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- EMDB-10499: Cryo-EM structure of AtNBR1-PB1 filament (L-type) -

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Basic information

Entry
Database: EMDB / ID: EMD-10499
TitleCryo-EM structure of AtNBR1-PB1 filament (L-type)
Map data
Sample
  • Complex: AtNBR1-PB1 (L-type)
    • Protein or peptide: Protein NBR1 homolog
Function / homology
Function and homology information


protein targeting to vacuole involved in autophagy / vacuole / protein polymerization / autophagosome / ubiquitin binding / protein transport / zinc ion binding / cytoplasm
Similarity search - Function
Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / PB1 domain / PB1 domain / PB1 domain / Zinc-binding domain, present in Dystrophin, CREB-binding protein. / Zinc finger, ZZ type / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. ...Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / PB1 domain / PB1 domain / PB1 domain / Zinc-binding domain, present in Dystrophin, CREB-binding protein. / Zinc finger, ZZ type / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. / UBA-like superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Protein NBR1 homolog
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodhelical reconstruction / cryo EM / Resolution: 3.9 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.
History
DepositionNov 17, 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: 2.53
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.53
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tgn
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tgn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10499.png

Downloads & links

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Map

FileDownload / File: emd_10499.map.gz / Format: CCP4 / Size: 5.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.386 Å
Density
Contour LevelBy AUTHOR: 2.53 / Movie #1: 2.53
Minimum - Maximum-3.2189863 - 9.525518
Average (Standard dev.)-0.00000001211 (±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-3.2199.526-0.000

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Supplemental data

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Additional map: Unfiltered map

Fileemd_10499_additional.map
AnnotationUnfiltered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AtNBR1-PB1 (L-type)

EntireName: AtNBR1-PB1 (L-type)
Components
  • Complex: AtNBR1-PB1 (L-type)
    • Protein or peptide: Protein NBR1 homolog

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Supramolecule #1: AtNBR1-PB1 (L-type)

SupramoleculeName: AtNBR1-PB1 (L-type) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Helical filament of AtNBR 1-PB1 domain (1-95)
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Protein NBR1 homolog

MacromoleculeName: Protein NBR1 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 10.198557 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MESTANALVV KVSYGGVLRR FRVPVKANGQ LDLEMAGLKE KIAALFNLSA DAELSLTYSD EDGDVVALVD DNDLFDVTNQ RLKFLKINV NAGVS

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMHEPES
150.0 mMSodium chlorideNaClSodium chloride
0.05 mMTCEP
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-7 / Average electron dose: 14.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPRING (ver. 0.85)
Final reconstructionApplied symmetry - Helical parameters - Δz: 6.721 Å
Applied symmetry - Helical parameters - Δ&Phi: -31.44 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPRING (ver. 0.85) / Number images used: 25387

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6tgn:
Cryo-EM structure of AtNBR1-PB1 filament (L-type)

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