|Entry||Database: EMDB / ID: 4119|
|Title||In situ TPPII 36mer|
|Map data||TPPII 36mer|
|Sample||Tripeptidyl peptidase II|
|Source||Rattus / Rat / mammal / image: Rattus norvegicus|
|Method||Cryo EM / subtomogram averaging / 27.8 Å resolution|
|Authors||Fukuda Y / Beck F / Baumeister W|
|Citation||Journal: 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.
|Date||Deposition: Sep 28, 2016 / Header (metadata) release: Nov 9, 2016 / Map release: Apr 12, 2017 / Last update: Nov 29, 2017|
Downloads & links
|File||emd_4119.map.gz (map file in CCP4 format, 32001 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 4.21 Å|
CCP4 map header:
-Entire Tripeptidyl peptidase II
|Entire||Name: Tripeptidyl peptidase II / Details: 36mer / Number of components: 1|
|Mass||Theoretical: 5 MDa|
-Component #1: protein, Tripeptidyl peptidase II
|Specimen||Specimen state: cell / Method: Cryo EM|
|Sample solution||pH: 7|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE MIXTURE / Temperature: 310 K / Humidity: 90 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.8 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 33000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500 - 1000 nm / Energy filter: GIF / Energy window: 0-20 eV|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt Angle: -60 - 60 deg. / Temperature: K ( 70 - 70 K)|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Image acquisition||Sampling size: 5 microns|
|Processing||Method: subtomogram averaging / Applied symmetry: D2 (2*2 fold dihedral)|
|3D reconstruction||Software: PyTom / Resolution: 27.8 Å / Resolution method: FSC 0.143 CUT-OFF / Euler angles: subtomogram averaging|
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