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- EMDB-5699: CryoEM structure of EGFR-specific virus (derived from sindbis virus) -

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Basic information

Entry
Database: EMDB / ID: 5699
TitleCryoEM structure of EGFR-specific virus (derived from sindbis virus)
Map dataCryoEM structure of EGFR-specific virus derived from sindbis virus
SampleEGFR-specific virus (derived from sindbis virus)
  • virus
KeywordsEGFR-specific virus / sindbis virus
SourceSynthetic construct
Methodsingle particle reconstruction / cryo EM / 12.2 Å resolution
AuthorsDai H / Liu Z / Jiang W / Kuhn RJ
CitationJournal: J. Virol. / Year: 2013
Title: Directed evolution of a virus exclusively utilizing human epidermal growth factor receptor as the entry receptor.
Authors: Hong-Sheng Dai / Zheng Liu / Wen Jiang / Richard J Kuhn
Abstract: Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. Here we describe a ...Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. Here we describe a directed-evolution strategy, named the tandem selection and enrichment system (TSES), and its use in generating virus with exclusive specificity for a particular cellular receptor. In TSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of receptor specificity and the other for enrichment of the fittest virus. By combining rational design and TSES, we generated human epidermal growth factor receptor (EGFR)-specific virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGF domain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGF chimera. ESV1 uses EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2-Å cryoelectron microscopic (cryoEM) reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation, with EGF sitting at the top of the E2 spike; The EGFR binding interface faces outward, and the EGF domain completely masks SINV receptor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modification. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.
DateDeposition: Jun 25, 2013 / Header (metadata) release: Aug 28, 2013 / Map release: Jul 30, 2014 / Last update: Oct 12, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_5699.map.gz (map file in CCP4 format, 132862 KB)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
324 pix
3.24 Å/pix.
= 1049.76 Å
324 pix
3.24 Å/pix.
= 1049.76 Å
324 pix
3.24 Å/pix.
= 1049.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.24 Å
Density
Contour Level:3.5 (by emdb), 3.5 (movie #1):
Minimum - Maximum-11.02840233 - 21.5696907
Average (Standard dev.)0.49513358 (1.9885757)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions324324324
Origin-162-162-162
Limit161161161
Spacing324324324
CellA=B=C: 1049.76 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.243.243.24
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z1049.7601049.7601049.760
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS-162-162-162
NC/NR/NS324324324
D min/max/mean-11.02821.5700.495

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Supplemental data

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Sample components

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Entire EGFR-specific virus (derived from sindbis virus)

EntireName: EGFR-specific virus (derived from sindbis virus) / Details: The sample was monodisperse. / Number of components: 1 / Oligomeric State: icosahedral

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Component #1: virus, synthetic construct

VirusName: synthetic construct / a.k.a: EGFR-specific Sindbis virus / Class: VIRION
Details: EGF domain was engrafted onto an icosahedral virus scaffold, and directed evolution was conducted to stabilize the chimeric virus.
Empty: No / Enveloped: Yes / Isolate: STRAIN
SpeciesSpecies: Synthetic construct / Strain: EGFR-specific no.1
Source (natural)Host Species: Homo sapiens / / human / Host category: VERTEBRATES
Shell #1Name of element: E1 and E2-EGF / Diameter: 375 Å / T number(triangulation number): 4

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.5 mg/ml / Buffer solution: 50 mM Tris, 200 mM NaCl, 1 mM EDTA / pH: 7.4
Support film400 mesh copper grid with thin carbon support, glow discharged in air
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 97.15 K / Humidity: 70 % / Method: Blot for 6 seconds before plunging.

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM200FEG / Date: Oct 12, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 38000 X (nominal), 39190 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 5000 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC / Temperature: 97 K ( 93.15 - 100.15 K)
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 166 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 3.24 microns / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 2290
3D reconstructionAlgorithm: projection matching / Software: jspr, EMAN2, EMAN / CTF correction: each particle / Details: Final maps were calculated from 2290 particles. / Resolution: 12.2 Å
Resolution method: FSC at 0.143 cut-off between two independently refined density maps, gold-standard

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Atomic model buiding

Modeling #1Software: veda / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3J0F
Modeling #2Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 1JL9

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