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- EMDB-10501: Cryo-EM structure of p62-PB1 filament (L-type) -

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Basic information

Entry
Database: EMDB / ID: EMD-10501
TitleCryo-EM structure of p62-PB1 filament (L-type)
Map data
Samplep62-PB1 domain filament (L-type):
Sequestosome-1
Function / homology
Function and homology information


protein localization to perinuclear region of cytoplasm / selective autophagy / regulation of Ras protein signal transduction / Lewy body / amphisome / response to mitochondrial depolarisation / aggrephagy / regulation of protein complex stability / mitophagy / endosome organization ...protein localization to perinuclear region of cytoplasm / selective autophagy / regulation of Ras protein signal transduction / Lewy body / amphisome / response to mitochondrial depolarisation / aggrephagy / regulation of protein complex stability / mitophagy / endosome organization / phagophore assembly site / K63-linked polyubiquitin modification-dependent protein binding / regulation of mitochondrion organization / autolysosome / immune system process / sperm midpiece / aggresome / regulation of I-kappaB kinase/NF-kappaB signaling / autophagy of mitochondrion / ionotropic glutamate receptor binding / autophagosome / inclusion body / sarcomere / negative regulation of protein ubiquitination / response to ischemia / mitochondrion organization / SH2 domain binding / ubiquitin binding / protein kinase C binding / protein localization / P-body / endosomal transport / positive regulation of long-term synaptic potentiation / positive regulation of protein localization to plasma membrane / macroautophagy / receptor tyrosine kinase binding / PML body / interleukin-1-mediated signaling pathway / autophagy / late endosome / ubiquitin-dependent protein catabolic process / cell differentiation / intracellular signal transduction / positive regulation of protein phosphorylation / positive regulation of apoptotic process / intracellular membrane-bounded organelle / apoptotic process / protein-containing complex binding / protein serine/threonine kinase activity / ubiquitin protein ligase binding / protein kinase binding / negative regulation of apoptotic process / endoplasmic reticulum / negative regulation of transcription by RNA polymerase II / enzyme binding / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / cytosol / cytoplasm
Zinc finger, ZZ-type / Sequestosome-1, UBA domain / PB1 domain / UBA-like superfamily / Ubiquitin-associated domain / Zinc finger, ZZ-type superfamily / Sequestosome-1, PB1 domain
Sequestosome-1
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHuber ST / Jakobi AJ / Mortensen SA / Sachse C
Funding support Germany, 3 items
OrganizationGrant numberCountry
European CommissionPIEF-GA-2012-331285
German Research FoundationEXC1074 Germany
European CommissionPCOFUND-GA-2008-229597
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: 6tgy

SummaryFull reportAbout validation report
History
DepositionNov 18, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseFeb 12, 2020-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tgy
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tgy
  • 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_10501.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 266.24 Å
1.04 Å/pix.
x 256 pix.
= 266.24 Å
1.04 Å/pix.
x 256 pix.
= 266.24 Å

Surface

Projections

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.0325 / Movie #1: 0.0325
Minimum - Maximum-0.0596657 - 0.11241907
Average (Standard dev.)0.001132681 (±0.006983475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0600.1120.001

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Supplemental data

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Segmentation: #1

Fileemd_10501_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unfiltered map

Fileemd_10501_additional.map
AnnotationUnfiltered map
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AxesZYX

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Half map: Unfiltered half map #2

Fileemd_10501_half_map_1.map
AnnotationUnfiltered half map #2
Projections & Slices
AxesZYX

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Density Histograms

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Half map: Unfiltered half map #1

Fileemd_10501_half_map_2.map
AnnotationUnfiltered half map #1
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AxesZYX

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Slices (1/2)
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Sample components

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Entire p62-PB1 domain filament (L-type)

EntireName: p62-PB1 domain filament (L-type) / Number of components: 2

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Component #1: protein, p62-PB1 domain filament (L-type)

ProteinName: p62-PB1 domain filament (L-type) / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #2: protein, Sequestosome-1

ProteinName: Sequestosome-1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 13.704609 kDa
SourceSpecies: Homo sapiens (human)
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: C2 (2 fold cyclic) / Delta z: 4.787 Å / Delta phi: 77.29 %deg;
Sample solutionBuffer solution: 50 mM TRIS (pH 7.5), 100 mM NaCl, 4 mM DTT / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283 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: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD / Defocus: 500.0 - 2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2277

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 2KKC
Output model

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