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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: EMD-5699
TitleCryoEM structure of EGFR-specific virus (derived from sindbis virus)
Map dataCryoEM structure of EGFR-specific virus derived from sindbis virus
Sample
  • Sample: EGFR-specific virus (derived from sindbis virus)
  • Virus: synthetic construct (others)
KeywordsEGFR-specific virus / sindbis virus
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.2 Å
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.
History
DepositionJun 25, 2013-
Header (metadata) releaseAug 28, 2013-
Map releaseJul 30, 2014-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
  • 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

emd_5699.png

Downloads & links

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Map

FileDownload / File: emd_5699.map.gz / Format: CCP4 / Size: 126.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of EGFR-specific virus derived from sindbis virus
Voxel sizeX=Y=Z: 3.24 Å
Density
Contour LevelBy EMDB: 3.5 / Movie #1: 3.5
Minimum - Maximum-11.02840233 - 21.569690699999999
Average (Standard dev.)0.49513358 (±1.9885757)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-162-162-162
Dimensions324324324
Spacing324324324
CellA=B=C: 1049.76 Å
α=β=γ: 90.0 °

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)
Components
  • Sample: EGFR-specific virus (derived from sindbis virus)
  • Virus: synthetic construct (others)

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Supramolecule #1000: EGFR-specific virus (derived from sindbis virus)

SupramoleculeName: EGFR-specific virus (derived from sindbis virus) / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: icosahedral / Number unique components: 1

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Supramolecule #1: synthetic construct

SupramoleculeName: synthetic construct / type: virus / ID: 1 / Name.synonym: EGFR-specific Sindbis virus
Details: EGF domain was engrafted onto an icosahedral virus scaffold, and directed evolution was conducted to stabilize the chimeric virus.
NCBI-ID: 32630 / Sci species name: synthetic construct / Sci species strain: EGFR-specific no.1 / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No / Syn species name: EGFR-specific Sindbis virus
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Virus shellShell ID: 1 / Name: E1 and E2-EGF / Diameter: 375.00 Å / T number (triangulation number): 4

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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

Concentration1.5 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris, 200 mM NaCl, 1 mM EDTA
GridDetails: 400 mesh copper grid with thin carbon support, glow discharged in air
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 97.15 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 6 seconds before plunging.

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39190 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 38000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 93.15 K / Max: 100.15 K / Average: 97 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateOct 12, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 3.24 µm / Number real images: 166 / Average electron dose: 20 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.2 Å / Resolution method: OTHER / Software - Name: jspr, EMAN2, EMAN / Details: Final maps were calculated from 2290 particles. / Number images used: 2290

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Atomic model buiding 1

Initial modelPDB ID:

3j0f

SoftwareName: veda
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1jl9

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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