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- EMDB-5288: The single particle reconstruction of the human Toll-like recepto... -

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Basic information

Entry
Database: EMDB / ID: EMD-5288
TitleThe single particle reconstruction of the human Toll-like receptor 5 ectodomain in detergent
Map dataThis is the volume of the hTLR5 ectodomain.
Sample
  • Sample: toll-like receptor 5 ectodomain
  • Protein or peptide: Toll-like receptor
KeywordsToll-like receptor 5 / ectodomain
Function / homologyToll-like receptor
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsZhou K / Kanai R / Lee P / Wang HW / Modis Y
CitationJournal: J Struct Biol / Year: 2012
Title: Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin.
Authors: Kaifeng Zhou / Ryuta Kanai / Phong Lee / Hong-Wei Wang / Yorgo Modis /
Abstract: The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain ...The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.
History
DepositionMay 2, 2011-
Header (metadata) releaseMay 5, 2011-
Map releaseDec 19, 2011-
UpdateDec 19, 2011-
Current statusDec 19, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.57
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.57
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5288.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the volume of the hTLR5 ectodomain.
Voxel sizeX=Y=Z: 2.14 Å
Density
Contour LevelBy AUTHOR: 3.57 / Movie #1: 3.57
Minimum - Maximum-5.61073 - 11.9316
Average (Standard dev.)0.000000000924672 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 214 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.142.142.14
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z214.000214.000214.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-5.61111.9320.000

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Supplemental data

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Sample components

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Entire : toll-like receptor 5 ectodomain

EntireName: toll-like receptor 5 ectodomain
Components
  • Sample: toll-like receptor 5 ectodomain
  • Protein or peptide: Toll-like receptor

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Supramolecule #1000: toll-like receptor 5 ectodomain

SupramoleculeName: toll-like receptor 5 ectodomain / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: dimer / Number unique components: 2
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa / Method: gel filtration

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Macromolecule #1: Toll-like receptor

MacromoleculeName: Toll-like receptor / type: protein_or_peptide / ID: 1 / Name.synonym: TLR5 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Sf9 insect cells / Organelle: membrane / Location in cell: plasma membrane
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Recombinant expressionOrganism: Sf9 insect cells / Recombinant plasmid: pAcGP67-A
SequenceInterPro: Toll-like receptor

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.005 mg/mL
BufferpH: 7.5 / Details: 10 mM TEA, 0.15 M NaCl
StainingType: NEGATIVE
Details: The protein solution was applied onto a thin-carbon-covered holey carbon copper grid and negatively stained in 1% uranyl-formate solution for 1 minute.
GridDetails: thin carbon covered home-made holey carbon on 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 52000
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
DateAug 1, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 50

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Image processing

Final two d classificationNumber classes: 100
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC SPIDER
Details: Final maps were calculated from all the particles by back-projection reconstruction.
Number images used: 3087

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