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- EMDB-2503: Cryo-EM Structure of Isomeric Molluscan Hemocyanin Type 1 trigger... -

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Basic information

Entry
Database: EMDB / ID: EMD-2503
TitleCryo-EM Structure of Isomeric Molluscan Hemocyanin Type 1 triggered by viral infection
Map dataReconstruction of isomeric Halitotis diversicolor hemocyanin type 1 triggered by AbSV
Sample
  • Sample: Isomeric Molluscan Hemocyanin triggered by AbSV infection
  • Protein or peptide: isomeric hemocyanin type 1
Keywordsisomeric molluscan hemocyanin / viral infection / AbSV / phenoloxidase
Function / homology
Function and homology information


: / oxygen carrier activity / oxidoreductase activity
Similarity search - Function
Haemocyanin beta-sandwich domain / Haemocyanin C-terminal domain superfamily / Haemocyanin beta-sandwich / Tyrosinase CuA-binding region signature. / Common central domain of tyrosinase / Tyrosinase and hemocyanins CuB-binding region signature. / Tyrosinase copper-binding domain / Di-copper centre-containing domain superfamily / Di-copper centre-containing domain superfamily
Similarity search - Domain/homology
Hemocyanin isoform 1
Similarity search - Component
Biological speciesHaliotis diversicolor (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsZhu HT / Zhuang J / Feng HL / Liang RF / Wang JY / Xie LH / Zhu P
CitationJournal: PLoS One / Year: 2014
Title: Cryo-EM structure of isomeric molluscan hemocyanin triggered by viral infection.
Authors: Hongtao Zhu / Jun Zhuang / Hongli Feng / Rongfeng Liang / Jiangyong Wang / Lianhui Xie / Ping Zhu /
Abstract: Hemocyanins (Hcs) of arthropods and mollusks function not only as oxygen transporters, but also as phenoloxidases (POs). In invertebrates, PO is an important component in the innate immune cascade, ...Hemocyanins (Hcs) of arthropods and mollusks function not only as oxygen transporters, but also as phenoloxidases (POs). In invertebrates, PO is an important component in the innate immune cascade, where it functions as the initiator of melanin synthesis, a pigment involved in encapsulating and killing of pathogenic microbes. Although structures of Hc from several species of invertebrates have been reported, the structural basis for how PO activity is triggered by structural changes of Hc in vivo remains poorly understood. Here, we report a 6.8 Å cryo-electron microscopy (cryo-EM) structure of the isomeric form of hemocyanin, which was isolated from Abalone Shriveling syndrome-associated Virus (AbSV) infected abalone (Halitotis diversicolor), and build a pseudoatomic model of isomeric H. diversicolor hemocyanin 1 (HdH1). Our results show that, compared with native form of HdH1, the architecture of isomeric HdH1 turns into a more relaxed form. The interactions between certain functional units (FUs) present in the native form of Hc either decreased or were totally abolished in the isomeric form of Hc. As a result of that, native state Hc switches to its isomeric form, enabling it to play its role in innate immune responses against invading pathogens.
History
DepositionOct 27, 2013-
Header (metadata) releaseNov 13, 2013-
Map releaseAug 27, 2014-
UpdateAug 27, 2014-
Current statusAug 27, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2503.png

Downloads & links

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Map

FileDownload / File: emd_2503.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of isomeric Halitotis diversicolor hemocyanin type 1 triggered by AbSV
Voxel sizeX=Y=Z: 1.196 Å
Density
Contour LevelBy AUTHOR: 1.7 / Movie #1: 1.7
Minimum - Maximum-5.1972332 - 10.29769993
Average (Standard dev.)0.00007128 (±1.00920987)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-200-200-200
Dimensions400400400
Spacing400400400
CellA=B=C: 478.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1961.1961.196
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z478.400478.400478.400
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-200-200-200
NC/NR/NS400400400
D min/max/mean-5.19710.2980.000

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Supplemental data

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Sample components

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Entire : Isomeric Molluscan Hemocyanin triggered by AbSV infection

EntireName: Isomeric Molluscan Hemocyanin triggered by AbSV infection
Components
  • Sample: Isomeric Molluscan Hemocyanin triggered by AbSV infection
  • Protein or peptide: isomeric hemocyanin type 1

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Supramolecule #1000: Isomeric Molluscan Hemocyanin triggered by AbSV infection

SupramoleculeName: Isomeric Molluscan Hemocyanin triggered by AbSV infection
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 8 MDa

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Macromolecule #1: isomeric hemocyanin type 1

MacromoleculeName: isomeric hemocyanin type 1 / type: protein_or_peptide / ID: 1 / Name.synonym: HdH1 / Recombinant expression: No
Source (natural)Organism: Haliotis diversicolor (invertebrata)
Molecular weightTheoretical: 8 MDa
SequenceUniProtKB: Hemocyanin isoform 1 / GO: GO: 0055114 / InterPro: Di-copper centre-containing domain superfamily

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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

Concentration0.5 mg/mL
BufferpH: 7.8 / Details: 20mM phosphate buffer, containing 2.5mM MgCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 125418 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125418 times magnification
DateDec 1, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 2379 / Average electron dose: 18 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: OTHER / Software - Name: EMAN2, Relion-1.1, Xmipp-2.4 / Number images used: 57043
DetailsEMAN2 was initially used for particle picking, 2D classification, projection refinement and 3D reconstruction for both normal and isomeric hemocyanins with C5 symmetry imposed. Subsequently, 57,043 isomeric hemocyanin particles were imported to Relion-1.1 for further refinement and reconstruction by applying D5 symmetry, using the reconstructed map from EMAN2 as the initial model. Xmipp-2.4 was used for the? bfactor correction to generate the final 3D reconstruction map of isomeric hemocyanin.

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