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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 dataUnsharpened map
Sample
  • Complex: 20S proteasome/PA28 complex.
    • Complex: Plasmodium falciparum 20S proteasome
    • Complex: 11S activator of the Plasmodium falciparum proteasome.
Function / homology
Function and homology information


AUF1 (hnRNP D0) binds and destabilizes mRNA / Cross-presentation of soluble exogenous antigens (endosomes) / ABC-family proteins mediated transport / MAPK6/MAPK4 signaling / : / Orc1 removal from chromatin / CDK-mediated phosphorylation and removal of Cdc6 / KEAP1-NFE2L2 pathway / UCH proteinases / Ub-specific processing proteases ...AUF1 (hnRNP D0) binds and destabilizes mRNA / Cross-presentation of soluble exogenous antigens (endosomes) / ABC-family proteins mediated transport / MAPK6/MAPK4 signaling / : / Orc1 removal from chromatin / CDK-mediated phosphorylation and removal of Cdc6 / KEAP1-NFE2L2 pathway / UCH proteinases / Ub-specific processing proteases / Neddylation / Antigen processing: Ubiquitination & Proteasome degradation / proteasome activator complex / Neutrophil degranulation / proteasome core complex / proteasomal ubiquitin-independent protein catabolic process / regulation of G1/S transition of mitotic cell cycle / endopeptidase activator activity / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / regulation of proteasomal protein catabolic process / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / endopeptidase activity / hydrolase activity / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Proteasome subunit beta 1 / Proteasome activator PA28 / Proteasome activator PA28, C-terminal domain / Proteasome activator superfamily / Proteasome activator PA28, C-terminal domain superfamily / Proteasome activator pa28 beta subunit / Proteasome subunit alpha 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit ...Proteasome subunit beta 1 / Proteasome activator PA28 / Proteasome activator PA28, C-terminal domain / Proteasome activator superfamily / Proteasome activator PA28, C-terminal domain superfamily / Proteasome activator pa28 beta subunit / Proteasome subunit alpha 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / 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 beta / Proteasome subunit alpha type-2, putative / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Proteasome subunit beta type-6, putative / Proteasome subunit beta / Proteasome activator 28 subunit beta, putative / Proteasome subunit beta / Proteasome subunit alpha type-6, putative / Proteasome subunit alpha type ...Proteasome subunit beta / Proteasome subunit alpha type-2, putative / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Proteasome subunit beta type-6, putative / Proteasome subunit beta / Proteasome activator 28 subunit beta, putative / Proteasome subunit beta / Proteasome subunit alpha type-6, putative / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit beta / Proteasome subunit alpha type-1, putative / Proteasome subunit beta
Similarity search - Component
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
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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)
AnnotationUnsharpened map
Voxel sizeX=Y=Z: 1.31 Å
Density
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.
Components
  • Complex: 20S proteasome/PA28 complex.
    • Complex: Plasmodium falciparum 20S proteasome
    • Complex: 11S activator of the Plasmodium falciparum proteasome.

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Supramolecule #1: 20S proteasome/PA28 complex.

SupramoleculeName: 20S proteasome/PA28 complex. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
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.
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)
Molecular weightTheoretical: 230 KDa

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Supramolecule #2: Plasmodium falciparum 20S proteasome

SupramoleculeName: Plasmodium falciparum 20S proteasome / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#14
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)

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Supramolecule #3: 11S activator of the Plasmodium falciparum proteasome.

SupramoleculeName: 11S activator of the Plasmodium falciparum proteasome.
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #15
Source (natural)Organism: Plasmodium falciparum 3D7 (eukaryote)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)

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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

Concentration0.7 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chlorideSodium Chloride
5.0 mMMgCl2Magnesium Chloride
1.0 mMDTTDithiothreitol
25.0 mMTris-HClTrisTris hydrochloride
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: wait time 0sec blot time 2sec blot force -1.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: 1.0 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 622 / Average exposure time: 10.0 sec. / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 18893 / Details: Relion autopick from 20S map
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER
Details: 60 A filtered map of single capped proteasome fixed.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 18893
DetailsImages were gain corrected
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6dfk

chain_id: M

6muw

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 81.36

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