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- EMDB-9257: The structure of the Plasmodium falciparum 20S proteasome in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-9257
TitleThe structure of the Plasmodium falciparum 20S proteasome in complex with two PA28 activators
Map data
Sample20S proteasome/PA28 complex.
  • Plasmodium falciparum 20S proteasome
  • 11S activator of the Plasmodium falciparum proteasome.
  • (20S proteasome alpha- ...) x 7
  • (20S proteasome beta- ...) x 7
  • Proteasome activator PA28
Function / homology
Function and homology information


Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / UCH proteinases / positive regulation of endopeptidase activity / proteasome activator complex / regulation of G1/S transition of mitotic cell cycle / endopeptidase activator activity / proteasome core complex, beta-subunit complex / proteasome core complex ...Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / UCH proteinases / positive regulation of endopeptidase activity / proteasome activator complex / regulation of G1/S transition of mitotic cell cycle / endopeptidase activator activity / proteasome core complex, beta-subunit complex / proteasome core complex / proteasomal ubiquitin-independent protein catabolic process / proteasome endopeptidase complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / regulation of proteasomal protein catabolic process / proteasomal protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / peptidase activity / ubiquitin-dependent protein catabolic process / endopeptidase activity / nucleoplasm / nucleus / cytoplasm
Proteasome subunit alpha 1 / Proteasome subunit alpha5 / Proteasome activator PA28 / Proteasome beta-type subunit, conserved site / Proteasome subunit beta 4 / Proteasome alpha-type subunit / Proteasome B-type subunit / Nucleophile aminohydrolases, N-terminal / Proteasome beta 3 subunit / Proteasome subunit alpha6 ...Proteasome subunit alpha 1 / Proteasome subunit alpha5 / Proteasome activator PA28 / Proteasome beta-type subunit, conserved site / Proteasome subunit beta 4 / Proteasome alpha-type subunit / Proteasome B-type subunit / Nucleophile aminohydrolases, N-terminal / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome, subunit alpha/beta / Proteasome subunit alpha2 / Proteasome subunit / Proteasome subunit beta 1 / Proteasome subunit beta 2 / Proteasome subunit beta 7 / Proteasome activator superfamily / Proteasome activator PA28, C-terminal domain superfamily / Proteasome activator PA28, C-terminal domain / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit beta Pre3 / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit profile. / Proteasome alpha-type subunit profile. / Proteasome beta-type subunits signature. / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome activator pa28 beta subunit / Proteasome subunit alpha 3
Proteasome endopeptidase complex / Proteasome endopeptidase complex / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Subunit of proteaseome activator complex,putative / Proteasome subunit beta ...Proteasome endopeptidase complex / Proteasome endopeptidase complex / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Subunit of proteaseome activator complex,putative / Proteasome subunit beta / Proteasome subunit beta type-6, putative / Proteasome subunit beta / Proteasome endopeptidase complex / Proteasome subunit beta / Proteasome subunit beta
Biological speciesPlasmodium falciparum 3D7 (eukaryote) / Plasmodium falciparum (isolate 3D7) (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHanssen E / Xie SC / Leis A / Metcalfe RD / Gillett DL / Tilley L / Griffin MDW
Funding support Australia, 2 items
OrganizationGrant numberCountry
National Health and Medical Research Council (Australia) Australia
Australian Research Council Australia
CitationJournal: Nat Microbiol / Year: 2019
Title: The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.
Authors: Stanley C Xie / Riley D Metcalfe / Eric Hanssen / Tuo Yang / David L Gillett / Andrew P Leis / Craig J Morton / Michael J Kuiper / Michael W Parker / Natalie J Spillman / Wilson Wong / Christopher Tsu / Lawrence R Dick / Michael D W Griffin / Leann Tilley /
Abstract: The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its ...The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Validation ReportPDB-ID: 6muv

