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- EMDB-7097: Doubly PafE-capped 20S core particle in Mycobacterium tuberculosis -

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Basic information

Entry
Database: EMDB / ID: EMD-7097
TitleDoubly PafE-capped 20S core particle in Mycobacterium tuberculosis
Map dataDoubly PafE-capped 20S CP
Sample
  • Complex: Doubly PafE-capped 20S CP
    • Protein or peptide: Proteasome subunit alpha
    • Protein or peptide: Bacterial proteasome activator
    • Protein or peptide: Proteasome subunit beta
KeywordsProtein degradation / HYDROLASE
Function / homology
Function and homology information


symbiont-mediated perturbation of host defenses / proteasome accessory complex / positive regulation of proteasomal protein catabolic process / zymogen binding / proteasome binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process ...symbiont-mediated perturbation of host defenses / proteasome accessory complex / positive regulation of proteasomal protein catabolic process / zymogen binding / proteasome binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / regulation of proteasomal protein catabolic process / proteasome complex / peptidoglycan-based cell wall / proteolysis involved in protein catabolic process / protein homooligomerization / modification-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / extracellular region / plasma membrane / cytoplasm
Similarity search - Function
Bacterial proteasome activator / Bacterial proteasome activator / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit ...Bacterial proteasome activator / Bacterial proteasome activator / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Proteasome subunit alpha / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit alpha / Bacterial proteasome activator / Bacterial proteasome activator
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLi H / Hu K
CitationJournal: J Biol Chem / Year: 2018
Title: Proteasome substrate capture and gate opening by the accessory factor PafE from .
Authors: Kuan Hu / Jordan B Jastrab / Susan Zhang / Amanda Kovach / Gongpu Zhao / K Heran Darwin / Huilin Li /
Abstract: In all domains of life, proteasomes are gated, chambered proteases that require opening by activators to facilitate protein degradation. Twelve proteasome accessory factor E (PafE) monomers assemble ...In all domains of life, proteasomes are gated, chambered proteases that require opening by activators to facilitate protein degradation. Twelve proteasome accessory factor E (PafE) monomers assemble into a single dodecameric ring that promotes proteolysis required for the full virulence of the human bacterial pathogen Whereas the best characterized proteasome activators use ATP to deliver proteins into a proteasome, PafE does not require ATP. Here, to unravel the mechanism of PafE-mediated protein targeting and proteasome activation, we studied the interactions of PafE with native substrates, including a newly identified proteasome substrate, the ParA-like protein, Rv3213c, and with proteasome core particles. We characterized the function of a highly conserved feature in bacterial proteasome activator proteins: a glycine-glutamine-tyrosine-leucine (GQYL) motif at their C termini that is essential for stimulating proteolysis. Using cryo-electron microscopy (cryo-EM), we found that the GQYL motif of PafE interacts with specific residues in the α subunits of the proteasome core particle to trigger gate opening and degradation. Finally, we also found that PafE rings have 40-Å openings lined with hydrophobic residues that form a chamber for capturing substrates before they are degraded, suggesting PafE has a previously unrecognized chaperone activity. In summary, we have identified the interactions between PafE and the proteasome core particle that cause conformational changes leading to the opening of the proteasome gate and have uncovered a mechanism of PafE-mediated substrate degradation. Collectively, our results provide detailed insights into the mechanism of ATP-independent proteasome degradation in bacteria.
History
DepositionOct 28, 2017-
Header (metadata) releaseJan 24, 2018-
Map releaseFeb 14, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00742
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.00742
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bgl
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7097.png

Downloads & links

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Map

FileDownload / File: emd_7097.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDoubly PafE-capped 20S CP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 400 pix.
= 436. Å		1.09 Å/pix.
x 400 pix.
= 436. Å		1.09 Å/pix.
x 400 pix.
= 436. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00742 / Movie #1: 0.00742
Minimum - Maximum-0.011481893 - 0.030812554
Average (Standard dev.)0.000029359235 (±0.0011126738)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 436.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z436.000436.000436.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0110.0310.000

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Supplemental data

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Sample components

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Entire : Doubly PafE-capped 20S CP

EntireName: Doubly PafE-capped 20S CP
Components
  • Complex: Doubly PafE-capped 20S CP
    • Protein or peptide: Proteasome subunit alpha
    • Protein or peptide: Bacterial proteasome activator
    • Protein or peptide: Proteasome subunit beta

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Supramolecule #1: Doubly PafE-capped 20S CP

SupramoleculeName: Doubly PafE-capped 20S CP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Macromolecule #1: Proteasome subunit alpha

MacromoleculeName: Proteasome subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
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: Bacterial proteasome activator

MacromoleculeName: Bacterial proteasome activator / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 18.963232 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MVIGLSTGSD DDDVEVIGGV DPRLIAVQEN DSDESSLTDL VEQPAKVMRI GTMIKQLLEE VRAAPLDEAS RNRLRDIHAT SIRELEDGL APELREELDR LTLPFNEDAV PSDAELRIAQ AQLVGWLEGL FHGIQTALFA QQMAARAQLQ QMRQGALPPG V GKSGQHGH GTGQYL

UniProtKB: Bacterial proteasome activator

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Macromolecule #3: Proteasome subunit beta

MacromoleculeName: Proteasome subunit beta / type: protein_or_peptide / ID: 3 / Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 25.274264 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TTIVALKYPG GVVMAGDRRS TQGNMISGRD VRKVYITDDY TATGIAGTAA VAVEFARLYA VELEHYEKLE GVPLTFAGKI NRLAIMVRG NLAAAMQGLL ALPLLAGYDI HASDPQSAGR IVSFDAAGGW NIEEEGYQAV GSGSLFAKSS MKKLYSQVTD G DSGLRVAV ...String:
TTIVALKYPG GVVMAGDRRS TQGNMISGRD VRKVYITDDY TATGIAGTAA VAVEFARLYA VELEHYEKLE GVPLTFAGKI NRLAIMVRG NLAAAMQGLL ALPLLAGYDI HASDPQSAGR IVSFDAAGGW NIEEEGYQAV GSGSLFAKSS MKKLYSQVTD G DSGLRVAV EALYDAADDD SATGGPDLVR GIFPTAVIID ADGAVDVPES RIAELARAII ESRSGADTFG SDGGEKHHHH HH

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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.9 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 reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51091
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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