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- PDB-8vym: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B... -

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Basic information

Entry
Database: PDB / ID: 8vym
TitleSoluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
Components
  • 1G2 Fab Heavy Chain
  • 1G2 Fab Light Chain
  • 7H3 Fab Heavy Chain
  • 7H3 Fab Light Chain
  • Envelope glycoprotein B
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / orthoherpesvirus / betaherpesvirus / cytomegalovirus / human betaherpesvirus 5 / human cytomegalovirus / HCMV / glycoprotein B / gB / HCMV gB / VIRAL PROTEIN / 7H3 / 1G2 / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


host cell Golgi membrane / host cell endosome membrane / symbiont entry into host cell / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane
Similarity search - Function
Herpesvirus Glycoprotein B, antigenic domain, N-terminal / Glycoprotein B N-terminal antigenic domain of HCMV / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B, PH-like domain 1 / Herpesvirus Glycoprotein B, PH-like domain 2 / Herpesvirus Glycoprotein B, PH-like domain 2 superfamily / Herpesvirus Glycoprotein B ectodomain / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B PH-like domain
Similarity search - Domain/homology
Envelope glycoprotein B
Similarity search - Component
Biological speciesHuman betaherpesvirus 5
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSponholtz, M.R. / Byrne, P.O. / McLellan, J.S.
Funding support1items
OrganizationGrant numberCountry
Other private
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.
Authors: Madeline R Sponholtz / Patrick O Byrne / Alison G Lee / Ajit R Ramamohan / Jory A Goldsmith / Ryan S McCool / Ling Zhou / Nicole V Johnson / Ching-Lin Hsieh / Megan Connors / Krithika P ...Authors: Madeline R Sponholtz / Patrick O Byrne / Alison G Lee / Ajit R Ramamohan / Jory A Goldsmith / Ryan S McCool / Ling Zhou / Nicole V Johnson / Ching-Lin Hsieh / Megan Connors / Krithika P Karthigeyan / Chelsea M Crooks / Adelaide S Fuller / John D Campbell / Sallie R Permar / Jennifer A Maynard / Dong Yu / Matthew J Bottomley / Jason S McLellan /
Abstract: Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, ...Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.
History
DepositionFeb 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 18, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Envelope glycoprotein B
B: Envelope glycoprotein B
C: Envelope glycoprotein B
D: 1G2 Fab Heavy Chain
E: 1G2 Fab Light Chain
F: 1G2 Fab Light Chain
G: 1G2 Fab Heavy Chain
H: 7H3 Fab Light Chain
I: 1G2 Fab Light Chain
J: 1G2 Fab Heavy Chain
K: 7H3 Fab Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)465,49337
Polymers456,00411
Non-polymers9,48926
Water00
1


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

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein Envelope glycoprotein B / gB


Mass: 88931.438 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: gB, UL55 / Production host: Homo sapiens (human) / References: UniProt: P13201

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Antibody , 4 types, 8 molecules DGJEFIHK

#2: Antibody 1G2 Fab Heavy Chain


Mass: 24206.172 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody 1G2 Fab Light Chain


Mass: 22853.125 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Antibody 7H3 Fab Light Chain


Mass: 22879.361 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody 7H3 Fab Heavy Chain


Mass: 25152.064 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Sugars , 3 types, 26 molecules

#6: 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.: 13
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#7: 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
#8: 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.: 10
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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

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Details

Has ligand of interestN
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: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 8
Details: 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% (w/v) sodium azide, 3% (v/v) glycerol, 0.25% CHAPS, and 0.1% (w/v) amphipol A8-35
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation, 1G2 Fab, and 7H3 Fab purified separately, mixed, and incubated for 30 minutes on ice ...Details: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation, 1G2 Fab, and 7H3 Fab purified separately, mixed, and incubated for 30 minutes on ice prior to grid preparation
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8679

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Processing

EM software
IDNameVersionCategory
2SerialEM3.9.0 betaimage acquisition
7PHENIXmodel fitting
12cryoSPARC4.2.03D reconstruction
13Cootmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 211132 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322599
ELECTRON MICROSCOPYf_angle_d0.40630683
ELECTRON MICROSCOPYf_dihedral_angle_d9.4148206
ELECTRON MICROSCOPYf_chiral_restr0.0423501
ELECTRON MICROSCOPYf_plane_restr0.0033860

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