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- PDB-9mnh: Angavokely virus (AngV) fusion (F) protein ectodomain in pre-fusi... -

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Basic information

Entry
Database: PDB / ID: 9mnh
TitleAngavokely virus (AngV) fusion (F) protein ectodomain in pre-fusion conformation
ComponentsFusion Protein
KeywordsVIRAL PROTEIN / Henipavirus / AngV-F protein / Pre-fusion F protein / Cryo-EM structure / Fusion Ectodomain
Biological speciesAngavokely henipavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsLella, M. / Acharya, P.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: bioRxiv / Year: 2025
Title: Structural and Antigenic Characterization of Novel and Diverse Glycoproteins.
Authors: Aaron J May / Muralikrishna Lella / Jared Lindenberger / Alex Berkman / Ujjwal Kumar / Moumita Dutta / Maggie Barr / Rob Parks / Amanda Newman / Xiao Huang / Kijun Song / Victor Ilevbare / ...Authors: Aaron J May / Muralikrishna Lella / Jared Lindenberger / Alex Berkman / Ujjwal Kumar / Moumita Dutta / Maggie Barr / Rob Parks / Amanda Newman / Xiao Huang / Kijun Song / Victor Ilevbare / Salam Sammour / Chan Soo Park / Radha Devkota Adhikari / Priyanka Devkota / Katarzyna Janowska / Yanshun Liu / Garrett Scapellato / Taylor N Spence / Katayoun Mansouri / Robert J Edwards / Kevin O Saunders / Barton F Haynes / Priyamvada Acharya /
Abstract: Henipaviruses, a genus within the family, include the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of deadly disease . Recent discoveries of several new species, ...Henipaviruses, a genus within the family, include the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of deadly disease . Recent discoveries of several new species, including the zoonotic Langya virus , have revealed much higher antigenic diversity than currently characterized and prompted the reorganization of these viruses into the and genera . Here, to explore the limits of structural and antigenic variation in both genera, collectively referred to here as HNVs, we constructed an expanded, antigenically diverse panel of HNV fusion and attachment glycoproteins from 56 unique HNV strains that better reflects global HNV diversity. We expressed and purified the fusion protein ectodomains and the attachment protein head domains and characterized their biochemical, biophysical and structural properties. We performed immunization experiments in mice leading to the elicitation of antibodies reactive to multiple HNV fusion proteins. Cryo-electron microscopy structures of diverse fusion proteins elucidated molecular determinants of differential pre-fusion state metastability and higher order contacts. A crystal structure of the Gamak virus attachment head domain revealed an additional domain added to the conserved 6-bladed, β-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the HNVs, reveal new cross-reactive epitopes within both genera and provide foundational data for the development of broadly reactive countermeasures.
History
DepositionDec 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Fusion Protein
B: Fusion Protein
C: Fusion Protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)181,71218
Polymers175,7633
Non-polymers5,94915
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Fusion Protein


Mass: 58587.699 Da / Num. of mol.: 3 / Fragment: Ectodomain
Source method: isolated from a genetically manipulated source
Details: Pre-fusion conformation. C-terminal foldon trimerization domain followed by HRV3C protease site, His-tag, and two streptavidin tags separated by GS linker.
Source: (gene. exp.) Angavokely henipavirus / Production host: Homo sapiens (human)
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 732.682 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1221m-1a_1-5]/1-1-2-3/a4-b1_a6-d1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

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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: AngV-F protein trimer in pre-fusion conformation / Type: COMPLEX
Details: AngV-F fusion ectodomain is expressed with following domains at C-terminus, foldon trimerization domain, HRV3C protease site, His-tag, and two streptavidin tags separated by GS linker
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.06 MDa / Experimental value: YES
Source (natural)Organism: Angavokely henipavirus / Cellular location: Outer Membrane
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: palphaH
Buffer solutionpH: 7.4
Buffer componentConc.: 1 1 / Name: Phosphate Buffer / Formula: Na2HPO4/NaH2PO4
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K
Details: Protein sample was applied to the grid and incubated for 30 seconds at >95% humidity. Excess protein was blotted away for 2.5 seconds before being plunge frozen into liquid ethane

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS / Details: Grid screening was performed manually
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1700 nm / Calibrated defocus min: 1700 nm / Calibrated defocus max: 2800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.8 sec. / Electron dose: 28 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 24000
Details: Images were collected in a movie -mode at 22 frames per second
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.5.3particle selection
4cryoSPARCv4.5.3CTF correction
7UCSF ChimeraX1.6.1model fitting
11cryoSPARCv4.5.3classification
12cryoSPARCv4.5.33D reconstruction
19PHENIX1.21.1_5286model refinement
Image processingDetails: Micrographs were motion-corrected and aligned micrographs were used for the processing
CTF correctionDetails: Micrographs were curated through contrast transfer function (CTF) where greater than 30 angstrom were discarded
Type: NONE
Particle selectionNum. of particles selected: 13247526
Details: Particles were picked using automated blob picker and extracted
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1106722
Details: Non-uniform refinement used for the final reconstruction
Num. of class averages: 63 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building
ID 3D fitting-IDChain-IDChain residue rangeSource nameType
11A22-482SwissModelin silico model
21B22-482SwissModelin silico model
31C22-482SwissModelin silico model
RefinementHighest resolution: 4.03 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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