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- PDB-6tax: Mouse RNF213 wild type protein -

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Basic information

Entry
Database: PDB / ID: 6tax
TitleMouse RNF213 wild type protein
ComponentsRNF213,E3 ubiquitin-protein ligase RNF213,E3 ubiquitin-protein ligase RNF213
KeywordsSIGNALING PROTEIN / RNF213 / E3 LIgase / AAA-Protein
Function / homology
Function and homology information


negative regulation of non-canonical Wnt signaling pathway / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / sprouting angiogenesis / protein autoubiquitination / RING-type E3 ubiquitin transferase / protein homooligomerization / ubiquitin-protein transferase activity / ubiquitin-dependent protein catabolic process / angiogenesis / protein ubiquitination ...negative regulation of non-canonical Wnt signaling pathway / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / sprouting angiogenesis / protein autoubiquitination / RING-type E3 ubiquitin transferase / protein homooligomerization / ubiquitin-protein transferase activity / ubiquitin-dependent protein catabolic process / angiogenesis / protein ubiquitination / ATPase activity / nucleolus / metal ion binding / cytosol / cytoplasm
Zinc finger, RING/FYVE/PHD-type / AAA+ ATPase domain / Zinc finger, RING-type / E3 ubiquitin-protein ligase RNF213 / P-loop containing nucleoside triphosphate hydrolase / Zinc finger, RING-type, conserved site
E3 ubiquitin-protein ligase RNF213
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAhel, J. / Meinhart, A. / Haselbach, D. / Clausen, T.
Funding support Austria, 1items
OrganizationGrant numberCountry
European Research Council (ERC)AdG 694978 Austria
CitationJournal: Elife / Year: 2020
Title: Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism.
Authors: Juraj Ahel / Anita Lehner / Antonia Vogel / Alexander Schleiffer / Anton Meinhart / David Haselbach / Tim Clausen /
Abstract: RNF213 is the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that often leads to brain stroke in adults and children. Characterization of disease-associated ...RNF213 is the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that often leads to brain stroke in adults and children. Characterization of disease-associated mutations has been complicated by the enormous size of RNF213. Here, we present the cryo-EM structure of mouse RNF213. The structure reveals the intricate fold of the 584 kDa protein, comprising an N-terminal stalk, a dynein-like core with six ATPase units, and a multidomain E3 module. Collaboration with UbcH7, a cysteine-reactive E2, points to an unexplored ubiquitin-transfer mechanism that proceeds in a RING-independent manner. Moreover, we show that pathologic MMD mutations cluster in the composite E3 domain, likely interfering with substrate ubiquitination. In conclusion, the structure of RNF213 uncovers a distinct type of an E3 enzyme, highlighting the growing mechanistic diversity in ubiquitination cascades. Our results also provide the molecular framework for investigating the emerging role of RNF213 in lipid metabolism, hypoxia, and angiogenesis.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 31, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: RNF213,E3 ubiquitin-protein ligase RNF213,E3 ubiquitin-protein ligase RNF213
hetero molecules


Theoretical massNumber of molelcules
Total (without water)528,3415
Polymers527,6781
Non-polymers6624
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1040 Å2
ΔGint-13 kcal/mol
Surface area185160 Å2
MethodPISA

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Components

#1: Protein RNF213,E3 ubiquitin-protein ligase RNF213,E3 ubiquitin-protein ligase RNF213 / Mysterin / RING finger protein 213 / RING-type E3 ubiquitin transferase RNF213


Mass: 527678.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Rnf213, Mystr / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: E9Q555, RING-type E3 ubiquitin transferase, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: full length RNF213 / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.58 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: BTI-Tn-5B1-4 / Plasmid: EMBacY
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2200 mMpotassium chlorideKCl1
30.25 mMTCEP1
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: SCD 005 Sputter Coater (BAL-TEC) grid sitting on a glass block during glow discharge
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Chamber temperature: 277 K
Details: sample incubated on the grid for 60 s prior to blotting. blotted for 2 seconds using using Whatman Filter Paper Grade 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 14 sec. / Electron dose: 58 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4749
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1SPHIREparticle selection
2SerialEM3.7.0image acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
8Omodel fitting
9PHENIXmodel fitting
11cryoSPARCv0initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 375000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 374683 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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