|Entry||Database: EMDB / ID: 5699|
|Title||CryoEM structure of EGFR-specific virus (derived from sindbis virus)|
|Map data||CryoEM structure of EGFR-specific virus derived from sindbis virus|
|Sample||EGFR-specific virus (derived from sindbis virus)|
|Keywords||EGFR-specific virus / sindbis virus|
|Method||single particle reconstruction / cryo EM / 12.2 Å resolution|
|Authors||Dai H / Liu Z / Jiang W / Kuhn RJ|
|Citation||Journal: 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.
|Date||Deposition: Jun 25, 2013 / Header (metadata) release: Aug 28, 2013 / Map release: Jul 30, 2014 / Last update: Oct 12, 2016|
|Structure viewer||EM map: |
Downloads & links
|File||emd_5699.map.gz (map file in CCP4 format, 132862 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 3.24 Å|
CCP4 map header:
-Entire EGFR-specific virus (derived from sindbis virus)
|Entire||Name: EGFR-specific virus (derived from sindbis virus) / Details: The sample was monodisperse. / Number of components: 1 / Oligomeric State: icosahedral|
-Component #1: virus, synthetic construct
|Virus||Name: 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
|Species||Species: Synthetic construct / Strain: EGFR-specific no.1|
|Source (natural)||Host Species: Homo sapiens / / human / Host category: VERTEBRATES|
|Shell #1||Name of element: E1 and E2-EGF / Diameter: 375 Å / T number(triangulation number): 4|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 1.5 mg/ml / Buffer solution: 50 mM Tris, 200 mM NaCl, 1 mM EDTA / pH: 7.4|
|Support film||400 mesh copper grid with thin carbon support, glow discharged in air|
|Vitrification||Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 97.15 K / Humidity: 70 % / Method: Blot for 6 seconds before plunging.|
-Electron microscopy imaging
|Imaging||Microscope: FEI/PHILIPS CM200FEG / Date: Oct 12, 2011|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Model: SIDE ENTRY, EUCENTRIC / Temperature: 97 K ( 93.15 - 100.15 K)|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 166 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 3.24 microns / Bit depth: 16|
|Processing||Method: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 2290|
|3D reconstruction||Algorithm: 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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