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- PDB-7lbe: CryoEM structure of the HCMV Trimer gHgLgO in complex with neutra... -

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Basic information

Entry
Database: PDB / ID: 7lbe
TitleCryoEM structure of the HCMV Trimer gHgLgO in complex with neutralizing fabs 13H11 and MSL-109
Components
  • (Envelope glycoprotein ...) x 3
  • Fab 13H11 heavy chain
  • Fab 13H11 light chain
  • Fab MSL-109 heavy chain
  • Fab MSL-109 light chain
KeywordsVIRAL PROTEIN/Immune System / virus / receptor / complex / neutralizing antibody / VIRAL PROTEIN / VIRAL PROTEIN-Immune System complex
Function / homology
Function and homology information


host cell endosome membrane / HCMV Late Events / HCMV Early Events / host cell Golgi apparatus / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / fusion of virus membrane with host plasma membrane / viral envelope / host cell plasma membrane / virion membrane / plasma membrane
Similarity search - Function
Betaherpesvirus glycoprotein L (gL) domain profile. / Herpesvirus UL74, glycoprotein / Herpes UL74 glycoproteins / Cytomegalovirus glycoprotein L / Cytomegalovirus glycoprotein L / 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
alpha-D-mannopyranose / Envelope glycoprotein L / Envelope glycoprotein H / Envelope glycoprotein O
Similarity search - Component
Biological speciesHuman cytomegalovirus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKschonsak, M. / Rouge, L. / Arthur, C.P. / Hoangdung, H. / Patel, N. / Kim, I. / Johnson, M. / Kraft, E. / Rohou, A.L. / Gill, A. ...Kschonsak, M. / Rouge, L. / Arthur, C.P. / Hoangdung, H. / Patel, N. / Kim, I. / Johnson, M. / Kraft, E. / Rohou, A.L. / Gill, A. / Martinez-Martin, N. / Payandeh, J. / Ciferri, C.
CitationJournal: Cell / Year: 2021
Title: Structures of HCMV Trimer reveal the basis for receptor recognition and cell entry.
Authors: Marc Kschonsak / Lionel Rougé / Christopher P Arthur / Ho Hoangdung / Nidhi Patel / Ingrid Kim / Matthew C Johnson / Edward Kraft / Alexis L Rohou / Avinash Gill / Nadia Martinez-Martin / ...Authors: Marc Kschonsak / Lionel Rougé / Christopher P Arthur / Ho Hoangdung / Nidhi Patel / Ingrid Kim / Matthew C Johnson / Edward Kraft / Alexis L Rohou / Avinash Gill / Nadia Martinez-Martin / Jian Payandeh / Claudio Ciferri /
Abstract: Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind ...Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFβR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFβR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFβR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.
History
DepositionJan 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 10, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 17, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: Envelope glycoprotein H
B: Envelope glycoprotein L
C: Envelope glycoprotein O
E: Fab 13H11 light chain
F: Fab 13H11 heavy chain
G: Fab MSL-109 light chain
H: Fab MSL-109 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,38127
Polymers284,7407
Non-polymers5,64120
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Eluted as a main peak with shoulder (monomer and dimer)
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Envelope glycoprotein ... , 3 types, 3 molecules ABC

#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 cytomegalovirus (strain Merlin) / Strain: Merlin / Gene: gH, UL75 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q6SW67
#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 cytomegalovirus (strain Merlin) / Strain: Merlin / Gene: gL, UL115 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: F5HCH8
#3: Protein Envelope glycoprotein O / UL74 / UL74 protein


Mass: 58298.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human cytomegalovirus / Gene: UL74 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q8BCU3

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Antibody , 4 types, 4 molecules EFGH

#4: 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)
#5: 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)
#6: Antibody Fab MSL-109 light chain


Mass: 28355.809 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 MSL-109 heavy chain


Mass: 27547.818 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 , 4 types, 20 molecules

#8: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D- ...alpha-D-mannopyranose-(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)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#9: 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.: 2
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
#10: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 16 / 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
#11: Sugar ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose / Mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1HCMV Trimer gHgLgO bound to neutralizing fabs 13H11 and MSL-109COMPLEX#1-#70RECOMBINANT
2HCMV Trimer gHgLgOCOMPLEX#1-#31RECOMBINANT
3neutralizing fabs 13H11 and MSL-109COMPLEX#4-#71RECOMBINANT
Molecular weightValue: 0.275 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Human cytomegalovirus10359
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Escherichia coli (E. coli)562
32Homo sapiens (human)9606
Buffer solutionpH: 7.5
Details: The sample was gently cross-linked with 0.025% (v/v) EM-grade glutaraldehyde for 10 min at RT and quenched with 9 mM Tris pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.3 Msodium chlorideNaClSodium chloride1
20.025 MHEPES1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample contained monomeric and dimeric protein complexes.
Specimen supportDetails: The grid was coated with Au/Pd 80/20 prior use. / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 2.5 seconds 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 FIELDBright-field microscopy / Nominal magnification: 165000 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14717 / Details: Images were collected in 50 frames every 0.2 s
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEM3.7.11image acquisition
4CTFFINDCTF correction4.1.13
7UCSF Chimera1.13.1model fitting
8Coot0.8.9model fitting
10PHENIX1.19model refinement
11ISOLDE1.1.0model refinement
12cisTEM1.02initial Euler assignment
13cisTEM1.02final Euler assignment
14RELION3.1classification
15cisTEM1.023D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1478640
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1350211
Details: Used score threshold of 0.25 for final 3D reconstruction. Map used for model building and refinements is a composite map after combining 3 focussed maps with PHENIX
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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