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- EMDB-22828: HCMV prefusion gB in complex with fusion inhibitor WAY-174865 -

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Basic information

Entry
Database: EMDB / ID: EMD-22828
TitleHCMV prefusion gB in complex with fusion inhibitor WAY-174865
Map dataHCMV gB in prefusion conformation
Sample
  • Complex: HCMV prefusion gB stabilized by a fusion inhibitor
    • Protein or peptide: Envelope glycoprotein B
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide
  • Ligand: CALCIUM ION
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 ectodomain / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B, PH-like domain 1 / Herpesvirus Glycoprotein B, PH-like domain 2 / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B PH-like domain / Herpesvirus Glycoprotein B, PH-like domain 2 superfamily
Similarity search - Domain/homology
Envelope glycoprotein B
Similarity search - Component
Biological speciesHuman cytomegalovirus (strain Towne) / HHV-5 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLiu Y / Heim PK / Che Y / Chi X / Qiu X / Han S / Dormitzer PR / 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 9, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 17, 2021-
Current statusMar 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kdp
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kdp
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22828.png

Downloads & links

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Map

FileDownload / File: emd_22828.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHCMV gB in prefusion conformation
Voxel sizeX=Y=Z: 1.376 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.18670075 - 0.31882638
Average (Standard dev.)0.00031225433 (±0.0054186704)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 385.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3761.3761.376
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z385.280385.280385.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1870.3190.000

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Supplemental data

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Sample components

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Entire : HCMV prefusion gB stabilized by a fusion inhibitor

EntireName: HCMV prefusion gB stabilized by a fusion inhibitor
Components
  • Complex: HCMV prefusion gB stabilized by a fusion inhibitor
    • Protein or peptide: Envelope glycoprotein B
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide
  • Ligand: CALCIUM ION

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Supramolecule #1: HCMV prefusion gB stabilized by a fusion inhibitor

SupramoleculeName: HCMV prefusion gB stabilized by a fusion inhibitor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: SM5-1 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)In ...Details: SM5-1 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)In the density map, the FAb is poorly resolved and not modeled
Source (natural)Organism: Human cytomegalovirus (strain Towne) / Strain: Towne
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Envelope glycoprotein B

MacromoleculeName: Envelope glycoprotein B / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: HHV-5 (virus) / Strain: Towne
Molecular weightTheoretical: 102.067688 KDa
SequenceString: MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKY PYRVCSMAQG TDLIRFERNI VCTSMKPINE DLDEGIMVVY KRNIVAHTFK VRVYQKVLTF RRSYAYIHTT Y LLGSNTEY ...String:
MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKY PYRVCSMAQG TDLIRFERNI VCTSMKPINE DLDEGIMVVY KRNIVAHTFK VRVYQKVLTF RRSYAYIHTT Y LLGSNTEY VAPPMWEIHH INSHSQCYSS YSRVIAGTVF VAYHRDSYEN KTMQLMPDDY SNTHSTRYVT VKDQWHSRGS TW LYRETCN LNCMVTITTA RSKYPYHFFA TSTGDVVDIS PFYNGTNRNA SYFGENADKF FIFPNYTIVS DFGRPNSALE THR LVAFLE RADSVISWDI QDEKNVTCQL TFWEASERTI RSEAEDSYHF SSAKMTATFL SKKQEVNMSD SALDCVRDEA INKL QQIFN TSYNQTYEKY GNVSVFETTG GLVVFWQGIK QKSLVELERL ANRSSLNLTH NRTKRSTDGN NATHLSNMES VHNLV YAQL QFTYDTLRGY INRALAQIAE AWCVDQRRTL EVFKELSKIN PSAILSAIYN KPIAARFMGD VLGLASCVTI NQTSVK VLR DMNVKESPGR CYSRPVVIFN FANSSYVQYG QLGEDNEILL GNHRTEECQL PSLKIFIAGN SAYEYVDYLF KRMIDLS SI STVDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV DPLPPYLKGL DDLMSGLG A AGKAVGVAIG AVGGAVASVV EGVATFLKNP FGAFTIILVA IAVVIIIYLI YTRQRRLCMQ PLQNLFPYLV SADGTTVTS GNTKDTSLQA PPSYEESVYN SGRKGPGPPS SDASTAAPPY TNEQAYQMLL ALVRLDAEQR AQQNGTDSLD GQTGTQDKGQ KPNLLDRLR HRKNGYRHLK DSDEEENV

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 30 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioy...

MacromoleculeName: N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide
type: ligand / ID: 3 / Number of copies: 3 / Formula: WCY
Molecular weightTheoretical: 518.498 Da
Chemical component information

ChemComp-WCY:
N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamothioyl)amino]phenyl}-1,3-thiazole-4-carboxamide

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.4 / Details: PBS, 0.1 % DDM, 1 mM EDTA, 2 mg/L WAY-174865
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II / Details: 4 second, -2 force.
Detailsthis sample was monodispersed

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-28 / Number grids imaged: 3 / Number real images: 7768 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 18000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailsthe movies are motion corrected dose weighted and averaged with and binned 2x in Fourier space with MotionCor2 program
Particle selectionNumber selected: 1906220
Details: a 20 angstrom low pass filtered postfusion structure was used to generate reference images for the particle auto picking
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5cxf

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1 beta) / Number images used: 129837
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 2.1 beta)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 1.8 degrees
Software - Name: RELION (ver. 2.1 beta)
Final 3D classificationNumber classes: 5 / Avg.num./class: 129837 / Software - Name: RELION (ver. 2.1 beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5cxf

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7kdp:
HCMV prefusion gB in complex with fusion inhibitor WAY-174865

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