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- PDB-6xye: Cryo-EM structure of the prefusion state of canine distemper viru... -

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Basic information

Entry
Database: PDB / ID: 6xye
TitleCryo-EM structure of the prefusion state of canine distemper virus fusion protein ectodomain
Components
  • Fusion glycoprotein F1
  • Fusion glycoprotein F2
KeywordsVIRAL PROTEIN / fusion protein / prefusion state
Function / homology
Function and homology information


membrane => GO:0016020 / fusion of virus membrane with host plasma membrane / viral envelope / host cell plasma membrane / virion membrane / plasma membrane
Similarity search - Function
Precursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0
Similarity search - Domain/homology
Fusion glycoprotein F0 / Fusion glycoprotein F0
Similarity search - Component
Biological speciesCanine morbillivirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsKalbermatter, D. / Fotiadis, D.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationSinergia, Ref. No. 183481 Switzerland
CitationJournal: J Struct Biol X / Year: 2020
Title: Cryo-EM structure of the prefusion state of canine distemper virus fusion protein ectodomain.
Authors: David Kalbermatter / Neeta Shrestha / Flavio M Gall / Marianne Wyss / Rainer Riedl / Philippe Plattet / Dimitrios Fotiadis /
Abstract: Measles virus (MeV) and canine distemper virus (CDV), two members of the genus, are still causing important global diseases of humans and animals, respectively. To enter target cells, ...Measles virus (MeV) and canine distemper virus (CDV), two members of the genus, are still causing important global diseases of humans and animals, respectively. To enter target cells, morbilliviruses rely on an envelope-anchored machinery, which is composed of two interacting glycoproteins: a tetrameric receptor binding (H) protein and a trimeric fusion (F) protein. To execute membrane fusion, the F protein initially adopts a metastable, prefusion state that refolds into a highly stable postfusion conformation as the result of a finely coordinated activation process mediated by the H protein. Here, we employed cryo-electron microscopy (cryo-EM) and single particle reconstruction to elucidate the structure of the prefusion state of the CDV F protein ectodomain (solF) at 4.3 Å resolution. Stabilization of the prefusion solF trimer was achieved by fusing the GCNt trimerization sequence at the C-terminal protein region, and expressing and purifying the recombinant protein in the presence of a morbilliviral fusion inhibitor class compound. The three-dimensional cryo-EM map of prefusion CDV solF in complex with the inhibitor clearly shows density for the ligand at the protein binding site suggesting common mechanisms of membrane fusion activation and inhibition employed by different morbillivirus members.
History
DepositionJan 30, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 22, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Fusion glycoprotein F2
B: Fusion glycoprotein F1
C: Fusion glycoprotein F2
D: Fusion glycoprotein F1
E: Fusion glycoprotein F2
F: Fusion glycoprotein F1


Theoretical massNumber of molelcules
Total (without water)172,9216
Polymers172,9216
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38470 Å2
ΔGint-287 kcal/mol
Surface area60710 Å2
MethodPISA

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Components

#1: Protein Fusion glycoprotein F2


Mass: 10128.781 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Canine morbillivirus / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9YKL7, UniProt: A0A0R5ZPI3*PLUS
#2: Protein Fusion glycoprotein F1


Mass: 47511.637 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Canine morbillivirus / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9YKL7

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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: Canine distemper virus fusion protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Canine morbillivirus
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-Amino-2-hydroxymethyl-propane-1,3-diolTRIS1
2100 mMsodium chlorideNaCl1
30.1 mM2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acidEDTA1
40.075 mMN-(3-cyanophenyl)-2-phenylacetamide3G1
SpecimenConc.: 0.23 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 73 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1604
Image scansMovie frames/image: 48

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2SerialEM3.7image acquisition
3FOCUS1.1.0image acquisition
5CTFFIND4CTF correction
8PHENIX1.16-3546model fittingDock in map tool
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14PHENIX1.16-3546model refinementReal-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 491315
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115248 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
15YZC

5yzc

A124-104
25YZC

5yzc

B1115-471

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