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- EMDB-5130: 3D cryo-EM Structure of Archaeal 20S Proteasome in Complex with t... -

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Basic information

Entry
Database: EMDB / ID: EMD-5130
Title3D cryo-EM Structure of Archaeal 20S Proteasome in Complex with the C-terminus of PAN
Map dataThis is the 3D volume of T.acidophilum 20S proteasome in complex with a hybrid activator of PA26 and PAN
Sample
  • Sample: Archaeal 20S proteasome in complex with a hybrid of PA26 and PAN
  • Protein or peptide: 20S proteasome, PA26-PANProteasome
KeywordsProteasome / proteasomal ATPase / protein degradation / AAA ATPase / x-ray crystallography / electron cryomicroscopy
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsYu Y / Smith DM / Kim H / Rodriguez V / Goldberg AL / Cheng Y
CitationJournal: EMBO J / Year: 2010
Title: Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome-ATPase interactions.
Authors: Yadong Yu / David M Smith / Ho Min Kim / Victor Rodriguez / Alfred L Goldberg / Yifan Cheng /
Abstract: Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on ...Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C-termini a conserved hydrophobic-tyrosine-X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C-terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C-terminal HbYX motif and the 20S alpha-subunits and indicates that the intersubunit pocket in the 20S undergoes an induced-fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening.
History
DepositionAug 18, 2009-
Header (metadata) releaseDec 21, 2009-
Map releaseDec 21, 2009-
UpdateSep 23, 2011-
Current statusSep 23, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5130_1.tif

Downloads & links

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Map

FileDownload / File: emd_5130.map.gz / Format: CCP4 / Size: 9.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the 3D volume of T.acidophilum 20S proteasome in complex with a hybrid activator of PA26 and PAN
Voxel sizeX: 1.79103 Å / Y: 1.79103 Å / Z: 1.79102 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-2.91739 - 3.55491
Average (Standard dev.)0.0676975 (±0.492819)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-58-58-98
Dimensions116116196
Spacing116116196
CellA: 207.76 Å / B: 207.76 Å / C: 351.04 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.79103448275861.79103448275861.7910204081633
M x/y/z116116196
origin x/y/z0.0000.0000.000
length x/y/z207.760207.760351.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-58-58-98
NC/NR/NS116116196
D min/max/mean-2.9173.5550.068

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Supplemental data

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Sample components

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Entire : Archaeal 20S proteasome in complex with a hybrid of PA26 and PAN

EntireName: Archaeal 20S proteasome in complex with a hybrid of PA26 and PAN
Components
  • Sample: Archaeal 20S proteasome in complex with a hybrid of PA26 and PAN
  • Protein or peptide: 20S proteasome, PA26-PANProteasome

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Supramolecule #1000: Archaeal 20S proteasome in complex with a hybrid of PA26 and PAN

SupramoleculeName: Archaeal 20S proteasome in complex with a hybrid of PA26 and PAN
type: sample / ID: 1000 / Details: Proteins were expressed from E.coli.
Oligomeric state: 20S proteasome is composed of 4 heptamer rings coaxially stacked to which heptamers of the PA26 PAN hybrid cap on both ends
Number unique components: 3
Molecular weightTheoretical: 1.1 MDa

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Macromolecule #1: 20S proteasome, PA26-PAN

MacromoleculeName: 20S proteasome, PA26-PAN / type: protein_or_peptide / ID: 1 / Name.synonym: 20S proteasome, PA26-PAN / Number of copies: 1
Oligomeric state: 2 heptamers of 20S alpha subunit, 2 heptamers of 20S beta subunit, and 2 heptamers of PA26-PAN
Recombinant expression: Yes
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 1.1 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pRSET-A, pET-15b

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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

Concentration0.3 mg/mL
BufferpH: 8.5
Details: 50 mM Tris pH 8.5, 1mM DTT, 10 mM MgCl2, 5% Glycerol
GridDetails: 400 mesh Cu grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Gatan CT-3500 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 90 K / Max: 90 K / Average: 90 K
Detailsdata was collected using Leginon
DateSep 17, 2007
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 20 e/Å2 / Details: images were recorded on CCD camera
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Details: D7 symmetry was applied / Number images used: 13020
DetailsThe particles were manually selected

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