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- PDB-6yej: Cryo-EM structure of the Full-length disease type human Huntingtin -

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Basic information

Entry
Database: PDB / ID: 6yej
TitleCryo-EM structure of the Full-length disease type human Huntingtin
ComponentsHuntingtin
KeywordsSTRUCTURAL PROTEIN / multivalent scaffold platform / PROTEIN BINDING
Function / homology
Function and homology information


regulation of cAMP-dependent protein kinase activity / regulation of phosphoprotein phosphatase activity / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / microtubule-based transport / vocal learning / regulation of CAMKK-AMPK signaling cascade / positive regulation of mitophagy / profilin binding / vesicle transport along microtubule / positive regulation of cilium assembly ...regulation of cAMP-dependent protein kinase activity / regulation of phosphoprotein phosphatase activity / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / microtubule-based transport / vocal learning / regulation of CAMKK-AMPK signaling cascade / positive regulation of mitophagy / profilin binding / vesicle transport along microtubule / positive regulation of cilium assembly / presynaptic cytosol / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / positive regulation of aggrephagy / postsynaptic cytosol / positive regulation of lipophagy / dynein intermediate chain binding / beta-tubulin binding / Golgi organization / dynactin binding / establishment of mitotic spindle orientation / Regulation of MECP2 expression and activity / autophagosome / inclusion body / heat shock protein binding / centriole / negative regulation of extrinsic apoptotic signaling pathway / protein destabilization / cytoplasmic vesicle membrane / kinase binding / p53 binding / late endosome / transmembrane transporter binding / early endosome / positive regulation of apoptotic process / axon / dendrite / apoptotic process / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / protein-containing complex / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Huntingtin / Huntingtin, middle-repeat / Huntingtin family / : / : / : / Huntingtin, N-terminal HEAT / Huntingtin, bridge / Huntingtin, N-terminal HEAT 1 / Huntingtin, C-terminal HEAT ...Huntingtin / Huntingtin, middle-repeat / Huntingtin family / : / : / : / Huntingtin, N-terminal HEAT / Huntingtin, bridge / Huntingtin, N-terminal HEAT 1 / Huntingtin, C-terminal HEAT / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 18.2 Å
AuthorsTame, G. / Jung, T. / Dal Perraro, M. / Hebert, H. / Song, J.
Funding support Korea, Republic Of, 2items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2016-K1A1A2912057 Korea, Republic Of
National Research Foundation (NRF, Korea)2016R1A2B3006293 Korea, Republic Of
CitationJournal: Structure / Year: 2020
Title: The Polyglutamine Expansion at the N-Terminal of Huntingtin Protein Modulates the Dynamic Configuration and Phosphorylation of the C-Terminal HEAT Domain.
Authors: Taeyang Jung / Baehyun Shin / Giorgio Tamo / Hyeongju Kim / Ravi Vijayvargia / Alexander Leitner / Maria J Marcaida / Juan Astorga-Wells / Roy Jung / Ruedi Aebersold / Matteo Dal Peraro / ...Authors: Taeyang Jung / Baehyun Shin / Giorgio Tamo / Hyeongju Kim / Ravi Vijayvargia / Alexander Leitner / Maria J Marcaida / Juan Astorga-Wells / Roy Jung / Ruedi Aebersold / Matteo Dal Peraro / Hans Hebert / Ihn Sik Seong / Ji-Joon Song /
Abstract: The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural ...The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT. These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.
History
DepositionMar 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Huntingtin


Theoretical massNumber of molelcules
Total (without water)355,2791
Polymers355,2791
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, cross-linking, gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area94910 Å2
MethodPISA

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Components

#1: Protein Huntingtin / / Huntington disease protein / HD protein


Mass: 355278.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HTT, HD, IT15 / Plasmid: pFASTBAC1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P42858

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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 human disease-type huntingtin / Type: ORGANELLE OR CELLULAR COMPONENT
Details: A disease type huntingtin with 78 polyglutamine repeat tract
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9 cell / Plasmid: pFASTBAC1
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaClSodium chloride1
220 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
SpecimenConc.: 0.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: HTT was mixed with final 0.05% of Octyl glucoside.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K / Details: 30 seconds incubation 8 seconds blotting

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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 / Calibrated magnification: 47170 X / Nominal defocus max: 400 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 33.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1500
EM imaging opticsEnergyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE
Image scansMovie frames/image: 24 / Used frames/image: 1-24

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment3D auto refine
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 45039 / Details: manual picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 18.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21781 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6EZ8

6ez8


Pdb chain-ID: A / Pdb chain residue range: 91-3198

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