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- PDB-8d87: Fitted crystal structure of the homotrimer of fusion glycoprotein... -

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Basic information

Entry
Database: PDB / ID: 8d87
TitleFitted crystal structure of the homotrimer of fusion glycoprotein E1 from SFV into subtomogram averaged CHIKV E1 glycoprotein density
ComponentsSpike glycoprotein E1
KeywordsVIRAL PROTEIN / envelope glycoprotein / membrane fusion / virus
Function / homology
Function and homology information


togavirin / T=4 icosahedral viral capsid / virion assembly / small molecule binding / host cell endosome / symbiont-mediated suppression of host toll-like receptor signaling pathway / clathrin-dependent endocytosis of virus by host cell / viral translational frameshifting / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane ...togavirin / T=4 icosahedral viral capsid / virion assembly / small molecule binding / host cell endosome / symbiont-mediated suppression of host toll-like receptor signaling pathway / clathrin-dependent endocytosis of virus by host cell / viral translational frameshifting / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / RNA binding / membrane
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
BROMIDE ION / HOLMIUM ATOM / PHOSPHATE ION / Structural polyprotein
Similarity search - Component
Biological speciesChikungunya virus strain S27-African prototype
MethodELECTRON MICROSCOPY / SYNCHROTRON / subtomogram averaging / MAD, MIR / cryo EM / Resolution: 27.2 Å
AuthorsMangala Prasad, V. / Lee, K.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM099989 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Nat Commun / Year: 2022
Title: Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery.
Authors: Vidya Mangala Prasad / Jelle S Blijleven / Jolanda M Smit / Kelly K Lee /
Abstract: Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though ...Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.
History
DepositionJun 8, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 31, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2022Group: Data collection / Refinement description
Category: pdbx_initial_refinement_model / refine ...pdbx_initial_refinement_model / refine / refine_ls_shell / reflns
Item: _refine.ls_d_res_high / _refine.ls_d_res_low ..._refine.ls_d_res_high / _refine.ls_d_res_low / _refine_ls_shell.d_res_high / _refine_ls_shell.d_res_low / _reflns.d_resolution_high / _reflns.d_resolution_low
Revision 1.2Oct 23, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature / refine_ls_shell
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _refine_ls_shell.d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein E1
B: Spike glycoprotein E1
C: Spike glycoprotein E1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,84714
Polymers128,0703
Non-polymers3,77711
Water2,504139
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Shape and size of density map matches that of known crystal structure of E1-ectodomain homotrimer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein Spike glycoprotein E1 / E1 envelope glycoprotein


Mass: 42690.125 Da / Num. of mol.: 3 / Fragment: SPIKE GLYCOPROTEIN E1 / Source method: isolated from a natural source
Details: The previously determined X-ray crystal structure PDB ID 1RER was rigidly docked into the sub-tomogram averaged map but no further refinement was performed
Source: (natural) Chikungunya virus strain S27-African prototype
References: UniProt: P03315

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Sugars , 3 types, 3 molecules

#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4) ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[beta-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1098.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-2DManpb1-3DManpb1-4DGlcpNAcb1-4[LFucpb1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1221m-1b_1-5]/1-1-2-2-1-3/a4-b1_a6-f1_b4-c1_c3-d1_d2-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][b-D-Manp]{[(2+1)][b-D-GlcpNAc]{}}}}[(6+1)][b-L-Fucp]{}}}LINUCSPDB-CARE
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 1260.157 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAca1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/5,7,6/[a2122h-1a_1-5_2*NCC/3=O][a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-2-3-4-2-4-5/a4-b1_a6-g1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE

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Non-polymers , 4 types, 147 molecules

#5: Chemical ChemComp-BR / BROMIDE ION


Mass: 79.904 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Br
#6: Chemical
ChemComp-HO / HOLMIUM ATOM


Mass: 164.930 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ho
#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 139 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY
Sequence detailsThe previously determined X-ray crystal structure PDB ID 1RER was rigidly docked into the sub- ...The previously determined X-ray crystal structure PDB ID 1RER was rigidly docked into the sub-tomogram averaged map but no further refinement was performed

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Sub-tomogram averaged map of post-fusion E1 glycoprotein trimer from Chikungunya virus
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Buffer solutionpH: 5.1 / Details: Hepes Buffer Saline
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sub-volumes picked from liposome membrane surface
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 7-8 seconds
CrystalDensity Matthews: 5.15 Å3/Da / Density % sol: 76.1 %
Crystal growpH: 4
Details: PEG 400, NABR, DETERGENT DDAO, HO3+, VAPOR DIFFUSION, HANGING DROP, PH 4, TEMPERATURE 277.0K
PH range: 4

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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 FIELD / Nominal magnification: 53000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2500 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 recordingElectron dose: 70 e/Å2 / Avg electron dose per subtomogram: 70 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Diffraction
IDMean temperature (K)Crystal-ID
11001
21
31
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONSLS X06SA11.53596, 1.53646, 1.18080
SYNCHROTRONESRF ID292
SYNCHROTRONESRF ID14-13
Detector
TypeIDDetectorDateDetails
MARRESEARCH1CCDMar 3, 2003SI(111) MONOCHROMATOR
2
3
Radiation
IDMonochromatorProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SAGITALLY FOCUSED SI(111) MONOCHROMATORMADMx-ray1
2x-ray1
3x-ray1
Radiation wavelength
IDWavelength (Å)Relative weight
11.535961
21.536461
31.18081
ReflectionNum. obs: 40912

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
SOLVEphasing
SHARPphasing
DMphasing
CNSrefinement
EM software
IDNameCategory
1PEETvolume selection
2Leginonimage acquisition
3SerialEMimage acquisition
5CTFPHASEFLIPCTF correction
8UCSF Chimeramodel fitting
13PEET3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 27.2 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 590 / Symmetry type: POINT
EM volume selectionDetails: low pass filtered map of post-fusion E1--homotrimer crystal structure
Num. of tomograms: 40 / Num. of volumes extracted: 591 / Reference model: crystal structure
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 1RER

1rer


Accession code: 1RER / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MAD, MIR / Resolution: 27.2→27.2 Å / σ(F): 2 / Stereochemistry target values: ENGH & HUBER
RfactorNum. reflection% reflectionSelection details
Rfree0.285 2075 -RANDOM. 5% OF THE REFLEXIONS USED IN THE RESOLUTION RANGE 20-3.2 ANGSTROM
Rwork0.265 ---
obs0.265 40912 94.2 %-
Refinement stepCycle: LAST / Resolution: 3.2→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8979 0 199 139 9317
LS refinement shellHighest resolution: 27.2 Å
RfactorNum. reflection% reflection
Rfree0.3396 21 -
Rwork0.4493 --
obs--94.6 %

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