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- PDB-6xob: CryoEM structure of Eastern Equine Encephalitis (EEEV) VLP with F... -

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Basic information

Entry
Database: PDB / ID: 6xob
TitleCryoEM structure of Eastern Equine Encephalitis (EEEV) VLP with Fab EEEV-143.
Components
  • (Togavirin) x 3
  • EEEV-143 Fab heavy chain
  • EEEV-143 Fab light chain
KeywordsVirus/Immune System / VLP / Fab / EEEV / VIRAL PROTEIN / Virus-Immune System complex
Function / homology
Function and homology information


togavirin / T=4 icosahedral viral capsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / host cell nucleus / virion attachment to host cell ...togavirin / T=4 icosahedral viral capsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / host cell nucleus / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / RNA binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Alphavirus E2 glycoprotein, domain B / Peptidase S3, togavirin / Alphavirus E2 glycoprotein / Alphavirus E3 spike glycoprotein / Alphavirus E1 glycoprotein / Alphavirus E2 glycoprotein, domain A / Alphavirus E2 glycoprotein, domain C / Alphavirus E2 glycoprotein / Alphavirus core protein / Alphavirus E3 glycoprotein ...Alphavirus E2 glycoprotein, domain B / Peptidase S3, togavirin / Alphavirus E2 glycoprotein / Alphavirus E3 spike glycoprotein / Alphavirus E1 glycoprotein / Alphavirus E2 glycoprotein, domain A / Alphavirus E2 glycoprotein, domain C / Alphavirus E2 glycoprotein / Alphavirus core protein / Alphavirus E3 glycoprotein / Alphavirus E1 glycoprotein / Alphavirus core protein (CP) domain profile. / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / Immunoglobulin E-set / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Structural polyprotein / Structural polyprotein
Similarity search - Component
Biological speciesEastern equine encephalitis virus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsBinshtein, E. / Crowe, J.E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI142790 United States
Defense Threat Reduction Agency (DTRA)HDTRA1-13-1-0034 United States
CitationJournal: Cell / Year: 2020
Title: Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.
Authors: Lauren E Williamson / Theron Gilliland / Pramod K Yadav / Elad Binshtein / Robin Bombardi / Nurgun Kose / Rachel S Nargi / Rachel E Sutton / Clarissa L Durie / Erica Armstrong / Robert H ...Authors: Lauren E Williamson / Theron Gilliland / Pramod K Yadav / Elad Binshtein / Robin Bombardi / Nurgun Kose / Rachel S Nargi / Rachel E Sutton / Clarissa L Durie / Erica Armstrong / Robert H Carnahan / Lauren M Walker / Arthur S Kim / Julie M Fox / Michael S Diamond / Melanie D Ohi / William B Klimstra / James E Crowe /
Abstract: Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has ...Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.
History
DepositionJul 6, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
  • Imaged by Jmol
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  • Biological unit as icosahedral 23 hexamer
  • Imaged by Jmol
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-22277
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  • Superimposition on EM map
  • EMDB-22277
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerMolecule:
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Assembly

Deposited unit
A: Togavirin
B: Togavirin
C: Togavirin
D: Togavirin
E: Togavirin
F: Togavirin
G: Togavirin
H: Togavirin
I: Togavirin
J: Togavirin
K: Togavirin
L: Togavirin
M: EEEV-143 Fab heavy chain
N: EEEV-143 Fab light chain
O: EEEV-143 Fab heavy chain
P: EEEV-143 Fab light chain
Q: EEEV-143 Fab heavy chain
R: EEEV-143 Fab light chain
S: EEEV-143 Fab heavy chain
T: EEEV-143 Fab light chain


Theoretical massNumber of molelcules
Total (without water)682,49220
Polymers682,49220
Non-polymers00
Water0
1
A: Togavirin
B: Togavirin
C: Togavirin
D: Togavirin
E: Togavirin
F: Togavirin
G: Togavirin
H: Togavirin
I: Togavirin
J: Togavirin
K: Togavirin
L: Togavirin
M: EEEV-143 Fab heavy chain
N: EEEV-143 Fab light chain
O: EEEV-143 Fab heavy chain
P: EEEV-143 Fab light chain
Q: EEEV-143 Fab heavy chain
R: EEEV-143 Fab light chain
S: EEEV-143 Fab heavy chain
T: EEEV-143 Fab light chain
x 60


Theoretical massNumber of molelcules
Total (without water)40,949,4951200
Polymers40,949,4951200
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Togavirin
B: Togavirin
C: Togavirin
D: Togavirin
E: Togavirin
F: Togavirin
G: Togavirin
H: Togavirin
I: Togavirin
J: Togavirin
K: Togavirin
L: Togavirin
M: EEEV-143 Fab heavy chain
N: EEEV-143 Fab light chain
O: EEEV-143 Fab heavy chain
P: EEEV-143 Fab light chain
Q: EEEV-143 Fab heavy chain
R: EEEV-143 Fab light chain
S: EEEV-143 Fab heavy chain
T: EEEV-143 Fab light chain
x 5


  • icosahedral pentamer
  • 3.41 MDa, 100 polymers
Theoretical massNumber of molelcules
Total (without water)3,412,458100
Polymers3,412,458100
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Togavirin
B: Togavirin
C: Togavirin
D: Togavirin
E: Togavirin
F: Togavirin
G: Togavirin
H: Togavirin
I: Togavirin
J: Togavirin
K: Togavirin
L: Togavirin
M: EEEV-143 Fab heavy chain
N: EEEV-143 Fab light chain
O: EEEV-143 Fab heavy chain
P: EEEV-143 Fab light chain
Q: EEEV-143 Fab heavy chain
R: EEEV-143 Fab light chain
S: EEEV-143 Fab heavy chain
T: EEEV-143 Fab light chain
x 6


  • icosahedral 23 hexamer
  • 4.09 MDa, 120 polymers
Theoretical massNumber of molelcules
Total (without water)4,094,949120
Polymers4,094,949120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
Togavirin /


Mass: 47961.215 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus / Production host: Homo sapiens (human) / References: UniProt: Q88678, togavirin
#2: Protein
Togavirin /


Mass: 47047.020 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus / Production host: Homo sapiens (human)
References: UniProt: Q88678, UniProt: Q4QXJ7*PLUS, togavirin
#3: Protein
Togavirin /


Mass: 29193.924 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus / Production host: Homo sapiens (human)
References: UniProt: Q88678, UniProt: Q4QXJ7*PLUS, togavirin
#4: Antibody
EEEV-143 Fab heavy chain


Mass: 23251.002 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody
EEEV-143 Fab light chain


Mass: 23169.734 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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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

ComponentName: Eastern equine encephalitis virusEastern equine encephalitis
Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Eastern equine encephalitis virus
Source (recombinant)Organism: Homo sapiens (human)
Details of virusEmpty: YES / Enveloped: YES / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Details: blot time 2.5s blot force 9

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4800

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13FREALIX3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3600
3D reconstructionResolution: 8.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1300 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00438264
ELECTRON MICROSCOPYf_angle_d0.78852468
ELECTRON MICROSCOPYf_dihedral_angle_d17.20411964
ELECTRON MICROSCOPYf_chiral_restr0.0456232
ELECTRON MICROSCOPYf_plane_restr0.0067008

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