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- PDB-6eqc: Cryo-EM reconstruction of a complex of a binding protein and huma... -

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Basic information

Entry
Database: PDB / ID: 6eqc
TitleCryo-EM reconstruction of a complex of a binding protein and human adenovirus C5 hexon
Components
  • Hexon protein
  • scFv of 9C12 antibody
KeywordsVIRAL PROTEIN / Antibody / Human Adenovirus C5 / gene therapy Viral Protein
Function / homology
Function and homology information


T=25 icosahedral viral capsid / microtubule-dependent intracellular transport of viral material towards nucleus / viral capsid / symbiont entry into host cell / host cell nucleus / structural molecule activity
Similarity search - Function
Adenovirus Pll, hexon, subdomain 2 / Adenovirus hexon protein / Adenovirus Pll, hexon, N-terminal / Adenovirus Pll, hexon, C-terminal / Hexon, adenovirus major coat protein, N-terminal domain / Hexon, adenovirus major coat protein, C-terminal domain / Group II dsDNA virus coat/capsid protein
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Human adenovirus 5
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsSchmid, M. / Ernst, P. / Honegger, A. / Suomalainen, M. / Zimmermann, M. / Braun, L. / Stauffer, S. / Thom, C. / Dreier, B. / Eibauer, M. ...Schmid, M. / Ernst, P. / Honegger, A. / Suomalainen, M. / Zimmermann, M. / Braun, L. / Stauffer, S. / Thom, C. / Dreier, B. / Eibauer, M. / Kipar, A. / Vogel, V. / Greber, U.F. / Medalia, O. / Plueckthun, A.
CitationJournal: Nat Commun / Year: 2018
Title: Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control.
Authors: Markus Schmid / Patrick Ernst / Annemarie Honegger / Maarit Suomalainen / Martina Zimmermann / Lukas Braun / Sarah Stauffer / Cristian Thom / Birgit Dreier / Matthias Eibauer / Anja Kipar / ...Authors: Markus Schmid / Patrick Ernst / Annemarie Honegger / Maarit Suomalainen / Martina Zimmermann / Lukas Braun / Sarah Stauffer / Cristian Thom / Birgit Dreier / Matthias Eibauer / Anja Kipar / Viola Vogel / Urs F Greber / Ohad Medalia / Andreas Plückthun /
Abstract: Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we ...Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.
History
DepositionOct 12, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2018Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Hexon protein
B: Hexon protein
C: Hexon protein
D: scFv of 9C12 antibody
E: scFv of 9C12 antibody
F: scFv of 9C12 antibody


Theoretical massNumber of molelcules
Total (without water)405,8966
Polymers405,8966
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56550 Å2
ΔGint-270 kcal/mol
Surface area129630 Å2
MethodPISA

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Components

#1: Protein Hexon protein / / CP-H / Protein II


Mass: 108042.469 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Human adenovirus 5 / References: UniProt: P04133
#2: Antibody scFv of 9C12 antibody


Mass: 27256.225 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Hexon protein-antibody complexCOMPLEXall0MULTIPLE SOURCES
2Hexon proteinCOMPLEX#11NATURAL
3scFv of 9C12 antibodyCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Human adenovirus 528285
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Details of virusEmpty: NO / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 0.4 sec. / Electron dose: 1 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
4CTFFIND4.0.17CTF correction
7NAMD2.12model fittingrun with MDFF 0.5
11RELION1.4classification
12RELION1.43D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1880 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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