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- PDB-7t4r: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in... -

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Basic information

Entry
Database: PDB / ID: 7t4r
TitleCryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with THBD and neutralizing fabs MSL-109 and 13H11
Components
  • (Envelope glycoprotein ...) x 3
  • (Envelope protein ...Viral envelope) x 2
  • Fab 13H11 heavy chain
  • Fab 13H11 light chain
  • Fab MSL-109 heavy chain
  • Fab MSL-109 light chain
  • Thrombomodulin
KeywordsVIRAL PROTEIN/Immune System / Human Cytomegalovirus / glycoprotein complex / antibody complex / thrombomodulin / VIRAL PROTEIN / VIRAL PROTEIN-Immune System complex
Function / homology
Function and homology information


blood coagulation, common pathway / negative regulation of blood coagulation / apicolateral plasma membrane / serine-type endopeptidase complex / zymogen activation / vacuolar membrane / negative regulation of platelet activation / response to X-ray / negative regulation of fibrinolysis / Common Pathway of Fibrin Clot Formation ...blood coagulation, common pathway / negative regulation of blood coagulation / apicolateral plasma membrane / serine-type endopeptidase complex / zymogen activation / vacuolar membrane / negative regulation of platelet activation / response to X-ray / negative regulation of fibrinolysis / Common Pathway of Fibrin Clot Formation / response to cAMP / host cell endosome membrane / female pregnancy / Cell surface interactions at the vascular wall / transmembrane signaling receptor activity / blood coagulation / signaling receptor activity / host cell Golgi apparatus / response to lipopolysaccharide / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / fusion of virus membrane with host plasma membrane / external side of plasma membrane / viral envelope / calcium ion binding / host cell plasma membrane / virion membrane / cell surface / proteolysis / extracellular space / membrane / plasma membrane
Similarity search - Function
Thrombomodulin-like, EGF-like / Thrombomodulin like fifth domain, EGF-like / Herpesvirus UL130, cytomegalovirus / HCMV glycoprotein pUL130 / 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 ...Thrombomodulin-like, EGF-like / Thrombomodulin like fifth domain, EGF-like / Herpesvirus UL130, cytomegalovirus / HCMV glycoprotein pUL130 / 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 / Complement Clr-like EGF domain / Complement Clr-like EGF-like / Calcium-binding EGF domain / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / Coagulation Factor Xa inhibitory site / C-type lectin-like/link domain superfamily / EGF-type aspartate/asparagine hydroxylation site / C-type lectin fold / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
Envelope glycoprotein H / Thrombomodulin / UL128 / UL130 / Envelope glycoprotein L / UL131A
Similarity search - Component
Biological speciesHomo sapiens (human)
Human betaherpesvirus 5
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thrombomodulin
B: Envelope glycoprotein H
C: Envelope glycoprotein L
D: Envelope protein UL128
E: Envelope glycoprotein UL130
F: Envelope protein UL131A
G: Fab 13H11 heavy chain
H: Fab 13H11 light chain
I: Fab MSL-109 light chain
J: Fab MSL-109 heavy chain
K: Envelope glycoprotein H
L: Envelope glycoprotein L
M: Envelope protein UL128
N: Envelope glycoprotein UL130
O: Envelope protein UL131A
P: Fab 13H11 heavy chain
Q: Fab 13H11 light chain
R: Fab MSL-109 light chain
S: Fab MSL-109 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)638,35633
Polymers635,25919
Non-polymers3,09714
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Envelope glycoprotein ... , 3 types, 6 molecules BKCLEN

#2: Protein Envelope glycoprotein H / gH


Mass: 87311.273 Da / Num. of mol.: 2
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
#3: Protein Envelope glycoprotein L / gL


Mass: 30846.492 Da / Num. of mol.: 2
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
#5: Protein Envelope glycoprotein UL130 / UL130


Mass: 28866.811 Da / Num. of mol.: 2
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, 4 molecules DMFO

#4: Protein Envelope protein UL128 / / UL128


Mass: 19746.023 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human betaherpesvirus 5 / Gene: UL128 / Production host: Homo sapiens (human) / References: UniProt: Q38LY2
#6: Protein Envelope protein UL131A / / Protein UL131A / UL131A


Mass: 15011.043 Da / Num. of mol.: 2
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 , 4 types, 8 molecules GPHQIRJS

#7: Antibody Fab 13H11 heavy chain


Mass: 26600.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#8: Antibody Fab 13H11 light chain


Mass: 25780.020 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#9: Antibody Fab MSL-109 light chain


Mass: 28355.809 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#10: Antibody Fab MSL-109 heavy chain


Mass: 27547.818 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Protein / Sugars , 2 types, 15 molecules A

#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.: 14 / 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
#1: Protein Thrombomodulin / / TM / Fetomodulin


Mass: 55128.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: THBD, THRM / Production host: Homo sapiens (human) / References: UniProt: P07204

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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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to 1x THBD and 2x fabs of human neutralizing antibodies MSL-109 and 13H11
Type: COMPLEX / Entity ID: #1-#10 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.59 MDa / Experimental value: NO
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaClSodium chloride1
225 mMHEPESHEPES1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10926

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEM3.7.11image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.89model fitting
9RELION3.1initial Euler assignment
11RELION3.1classification
12cisTEM1.023D reconstruction
13PHENIX1.19.2model refinement
Image processingDetails: Movie frames were corrected for motion and aligned. Images with a CTF fit resolution of 5.0 A or better were selected for particle picking.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10680163
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 504492
Details: A composite map was generated from the three individual focused 3D maps.
Symmetry type: POINT

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