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- EMDB-0755: Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-ter... -

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Basic information

Entry
Database: EMDB / ID: EMD-0755
TitleRecombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-terminal truncation.
Map dataPbaA/PF0014 complex with 30 residue C-terminal truncation at 7.3A
Sample
  • Complex: Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-terminal truncation.
Biological speciesPyrococcus furiosus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsBurton-Smith RN / Song C / Murata K
Funding support Japan, 1 items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan) Japan
CitationJournal: Sci Rep / Year: 2020
Title: Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation.
Authors: Maho Yagi-Utsumi / Arunima Sikdar / Chihong Song / Jimin Park / Rintaro Inoue / Hiroki Watanabe / Raymond N Burton-Smith / Toshiya Kozai / Tatsuya Suzuki / Atsuji Kodama / Kentaro Ishii / ...Authors: Maho Yagi-Utsumi / Arunima Sikdar / Chihong Song / Jimin Park / Rintaro Inoue / Hiroki Watanabe / Raymond N Burton-Smith / Toshiya Kozai / Tatsuya Suzuki / Atsuji Kodama / Kentaro Ishii / Hirokazu Yagi / Tadashi Satoh / Susumu Uchiyama / Takayuki Uchihashi / Keehyoung Joo / Jooyoung Lee / Masaaki Sugiyama / Kazuyoshi Murata / Koichi Kato /
Abstract: Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a ...Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.
History
DepositionAug 21, 2019-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 19, 2020-
UpdateFeb 19, 2020-
Current statusFeb 19, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0755.png

Downloads & links

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Map

FileDownload / File: emd_0755.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPbaA/PF0014 complex with 30 residue C-terminal truncation at 7.3A
Voxel sizeX=Y=Z: 0.93 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.004
Minimum - Maximum-0.002092986 - 0.020782728
Average (Standard dev.)0.0000386074 (±0.0014761806)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 306.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.930.930.93
M x/y/z330330330
origin x/y/z0.0000.0000.000
length x/y/z306.900306.900306.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS330330330
D min/max/mean-0.0020.0210.000

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Supplemental data

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Sample components

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Entire : Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-ter...

EntireName: Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-terminal truncation.
Components
  • Complex: Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-terminal truncation.

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Supramolecule #1: Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-ter...

SupramoleculeName: Recombinant Pyrococcus furiosus PbaA/PF0014 with 30 residue C-terminal truncation.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 370 KDa

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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: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.5) / Number images used: 10251
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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