPQSAGRIV SFDAAGGWNI EEEGYQAVGS GSLFAKSSMK KLYSQVTDGD SGLRVAVEAL YDAADDDSAT GG PDLVRGI FPTAVIIDAD GAVDVPESRI AELARAIIES RSGADTFGSD GGEK

UniProtKB: Proteasome subunit beta

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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-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 860718
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio cryoSPARC
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 222719
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 5 / Software - Name: RELION

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Atomic model buiding 1

Initial modelPDB ID:

5kwa


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7pxa:
Open-gate mycobacterium 20S CP proteasome in complex MPA - global 3D refinement

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