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- PDB-7lbf: CryoEM structure of the HCMV Trimer gHgLgO in complex with human ... -

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Entry
Database: PDB / ID: 7lbf
TitleCryoEM structure of the HCMV Trimer gHgLgO in complex with human Platelet-derived growth factor receptor alpha and 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
  • Isoform 3 of Platelet-derived growth factor receptor alpha
KeywordsVIRAL PROTEIN/Immune System / virus / receptor / complex / neutralizing antibody / VIRAL PROTEIN / VIRAL PROTEIN-Immune System complex
Function / homology
Function and homology information


platelet-derived growth factor alpha-receptor activity / platelet-derived growth factor receptor-alpha signaling pathway / Imatinib-resistant PDGFR mutants / Sunitinib-resistant PDGFR mutants / Regorafenib-resistant PDGFR mutants / Sorafenib-resistant PDGFR mutants / PDGFR mutants bind TKIs / metanephric glomerular capillary formation / regulation of mesenchymal stem cell differentiation / luteinization ...platelet-derived growth factor alpha-receptor activity / platelet-derived growth factor receptor-alpha signaling pathway / Imatinib-resistant PDGFR mutants / Sunitinib-resistant PDGFR mutants / Regorafenib-resistant PDGFR mutants / Sorafenib-resistant PDGFR mutants / PDGFR mutants bind TKIs / metanephric glomerular capillary formation / regulation of mesenchymal stem cell differentiation / luteinization / positive regulation of cell proliferation by VEGF-activated platelet derived growth factor receptor signaling pathway / platelet-derived growth factor binding / retina vasculature development in camera-type eye / embryonic skeletal system morphogenesis / vascular endothelial growth factor binding / cardiac myofibril assembly / embryonic digestive tract morphogenesis / vascular endothelial growth factor receptor activity / Leydig cell differentiation / male genitalia development / cell activation / positive regulation of chemotaxis / signal transduction involved in regulation of gene expression / Signaling by PDGF / embryonic cranial skeleton morphogenesis / platelet-derived growth factor receptor binding / face morphogenesis / estrogen metabolic process / adrenal gland development / roof of mouth development / odontogenesis of dentin-containing tooth / microvillus / negative regulation of platelet activation / platelet-derived growth factor receptor signaling pathway / white fat cell differentiation / hematopoietic progenitor cell differentiation / positive regulation of phospholipase C activity / positive regulation of calcium-mediated signaling / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / transmembrane receptor protein tyrosine kinase activity / extracellular matrix organization / cell chemotaxis / Downstream signal transduction / host cell endosome membrane / HCMV Late Events / regulation of actin cytoskeleton organization / cellular response to amino acid stimulus / lung development / wound healing / cilium / receptor protein-tyrosine kinase / platelet aggregation / cellular response to reactive oxygen species / HCMV Early Events / peptidyl-tyrosine phosphorylation / cell surface receptor protein tyrosine kinase signaling pathway / Constitutive Signaling by Aberrant PI3K in Cancer / positive regulation of fibroblast proliferation / PIP3 activates AKT signaling / cell junction / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / host cell Golgi apparatus / RAF/MAP kinase cascade / in utero embryonic development / entry receptor-mediated virion attachment to host cell / protein autophosphorylation / positive regulation of ERK1 and ERK2 cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor complex / protein kinase activity / positive regulation of cell migration / symbiont entry into host cell / fusion of virus membrane with host plasma membrane / external side of plasma membrane / viral envelope / positive regulation of cell population proliferation / protein-containing complex binding / endoplasmic reticulum membrane / host cell plasma membrane / virion membrane / Golgi apparatus / protein homodimerization activity / protein-containing complex / nucleoplasm / ATP binding / membrane / nucleus / plasma membrane / cytoplasm
Similarity search - Function
Betaherpesvirus glycoprotein L (gL) domain profile. / Herpesvirus UL74, glycoprotein / Herpes UL74 glycoproteins / Platelet-derived growth factor receptor alpha / 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 ...Betaherpesvirus glycoprotein L (gL) domain profile. / Herpesvirus UL74, glycoprotein / Herpes UL74 glycoproteins / Platelet-derived growth factor receptor alpha / 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 / Tyrosine-protein kinase, receptor class III, conserved site / Receptor tyrosine kinase class III signature. / Immunoglobulin I-set / Immunoglobulin I-set domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Immunoglobulin subtype / Immunoglobulin / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Envelope glycoprotein L / Platelet-derived growth factor receptor alpha / Envelope glycoprotein H / Envelope glycoprotein O
Similarity search - Component
Biological speciesHuman cytomegalovirus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 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
D: Isoform 3 of Platelet-derived growth factor receptor alpha
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)349,96029
Polymers343,8958
Non-polymers6,06621
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Eluted as monodispersed peak
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 / Production host: Homo sapiens (human) / References: UniProt: Q8BCU3

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Protein , 1 types, 1 molecules D

#4: Protein Isoform 3 of Platelet-derived growth factor receptor alpha / PDGFR-alpha / Alpha platelet-derived growth factor receptor / Alpha-type platelet-derived growth ...PDGFR-alpha / Alpha platelet-derived growth factor receptor / Alpha-type platelet-derived growth factor receptor / CD140 antigen-like family member A / CD140a antigen / Platelet-derived growth factor alpha receptor / Platelet-derived growth factor receptor 2 / PDGFR-2


Mass: 59154.660 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PDGFRA, PDGFR2, RHEPDGFRA / Production host: Homo sapiens (human)
References: UniProt: P16234, receptor protein-tyrosine kinase

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Antibody , 4 types, 4 molecules EFGH

#5: 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)
#6: 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)
#7: 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)
#8: 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 , 3 types, 21 molecules

#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-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
#10: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-3)-alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[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: 1235.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-3DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1_f3-g1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-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]{[(3+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#11: 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.: 18 / 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

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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 human receptor PDGFRa and neutralizing fabs 13H11 and MSL-109COMPLEX#1-#80MULTIPLE SOURCES
2HCMV Trimer gHgLgOCOMPLEX#1-#31RECOMBINANT
3PDGFRa and neutralizing fabs 13H11 and MSL-109COMPLEX#4-#81RECOMBINANT
Molecular weightValue: 0.335 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Human cytomegalovirus10359
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
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 was monodisperse.
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: 2 / Num. of real images: 34829 / Details: Images were collected in 50 frames every 0.2 s
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatch0.56particle selection
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: 4151085
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3560620
Details: Used score threshold of 0.25 for final 3D reconstruction. Map used for model 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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