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- PDB-7kdd: HCMV postfusion gB in complex with SM5-1 Fab -

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Basic information

Entry
Database: PDB / ID: 7kdd
TitleHCMV postfusion gB in complex with SM5-1 Fab
Components
  • Envelope glycoprotein B
  • SM5-1 Fab antibody heavy chain
  • SM5-1 Fab antibody light chain
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / fusogen / postfusion / HCMV / gB / antibody / VIRAL PROTEIN / 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 speciesHomo sapiens (human)
Human cytomegalovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLiu, Y. / Heim, P.K. / Che, Y. / Chi, X. / Qiu, X. / Han, S. / Dormitzer, P.R. / Yang, X.
CitationJournal: Sci Adv / Year: 2021
Title: Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion.
Authors: Yuhang Liu / Kyle P Heim / Ye Che / Xiaoyuan Chi / Xiayang Qiu / Seungil Han / Philip R Dormitzer / Xinzhen Yang /
Abstract: Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to ...Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.
History
DepositionOct 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Envelope glycoprotein B
D: SM5-1 Fab antibody heavy chain
E: SM5-1 Fab antibody light chain
B: Envelope glycoprotein B
F: SM5-1 Fab antibody heavy chain
G: SM5-1 Fab antibody light chain
C: Envelope glycoprotein B
H: SM5-1 Fab antibody heavy chain
I: SM5-1 Fab antibody light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)463,39840
Polymers456,7229
Non-polymers6,67631
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area49210 Å2
ΔGint-161 kcal/mol
Surface area120550 Å2

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Components

#1: Protein Envelope glycoprotein B / gB


Mass: 102067.688 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Human cytomegalovirus (strain Towne) / Strain: Towne / References: UniProt: P13201
#2: Antibody SM5-1 Fab antibody heavy chain


Mass: 27534.768 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pLEV123 / Cell line (production host): 293 / Production host: Scombridae gen. sp. (tuna)
#3: Antibody SM5-1 Fab antibody light chain


Mass: 22638.217 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Strain: Towne / Plasmid: pLEV123 / Cell line (production host): 293 / Production host: Scombridae gen. sp. (tuna)
#4: 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.: 30 / 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
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1HCMV postfusion gB in complex with a neutralizing antibody Fab SM5-1COMPLEXSM5-1 FAb is recombinant expressed with 6xHis and Strep sequence at C-terminal as purification tags. The FAb was used to purify the gB protein from lab cultured HCMV virus (Towne strain)#1-#30MULTIPLE SOURCES
2Envelope glycoprotein BCOMPLEX#11NATURAL
3SM5-1 Fab antibody heavy chain, SM5-1 Fab antibody light chainCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Human cytomegalovirus (strain Towne)10363Towne
23Homo sapiens (human)9606
Source (recombinant)Organism: Scombridae gen. sp. (tuna)
Buffer solutionpH: 7.4 / Details: PBS, 0.1 % DDM, 1 mM EDTA, 2 mg/L WAY-174865
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: this sample was monodispersed
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 4 second, -2 force

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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: 18000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 7768
Image scansMovie frames/image: 28 / Used frames/image: 1-28

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Processing

SoftwareName: PHENIX / Version: dev_3965: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.06CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION2.1 betainitial Euler assignment
11RELION2.1 betafinal Euler assignment
12RELION2.1 betaclassification
13RELION2.1 beta3D reconstruction
Image processingDetails: the movies are motion corrected dose weighted and averaged with and binned 2x in Fourier space with MotionCor2 program
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1906220
Details: a 20 angstrom low pass filtered postfusion structure was used to generate reference images for the particle auto picking
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198170 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
15CXF

5cxf

1
21
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 76.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00420166
ELECTRON MICROSCOPYf_angle_d0.60427339
ELECTRON MICROSCOPYf_dihedral_angle_d4.4972700
ELECTRON MICROSCOPYf_chiral_restr0.0443105
ELECTRON MICROSCOPYf_plane_restr0.0043459

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