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- EMDB-1732: Three-dimensional structure of Tripeptidyl peptidase II from Dros... -

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Basic information

Entry
Database: EMDB / ID: EMD-1732
TitleThree-dimensional structure of Tripeptidyl peptidase II from Drosophila melanogaster - a spindle shaped homo-40mer
Map dataEM density map of TPPII from Drosophila melanogaster
Sample
  • Sample: Tripeptidyl peptidase II complex of Drosophila melanogaster
  • Protein or peptide: Tripeptidyl peptidase II
KeywordsProtease / subtilase
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsChuang CK / Rockel B / Seyit G / Walian PJ / Schoenegge A-M / Peters J / Zwart PH / Baumeister W / Jap BK
CitationJournal: Nat Struct Mol Biol / Year: 2010
Title: Hybrid molecular structure of the giant protease tripeptidyl peptidase II.
Authors: Crystal K Chuang / Beate Rockel / Gönül Seyit / Peter J Walian / Anne-Marie Schönegge / Jürgen Peters / Petrus H Zwart / Wolfgang Baumeister / Bing K Jap /
Abstract: Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, ...Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach. We solved the structure of the dimer by X-ray crystallography and docked it into the three-dimensional map of the holocomplex, which we obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.
History
DepositionMay 21, 2010-
Header (metadata) releaseJun 16, 2010-
Map releaseAug 6, 2010-
UpdateNov 1, 2010-
Current statusNov 1, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.16
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd1732.png

Downloads & links

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Map

FileDownload / File: emd_1732.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM density map of TPPII from Drosophila melanogaster
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 320 pix.
= 691.2 Å		2.16 Å/pix.
x 320 pix.
= 691.2 Å		2.16 Å/pix.
x 320 pix.
= 691.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.16 / Movie #1: 2.16
Minimum - Maximum-7.54674 - 12.714399999999999
Average (Standard dev.)-0.000000000183617 (±0.99993)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 691.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z691.200691.200691.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-100-100-100
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-7.54712.714-0.000

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Supplemental data

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Sample components

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Entire : Tripeptidyl peptidase II complex of Drosophila melanogaster

EntireName: Tripeptidyl peptidase II complex of Drosophila melanogaster
Components
  • Sample: Tripeptidyl peptidase II complex of Drosophila melanogaster
  • Protein or peptide: Tripeptidyl peptidase II

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Supramolecule #1000: Tripeptidyl peptidase II complex of Drosophila melanogaster

SupramoleculeName: Tripeptidyl peptidase II complex of Drosophila melanogaster
type: sample / ID: 1000 / Oligomeric state: 40-mer / Number unique components: 1
Molecular weightTheoretical: 6 MDa

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Macromolecule #1: Tripeptidyl peptidase II

MacromoleculeName: Tripeptidyl peptidase II / type: protein_or_peptide / ID: 1 / Name.synonym: TPPII / Number of copies: 40 / Oligomeric state: 40-mer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit Fly / Location in cell: Cytoplasmic
Molecular weightTheoretical: 150 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 7.8 / Details: 40 mM KPO4, pH 7.8, 2 mM DTT, 5% glycerol
GridDetails: Holey carbon 200 mesh copper grids covered with thin carbon film
VitrificationCryogen name: ETHANE / Instrument: OTHER
Method: Sample was applied on grid, blotted briefly and washed twice with buffer (40 mM ammonium sulfate, pH 7.5)

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 15 e/Å2
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.7 µm
Sample stageSpecimen holder: Side-entry / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each CCD frame
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 37195

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