SummaryFull reportAbout validation report
History
DepositionOct 23, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseAug 7, 2019-
UpdateAug 14, 2019-
Current statusAug 14, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6muv
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9257.map.gz / Format: CCP4 / Size: 155.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.31 Å/pix.
x 344 pix.
= 450.64 Å
1.31 Å/pix.
x 344 pix.
= 450.64 Å
1.31 Å/pix.
x 344 pix.
= 450.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 6.0 / Movie #1: 4
Minimum - Maximum-16.918745000000001 - 29.237331000000001
Average (Standard dev.)0.000000000004003 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions344344344
Spacing344344344
CellA=B=C: 450.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z344344344
origin x/y/z0.0000.0000.000
length x/y/z450.640450.640450.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ344344344
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS344344344
D min/max/mean-16.91929.2370.000

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Supplemental data

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Sample components

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Entire 20S proteasome/PA28 complex.

EntireName: 20S proteasome/PA28 complex.
Details: Sample was a mixture of unbound 20S proteasome, 20S proteasome in complex with one PA28 cap, and 20S proteasome in complex with two PA28 caps.
Number of components: 18

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Component #1: protein, 20S proteasome/PA28 complex.

ProteinName: 20S proteasome/PA28 complex.
Details: Sample was a mixture of unbound 20S proteasome, 20S proteasome in complex with one PA28 cap, and 20S proteasome in complex with two PA28 caps.
Recombinant expression: No
MassTheoretical: 230 kDa
SourceSpecies: Plasmodium falciparum 3D7 (eukaryote)

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Component #2: protein, Plasmodium falciparum 20S proteasome

ProteinName: Plasmodium falciparum 20S proteasome / Recombinant expression: No
SourceSpecies: Plasmodium falciparum 3D7 (eukaryote)

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Component #3: protein, 11S activator of the Plasmodium falciparum proteasome.

ProteinName: 11S activator of the Plasmodium falciparum proteasome.
Recombinant expression: No
SourceSpecies: Plasmodium falciparum 3D7 (eukaryote)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #4: protein, 20S proteasome alpha-1 subunit

ProteinName: 20S proteasome alpha-1 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 29.531656 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #5: protein, 20S proteasome alpha-2 subunit

ProteinName: 20S proteasome alpha-2 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 26.556391 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #6: protein, 20S proteasome alpha-3 subunit

ProteinName: 20S proteasome alpha-3 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 27.977664 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #7: protein, 20S proteasome alpha-4 subunit

ProteinName: 20S proteasome alpha-4 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 27.263285 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #8: protein, 20S proteasome alpha-5 subunit

ProteinName: 20S proteasome alpha-5 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 28.417367 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #9: protein, 20S proteasome alpha-6 subunit

ProteinName: 20S proteasome alpha-6 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 28.871697 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #10: protein, 20S proteasome alpha-7 subunit

ProteinName: 20S proteasome alpha-7 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 29.324295 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #11: protein, 20S proteasome beta-1 subunit

ProteinName: 20S proteasome beta-1 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 29.143936 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #12: protein, 20S proteasome beta-2 subunit

ProteinName: 20S proteasome beta-2 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 25.104885 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #13: protein, 20S proteasome beta-3 subunit

ProteinName: 20S proteasome beta-3 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 24.533131 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #14: protein, 20S proteasome beta-4 subunit

ProteinName: 20S proteasome beta-4 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 22.889105 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #15: protein, 20S proteasome beta-5 subunit

ProteinName: 20S proteasome beta-5 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 23.620646 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #16: protein, 20S proteasome beta-6 subunit

ProteinName: 20S proteasome beta-6 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 27.301203 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #17: protein, 20S proteasome beta-7 subunit

ProteinName: 20S proteasome beta-7 subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 30.909893 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7

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Component #18: protein, Proteasome activator PA28

ProteinName: Proteasome activator PA28 / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 33.178773 kDa
SourceSpecies: Plasmodium falciparum (isolate 3D7) (eukaryote) / Strain: isolate 3D7
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.7 mg/mL / pH: 7.4
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 % / Details: wait time 0sec blot time 2sec blot force -1.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 32 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 100000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5200 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 27516 / Details: Images were gain corrected
3D reconstructionSoftware: RELION / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 6DFK
Chain ID: M

Overall bvalue: 99.23
Output model

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