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- EMDB-20073: Reconstruction of the Plasmodium falciparum 20S proteasome in com... -

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Basic information

Entry
Database: EMDB / ID: EMD-20073
TitleReconstruction of the Plasmodium falciparum 20S proteasome in complex with one PA28 activator prepared without chemical stabilisation
Map data
Sample20S proteasome/PA28 complex.
  • Plasmodium falciparum 20S proteasome
  • 11S activator of the Plasmodium falciparum proteasome.
Biological speciesPlasmodium falciparum 3D7 (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 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 / ...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.
History
Header (metadata) releaseNov 7, 2018-
DepositionApr 8, 2019-
Map releaseAug 7, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20073.png

Downloads & links

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Map

FileDownload / File: emd_20073.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
AxesZ (Sec.)Y (Row.)X (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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.014002914 - 0.041436154
Average (Standard dev.)0.00007986513 (±0.0021799023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS344344344
D min/max/mean-0.0140.0410.000

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Supplemental data

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Half map: Half-map 1

Fileemd_20073_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_20073_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: 3

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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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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
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: 622 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 18893 / Details: Images were gain corrected
3D reconstructionSoftware: RELION / Resolution: 4.6 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, 6MUW

6dfk

6muw


Chain ID: M

Overall bvalue: 81.36

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