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- EMDB-2568: Human full-length coagulation factor X and its isolated GLA domai... -

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Basic information

Entry
Database: EMDB / ID: EMD-2568
TitleHuman full-length coagulation factor X and its isolated GLA domain bind to different regions of the adenovirus serotype 5 hexon capsomer
Map dataOnly 1/8 of the map to see the hexons
Sample
  • Sample: HAdV-C5 virions complexed with GLAmim
  • Virus: Human adenovirus 5
  • Ligand: GLAmim peptide
KeywordsAdenovirus / Hexon / Scavenger receptors / Factor X / GLA domain / Peptidomimetic / Vectors / Targeting
Biological speciessynthetic construct (others) / Human adenovirus 5
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.0 Å
AuthorsSumarheni S / Hong SS / Josserand V / Coll J-L / Boulanger P / Schoehn G / Fender P
CitationJournal: Hum Gene Ther / Year: 2014
Title: Human full-length coagulation factor X and a GLA domain-derived 40-mer polypeptide bind to different regions of the adenovirus serotype 5 hexon capsomer.
Authors: Sudir Sumarheni / Saw See Hong / Véronique Josserand / Jean-Luc Coll / Pierre Boulanger / Guy Schoehn / Pascal Fender /
Abstract: The interaction of human adenovirus (HAdV)-C5 and many other adenoviruses with blood coagulation factors (e.g., human factor X, FX) involves the binding of their GLA domain to the hexon capsomers, ...The interaction of human adenovirus (HAdV)-C5 and many other adenoviruses with blood coagulation factors (e.g., human factor X, FX) involves the binding of their GLA domain to the hexon capsomers, resulting in high levels of hepatotropism and potential hepatotoxicity. In this study, we tested the possibility of preventing these undesirable effects by using a GLA-mimicking peptide as a competitor. An FX GLA domain-derived, 40-mer polypeptide carrying 12 carboxyglutamate residues was synthesized (GLA(mim)). Surface plasmon resistance (SPR) analysis showed that GLA(mim) reacted with free and capsid-embedded hexon with a nanomolar affinity. Unexpectedly, GLA(mim) failed to compete with FX for hexon binding, and instead significantly increased the formation of FX-hexon or FX-adenovirion complexes. This observation was confirmed by in vitro cell transduction experiments using HAdV-C5-Luciferase vector (HAdV5-Luc), as preincubation of HAdV5-Luc with GLA(mim) before FX addition resulted in a higher transgene expression compared with FX alone. HAdV-C5 virions complexed with GLA(mim) were analyzed by cryoelectron microscopy. Image reconstruction demonstrated the bona fide hexon-GLA(mim) interaction, as for the full-length FX, although with considerable differences in stoichiometry and relative location on the hexon capsomer. Three extra densities were found at the periphery of each hexon, whereas one single FX molecule occupied the central cavity of the hexon trimeric capsomer. A refined analysis indicated that each extra density is found at the expected location of one highly variable loop 1 of the hexon, involved in scavenger receptor recognition. HAdV5-Luc complexed with a bifunctional GLA(mim)RGD peptide showed a lesser hepatotropism, compared with control HAdV5-Luc alone, and efficiently targeted αβ-integrin-overexpressing tumor cells in an in vivo mouse tumor model. Collectively, our findings open new perspectives in the design of adenoviral vectors for biotherapy.
History
DepositionJan 29, 2014-
Header (metadata) releaseMar 12, 2014-
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: 160
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 160
  • 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_2568.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOnly 1/8 of the map to see the hexons
Voxel sizeX=Y=Z: 2.26 Å
Density
Contour LevelBy AUTHOR: 160.0 / Movie #1: 160
Minimum - Maximum-1139.0 - 1311.0
Average (Standard dev.)7.57692862 (±161.530014039999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-223-223-223
Dimensions250250250
Spacing250250250
CellA=B=C: 565.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.262.262.26
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z565.000565.000565.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-223-223-223
NC/NR/NS250250250
D min/max/mean-1139.0001311.0007.577

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Supplemental data

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Sample components

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Entire : HAdV-C5 virions complexed with GLAmim

EntireName: HAdV-C5 virions complexed with GLAmim
Components
  • Sample: HAdV-C5 virions complexed with GLAmim
  • Virus: Human adenovirus 5
  • Ligand: GLAmim peptide

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Supramolecule #1000: HAdV-C5 virions complexed with GLAmim

SupramoleculeName: HAdV-C5 virions complexed with GLAmim / type: sample / ID: 1000 / Number unique components: 2
Molecular weightExperimental: 150 MDa / Theoretical: 150 MDa

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Supramolecule #1: Human adenovirus 5

SupramoleculeName: Human adenovirus 5 / type: virus / ID: 1 / NCBI-ID: 28285 / Sci species name: Human adenovirus 5 / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No / Sci species serotype: 5
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightExperimental: 150 MDa / Theoretical: 150 MDa
Virus shellShell ID: 1 / Diameter: 1000 Å / T number (triangulation number): 25

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Macromolecule #1: GLAmim peptide

MacromoleculeName: GLAmim peptide / type: ligand / ID: 1
Details: GLAmim peptide contains 12 gamma-carboxyglutamate residues and carries a biotinyl group at its C-terminal end.
Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: synthetic construct (others)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: 10mM Hepes buffer 2 mm CaCl2
StainingType: NEGATIVE / Details: cryo
GridDetails: Quantifoil R2/1 holey grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.3 mm / Nominal defocus max: 0.004 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: OTHER
DateSep 10, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 33 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: flipmix
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: pft2, em3dr2 / Number images used: 4762
Detailspft2 em3dr2

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