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- EMDB-3244: Electron negative-staining microscopy of an aerolysin-like protein -

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Basic information

Entry
Database: EMDB / ID: EMD-3244
TitleElectron negative-staining microscopy of an aerolysin-like protein
Map dataReconstruction of mutant aerolysin-like protein
Sample
  • Sample: Dln1 (a zebrafish aerolysin-like protein) oligomer on phospholipid monolayer
  • Protein or peptide: Dln1
Function / homology
Function and homology information


disaccharide binding / pore complex / mannose binding / defense response to bacterium / identical protein binding / plasma membrane
Similarity search - Function
Jacalin-like lectin domain / Jacalin-type lectin domain profile. / Jacalin-like lectin domain superfamily
Similarity search - Domain/homology
Aerolysin-like protein
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodelectron crystallography / negative staining / Resolution: 20.0 Å
AuthorsJia N / Liu N / Cheng W / Jiang YL / Sun H / Chen LL / Peng JH / Zhang YH / Zhang ZH / Wang XJ ...Jia N / Liu N / Cheng W / Jiang YL / Sun H / Chen LL / Peng JH / Zhang YH / Zhang ZH / Wang XJ / Cai G / Wang JF / Zhang ZY / Wu H / Wang HW / Chen YX / Zhou CZ
CitationJournal: EMBO Rep / Year: 2016
Title: Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.
Authors: Ning Jia / Nan Liu / Wang Cheng / Yong-Liang Jiang / Hui Sun / Lan-Lan Chen / Junhui Peng / Yonghui Zhang / Yue-He Ding / Zhi-Hui Zhang / Xuejuan Wang / Gang Cai / Junfeng Wang / Meng-Qiu ...Authors: Ning Jia / Nan Liu / Wang Cheng / Yong-Liang Jiang / Hui Sun / Lan-Lan Chen / Junhui Peng / Yonghui Zhang / Yue-He Ding / Zhi-Hui Zhang / Xuejuan Wang / Gang Cai / Junfeng Wang / Meng-Qiu Dong / Zhiyong Zhang / Hui Wu / Hong-Wei Wang / Yuxing Chen / Cong-Zhao Zhou /
Abstract: Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains ...Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.
History
DepositionNov 13, 2015-
Header (metadata) releaseNov 25, 2015-
Map releaseJan 13, 2016-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 55
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3244_Dln...

Downloads & links

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Map

FileDownload / File: emd_3244.map.gz / Format: CCP4 / Size: 30.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mutant aerolysin-like protein
Voxel sizeX=Y=Z: 0.625 Å
Density
Contour LevelBy EMDB: 55.0 / Movie #1: 55
Minimum - Maximum-234.948883059999986 - 250.0
Average (Standard dev.)-0.78882813 (±51.022731780000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-55-60-55
Dimensions198207201
Spacing198207201
CellA: 129.375 Å / B: 123.75 Å / C: 125.625 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6250.6250.625
M x/y/z207198201
origin x/y/z0.0000.0000.000
length x/y/z129.375123.750125.625
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-60-55-55
NC/NR/NS207198201
D min/max/mean-234.949250.000-0.789

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Supplemental data

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Sample components

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Entire : Dln1 (a zebrafish aerolysin-like protein) oligomer on phospholipi...

EntireName: Dln1 (a zebrafish aerolysin-like protein) oligomer on phospholipid monolayer
Components
  • Sample: Dln1 (a zebrafish aerolysin-like protein) oligomer on phospholipid monolayer
  • Protein or peptide: Dln1

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Supramolecule #1000: Dln1 (a zebrafish aerolysin-like protein) oligomer on phospholipi...

SupramoleculeName: Dln1 (a zebrafish aerolysin-like protein) oligomer on phospholipid monolayer
type: sample / ID: 1000
Oligomeric state: Octameric Dln1 reconstituted on lipid monolayer
Number unique components: 1
Molecular weightExperimental: 280 KDa / Theoretical: 280 KDa

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Macromolecule #1: Dln1

MacromoleculeName: Dln1 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Oligomeric state: Octamer / Recombinant expression: Yes
Source (natural)Organism: Danio rerio (zebrafish) / synonym: zebrafish
Molecular weightExperimental: 280 KDa / Theoretical: 280 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Aerolysin-like protein

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 5.5 / Details: 50 mM MES, 150 mM NaC
StainingType: NEGATIVE
Details: Grids attached with two-dimensional protein crystal on lipid monolayer were gently washed, followed by negative staining with 2% w/v uranyl acetate for about 30 seconds.
GridDetails: 200 mesh copper grid with thin holey carbon support. The grids were not glow discharged.
VitrificationCryogen name: NONE / Instrument: OTHER
DetailsCrystals grown on a lipid-monolayer
Crystal formationDetails: Crystals grown on a lipid-monolayer

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 98000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 6.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.588 µm / Nominal magnification: 98000
Sample stageSpecimen holder model: OTHER / Tilt angle max: 40 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 40 °
DateOct 9, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 45
Tilt angle min0

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Image processing

Crystal parametersUnit cell - A: 125 Å / Unit cell - B: 125 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 4
Final reconstructionResolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: 2dx
DetailsImages were unbent using 2dx.

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