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- PDB-7t4q: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in... -

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Basic information

Entry
Database: PDB / ID: 7t4q
TitleCryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with neutralizing fabs 2C12, 7I13 and 13H11
Components
  • (Envelope glycoprotein ...) x 3
  • (Envelope protein ...) x 2
  • Fab 13H11 heavy chain
  • Fab 13H11 light chain
  • Fab 2C12 heavy chain
  • Fab 2C12 light chain
  • Fab 7I13 heavy chain
  • Fab 7I13 light chain
KeywordsVIRAL PROTEIN/Immune System / Human Cytomegalovirus / glycoprotein complex / antibody complex / VIRAL PROTEIN / VIRAL PROTEIN-Immune System complex
Function / homology
Function and homology information


host cell endosome / host cell Golgi apparatus / entry receptor-mediated virion attachment to host cell / fusion of virus membrane with host plasma membrane / viral envelope / symbiont entry into host cell / host cell plasma membrane / virion membrane / membrane
Similarity search - Function
Cytomegalovirus UL128 protein / Herpesvirus UL130, cytomegalovirus / HCMV glycoprotein pUL130 / Cytomegalovirus glycoprotein L / Cytomegalovirus glycoprotein L / Betaherpesvirus glycoprotein L (gL) domain profile. / Herpesvirus glycoprotein H main domain / Herpesvirus glycoprotein H / Herpesvirus glycoprotein H, C-terminal / Herpesvirus glycoprotein H, C-terminal domain superfamily / Herpesvirus glycoprotein H C-terminal domain
Similarity search - Domain/homology
Envelope glycoprotein H / UL128 / UL130 / Envelope glycoprotein L / UL131A
Similarity search - Component
Biological speciesHuman betaherpesvirus 5
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKschonsak, M. / Johnson, M.C. / Schelling, R. / Green, E.M. / Rouge, L. / Ho, H. / Patel, N. / Kilic, C. / Kraft, E. / Arthur, C.P. ...Kschonsak, M. / Johnson, M.C. / Schelling, R. / Green, E.M. / Rouge, L. / Ho, H. / Patel, N. / Kilic, C. / Kraft, E. / Arthur, C.P. / Rohou, A.L. / Comps-Agrar, L. / Martinez-Martin, N. / Perez, L. / Payandeh, J. / Ciferri, C.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Sci Adv / Year: 2022
Title: Structural basis for HCMV Pentamer receptor recognition and antibody neutralization.
Authors: Marc Kschonsak / Matthew C Johnson / Rachel Schelling / Evan M Green / Lionel Rougé / Hoangdung Ho / Nidhi Patel / Cem Kilic / Edward Kraft / Christopher P Arthur / Alexis L Rohou / ...Authors: Marc Kschonsak / Matthew C Johnson / Rachel Schelling / Evan M Green / Lionel Rougé / Hoangdung Ho / Nidhi Patel / Cem Kilic / Edward Kraft / Christopher P Arthur / Alexis L Rohou / Laetitia Comps-Agrar / Nadia Martinez-Martin / Laurent Perez / Jian Payandeh / Claudio Ciferri /
Abstract: Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial ...Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.
History
DepositionDec 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 23, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 13, 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_initial_refinement_model / pdbx_modification_feature
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Envelope glycoprotein H
B: Envelope glycoprotein L
C: Envelope protein UL128
D: Envelope glycoprotein UL130
E: Envelope protein UL131A
F: Fab 2C12 heavy chain
G: Fab 2C12 light chain
H: Fab 7I13 light chain
I: Fab 7I13 heavy chain
J: Fab 13H11 heavy chain
K: Fab 13H11 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)340,97717
Polymers339,65011
Non-polymers1,3276
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Elutes as single, monodispersed peak
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Envelope glycoprotein ... , 3 types, 3 molecules ABD

#1: Protein Envelope glycoprotein H / gH


Mass: 87311.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: UL75, gH / Production host: Homo sapiens (human) / References: UniProt: F5H9T3
#2: Protein Envelope glycoprotein L / gL


Mass: 30846.492 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: UL115, gL / Production host: Homo sapiens (human) / References: UniProt: Q71DN9
#4: Protein Envelope glycoprotein UL130 / UL130


Mass: 28866.811 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: UL130 / Production host: Homo sapiens (human) / References: UniProt: Q38M07

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Envelope protein ... , 2 types, 2 molecules CE

#3: Protein Envelope protein UL128 / UL128


Mass: 19746.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: UL128 / Production host: Homo sapiens (human) / References: UniProt: Q38LY2
#5: Protein Envelope protein UL131A / Protein UL131A / UL131A


Mass: 15011.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: UL131A, HHV5wtgp112 / Production host: Homo sapiens (human) / References: UniProt: Q8AZ45

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Antibody , 6 types, 6 molecules FGHIJK

#6: Antibody Fab 2C12 heavy chain


Mass: 27001.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#7: Antibody Fab 2C12 light chain


Mass: 25594.654 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#8: Antibody Fab 7I13 light chain


Mass: 25844.900 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#9: Antibody Fab 7I13 heavy chain


Mass: 27046.506 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#10: Antibody Fab 13H11 heavy chain


Mass: 26600.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#11: Antibody Fab 13H11 light chain


Mass: 25780.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Sugars , 1 types, 6 molecules

#12: 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.: 6 / Source method: obtained synthetically / 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: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to fabs of human neutralizing antibodies 2C12, 7I13 and 13H11
Type: COMPLEX / Entity ID: #1-#11 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.32 MDa / Experimental value: NO
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaCl1
225 mMHEPESHEPES1
SpecimenConc.: 1.77 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse.
Specimen supportDetails: The grid was incubated with a thiol reactive self-assembling reaction mixture of 4mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol, (SPT-0011P6, SensoPath Technologies, ...Details: The grid was incubated with a thiol reactive self-assembling reaction mixture of 4mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol, (SPT-0011P6, SensoPath Technologies, Inc., Bozeman, MT)[23]. Grids were incubated with this self-assembled, monolayer (SAM) solution for 24 hours. Prior to grid freezing, grids were removed from the SAM solution and rinsed with EtOH.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: blot for 3.5s before plunging

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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: 165000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 58 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18326
Details: Images were collected in movie-mode at 5 frames/second.
Image scansMovie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEM3.7.11image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.89model fitting
9PHENIX1.19.2model refinement
10cisTEM1.02initial Euler assignment
11cisTEM1.02final Euler assignment
12RELION3.1classification
13cisTEM1.023D reconstruction
Image processingDetails: Movie frames were corrected for motion and aligned. Images with a CTF fit resolution of 8.0 A or better were selected for particle picking.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5128264 / Details: template-matching particle picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4792984
Details: A composite map was generated from the three individual focused 3D maps using phenix combine_focused_maps
Num. of class averages: 39 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5VOB

5vob


Accession code: 5VOB / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315833
ELECTRON MICROSCOPYf_angle_d0.62421524
ELECTRON MICROSCOPYf_dihedral_angle_d7.062182
ELECTRON MICROSCOPYf_chiral_restr0.0432441
ELECTRON MICROSCOPYf_plane_restr0.0072748

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