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- EMDB-4944: Cryo-EM 3D map of the N-terminal GFP tagged normal type Huntingtin -

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Basic information

Entry
Database: EMDB / ID: EMD-4944
TitleCryo-EM 3D map of the N-terminal GFP tagged normal type Huntingtin
Map dataThe cryo-EM map of the N-terminal GFP tagged normal Huntingtin.
Sample
  • Organelle or cellular component: N-terminal GFP tagged normal type huntingtin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.3 Å
AuthorsJung T / Tamo G / Dal Perraro M / Hebert H / Song J
Funding support Korea, Republic Of, 2 items
OrganizationGrant numberCountry
National Research Foundation (Korea)2016-K1A1A2912057 Korea, Republic Of
National Research Foundation (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
DepositionMay 8, 2019-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4944.png

Downloads & links

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Map

FileDownload / File: emd_4944.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe cryo-EM map of the N-terminal GFP tagged normal Huntingtin.
Voxel sizeX=Y=Z: 2.20833 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.014368322 - 0.05213319
Average (Standard dev.)0.00010867433 (±0.0052492046)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin333
Dimensions120120120
Spacing120120120
CellA=B=C: 264.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.20833333333332.20833333333332.2083333333333
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z265.000265.000265.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS333
NC/NR/NS120120120
D min/max/mean-0.0140.0520.000

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Supplemental data

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Sample components

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Entire : N-terminal GFP tagged normal type huntingtin

EntireName: N-terminal GFP tagged normal type huntingtin
Components
  • Organelle or cellular component: N-terminal GFP tagged normal type huntingtin

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Supramolecule #1: N-terminal GFP tagged normal type huntingtin

SupramoleculeName: N-terminal GFP tagged normal type huntingtin / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: GFP inserted at N-terminal at normal Huntingtin.
Source (natural)Organism: Homo sapiens (human)
Molecular weightExperimental: 350 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 cell / Recombinant plasmid: pFASTBAC1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.08 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 15 K / Instrument: FEI VITROBOT MARK II / Details: 30 seconds incubation 9 seconds blotting.
DetailsHTT was mixed with final 0.05% of Octyl glucoside.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47170 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.6 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-40 / Number real images: 2226 / Average exposure time: 8.0 sec. / Average electron dose: 47.72 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 56538 / Details: manual picking
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: INSILICO MODEL / In silico model: in RELION 3D initial model building
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 1 / Avg.num./class: 17689 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1) / Software - details: 3D auto refine
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 15.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 17689
FSC plot (resolution estimation)

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