+Open data
-Basic information
Entry | Database: PDB / ID: 6tgn | ||||||||||||
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Title | Cryo-EM structure of AtNBR1-PB1 filament (L-type) | ||||||||||||
Components | Protein NBR1 homolog | ||||||||||||
Keywords | SIGNALING PROTEIN / Autophagy / helical filament | ||||||||||||
Function / homology | Function and homology information protein targeting to vacuole involved in autophagy / phagophore assembly site / vacuole / protein polymerization / autophagosome / ubiquitin binding / macroautophagy / zinc ion binding / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Arabidopsis thaliana (thale cress) | ||||||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å | ||||||||||||
Authors | Jakobi, A.J. / Sachse, C. | ||||||||||||
Funding support | Germany, 3items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6tgn.cif.gz | 30.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6tgn.ent.gz | 17.4 KB | Display | PDB format |
PDBx/mmJSON format | 6tgn.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6tgn_validation.pdf.gz | 803 KB | Display | wwPDB validaton report |
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Full document | 6tgn_full_validation.pdf.gz | 802.6 KB | Display | |
Data in XML | 6tgn_validation.xml.gz | 9.1 KB | Display | |
Data in CIF | 6tgn_validation.cif.gz | 11.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/tg/6tgn ftp://data.pdbj.org/pub/pdb/validation_reports/tg/6tgn | HTTPS FTP |
-Related structure data
Related structure data | 10499MC 6tgpC 6tgsC 6tgyC 6th3C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 10198.557 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: NBR1, At4g24690, F22K18.110 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9SB64 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: AtNBR1-PB1 (L-type) / Type: COMPLEX / Details: Helical filament of AtNBR 1-PB1 domain (1-95) / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Arabidopsis thaliana (thale cress) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C | ||||||||||||||||||||
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 59000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 14 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) |
Image scans | Movie frames/image: 7 / Used frames/image: 1-7 |
-Processing
Software | Name: REFMAC / Version: 5.8.0238 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -31.44 ° / Axial rise/subunit: 6.721 Å / Axial symmetry: C2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25387 / Symmetry type: HELICAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3.9→3.9 Å / Cor.coef. Fo:Fc: 0.916 / SU B: 46.351 / SU ML: 0.622 / ESU R: 5.547 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 139.102 Å2
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Refinement step | Cycle: 1 / Total: 672 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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