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- EMDB-4119: In situ TPPII 36mer -

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Basic information

Entry
Database: EMDB / ID: EMD-4119
TitleIn situ TPPII 36mer
Map dataTPPII 36mer
Sample
  • Complex: Tripeptidyl peptidase II
Biological speciesRattus (rat)
Methodsubtomogram averaging / cryo EM / Resolution: 27.8 Å
AuthorsFukuda Y / Beck F / Baumeister W
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: In situ structural studies of tripeptidyl peptidase II (TPPII) reveal spatial association with proteasomes.
Authors: Yoshiyuki Fukuda / Florian Beck / Jürgen M Plitzko / Wolfgang Baumeister /
Abstract: Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies ...Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a two-stranded linear polymer that assembles to form a spindle-shaped complex of ∼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII's role in postproteasomal degradation.
History
DepositionSep 28, 2016-
Header (metadata) releaseNov 9, 2016-
Map releaseApr 12, 2017-
UpdateAug 26, 2020-
Current statusAug 26, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4119.jpg

emd_4119.png

Downloads & links

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Map

FileDownload / File: emd_4119.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTPPII 36mer
Voxel sizeX=Y=Z: 4.21 Å
Density
Contour LevelBy AUTHOR: 4.1 / Movie #1: 4.1
Minimum - Maximum-0.65250033 - 0.9482326
Average (Standard dev.)0.0035667506 (±0.09438034)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 842.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.214.214.21
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z842.000842.000842.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-6.95110.0090.000

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Supplemental data

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Sample components

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Entire : Tripeptidyl peptidase II

EntireName: Tripeptidyl peptidase II
Components
  • Complex: Tripeptidyl peptidase II

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Supramolecule #1: Tripeptidyl peptidase II

SupramoleculeName: Tripeptidyl peptidase II / type: complex / ID: 1 / Parent: 0 / Details: 36mer
Source (natural)Organism: Rattus (rat) / Organ: brain / Tissue: hippocampus
Molecular weightTheoretical: 5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1/4 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 1000.0 nm
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV
Detailsin situ

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 33000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-10 / Number grids imaged: 2 / Average exposure time: 1.4 sec. / Average electron dose: 1.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 70 / Number images used: 81 / Reference model: emdb 2036 / Method: template matching / Software - Name: PyTom (ver. 0.97)
Final angle assignmentType: OTHER / Software - Name: PyTom (ver. 0.97) / Details: subtomogram averaging
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 27.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PyTom (ver. 0.97) / Number subtomograms used: 81

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