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- EMDB-10500: Cryo-EM structure of AtNBR1-PB1 filament (S-type) -

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Basic information

Entry
Database: EMDB / ID: EMD-10500
TitleCryo-EM structure of AtNBR1-PB1 filament (S-type)
Map data
SampleAtNBR1-PB1 (S-type):
Protein NBR1 homolog
Function / homology
Function and homology information


vacuole / protein polymerization / ubiquitin binding / autophagy / protein transport / zinc ion binding / cytoplasm
Zinc finger, ZZ-type / PB1 domain / UBA-like superfamily / Immunoglobulin-like fold / Next to BRCA1, central domain
Protein NBR1 homolog
Biological speciesArabidopsis thaliana (thale cress)
Methodhelical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsJakobi AJ / Sachse C
Funding support Germany, 3 items
OrganizationGrant numberCountry
European CommissionPIEF-GA-2012-331285 Germany
German Research FoundationEXC1074 Germany
European CommissionPCOFUND-GA-2008-229597 Germany
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.
Authors: Arjen J Jakobi / Stefan T Huber / Simon A Mortensen / Sebastian W Schultz / Anthimi Palara / Tanja Kuhm / Birendra Kumar Shrestha / Trond Lamark / Wim J H Hagen / Matthias Wilmanns / Terje ...Authors: Arjen J Jakobi / Stefan T Huber / Simon A Mortensen / Sebastian W Schultz / Anthimi Palara / Tanja Kuhm / Birendra Kumar Shrestha / Trond Lamark / Wim J H Hagen / Matthias Wilmanns / Terje Johansen / Andreas Brech / Carsten Sachse /
Abstract: p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 ...p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
Validation ReportPDB-ID: 6tgp

SummaryFull reportAbout validation report
History
DepositionNov 17, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseFeb 19, 2020-
UpdateFeb 19, 2020-
Current statusFeb 19, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tgp
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tgp
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10500.map.gz / Format: CCP4 / Size: 5.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 140 pix.
= 194.04 Å
1.39 Å/pix.
x 100 pix.
= 138.6 Å
1.39 Å/pix.
x 100 pix.
= 138.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.386 Å
Density
Contour LevelBy AUTHOR: 2.1 / Movie #1: 1.8
Minimum - Maximum-2.8809712 - 3.9452217
Average (Standard dev.)0.00000000199 (±0.99999964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100140
Spacing100100140
CellA: 138.6 Å / B: 138.6 Å / C: 194.04001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3861.3861.386
M x/y/z100100140
origin x/y/z0.0000.0000.000
length x/y/z138.600138.600194.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100140
D min/max/mean-2.8813.9450.000

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Supplemental data

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Sample components

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Entire AtNBR1-PB1 (S-type)

EntireName: AtNBR1-PB1 (S-type) / Details: Helical filament of AtNBR1-PB1 domain (1-95) / Number of components: 2

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Component #1: protein, AtNBR1-PB1 (S-type)

ProteinName: AtNBR1-PB1 (S-type) / Details: Helical filament of AtNBR1-PB1 domain (1-95) / Recombinant expression: No
SourceSpecies: Arabidopsis thaliana (thale cress)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #2: protein, Protein NBR1 homolog

ProteinName: Protein NBR1 homolog / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 10.198557 kDa
SourceSpecies: Arabidopsis thaliana (thale cress)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Filament / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 5.905 Å / Delta phi: -31.17 %deg;
Sample solutionSpecimen conc.: 0.4 mg/mL / pH: 7.5
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 288 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 14 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 4500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 684

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: SPRING / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Correlation coefficient / Refinement space: REAL
Output model

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