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- EMDB-30864: Unliganded EGFR averaged cluster 21 -

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Basic information

Entry
Database: EMDB / ID: EMD-30864
TitleUnliganded EGFR averaged cluster 21
Map data
Sample
  • Complex: Unliganded EGFR averaged cluster 21
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM / Resolution: 15.0 Å
AuthorsPurba ER / Saita EI / Akhouri RR / Ofverstedt LG / Wilken G / Skoglund U / Maruyama IN
CitationJournal: Front Endocrinol (Lausanne) / Year: 2022
Title: Allosteric activation of preformed EGF receptor dimers by a single ligand binding event.
Authors: Endang R Purba / Ei-Ichiro Saita / Reetesh R Akhouri / Lars-Goran Öfverstedt / Gunnar Wilken / Ulf Skoglund / Ichiro N Maruyama /
Abstract: Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is ...Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor . Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.
History
DepositionJan 7, 2021-
Header (metadata) releaseJun 23, 2021-
Map releaseJun 23, 2021-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.54
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.54
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30864.png

Downloads & links

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Map

FileDownload / File: emd_30864.map.gz / Format: CCP4 / Size: 5.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.258 Å
Density
Contour LevelMovie #1: 0.54
Minimum - Maximum0.1677199 - 0.80406636
Average (Standard dev.)0.28988537 (±0.13262056)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions110110110
Spacing110110110
CellA=B=C: 248.38002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.2582.2582.258
M x/y/z110110110
origin x/y/z0.0000.0000.000
length x/y/z248.380248.380248.380
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ847791
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS110110110
D min/max/mean0.1680.8040.290

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Supplemental data

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Sample components

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Entire : Unliganded EGFR averaged cluster 21

EntireName: Unliganded EGFR averaged cluster 21
Components
  • Complex: Unliganded EGFR averaged cluster 21

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Supramolecule #1: Unliganded EGFR averaged cluster 21

SupramoleculeName: Unliganded EGFR averaged cluster 21 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: DH5a / Location in cell: cellular membrane
Molecular weightTheoretical: 300 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris-HCL
200.0 mMSodium chloride
0.2 mMDodecyl maltocide
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 277.0 K / Instrument: FEI VITROBOT MARK IV
Detailsthe sample was monodisperse
Cryo protectantNo
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 134 / Average exposure time: 1.8 sec. / Average electron dose: 90.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 1.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 37000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Number images used: 134

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Atomic model buiding 1

Initial model
PDB IDChain

1nql

chain_id: A, residue_range: 3-614

2m0b

chain_id: A, residue_range: 634-677

2m0b

chain_id: B, residue_range: 634-677

3gt8

chain_id: A, residue_range: 676-990

3gt8

chain_id: B, residue_range: 677-989
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient

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