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- EMDB-43671: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B... -

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Entry
Database: EMDB / ID: EMD-43671
TitleSoluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3, local refinement
Map datasharpened
Sample
  • Complex: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3
    • Complex: human cytomegalovirus (HCMV) glycoprotein B (gB) ectodomain
    • Complex: Fab 1G2
    • Complex: Fab 7H3
Keywordsorthoherpesvirus / betaherpesvirus / cytomegalovirus / human betaherpesvirus 5 / human cytomegalovirus / HCMV / glycoprotein B / gB / HCMV gB / prefusion-stabilized / disulfide / viral protein / 1G2
Biological speciesHuman betaherpesvirus 5 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSponholtz MR / Byrne PO / McLellan JS
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.
Authors: Madeline R Sponholtz / Patrick O Byrne / Alison G Lee / Ajit R Ramamohan / Jory A Goldsmith / Ryan S McCool / Ling Zhou / Nicole V Johnson / Ching-Lin Hsieh / Megan Connors / Krithika P ...Authors: Madeline R Sponholtz / Patrick O Byrne / Alison G Lee / Ajit R Ramamohan / Jory A Goldsmith / Ryan S McCool / Ling Zhou / Nicole V Johnson / Ching-Lin Hsieh / Megan Connors / Krithika P Karthigeyan / Chelsea M Crooks / Adelaide S Fuller / John D Campbell / Sallie R Permar / Jennifer A Maynard / Dong Yu / Matthew J Bottomley / Jason S McLellan /
Abstract: Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, ...Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.
History
DepositionFeb 9, 2024-
Header (metadata) releaseSep 18, 2024-
Map releaseSep 18, 2024-
UpdateSep 18, 2024-
Current statusSep 18, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43671.png

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Map

FileDownload / File: emd_43671.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 399.936 Å		0.83 Å/pix.
x 480 pix.
= 399.936 Å		0.83 Å/pix.
x 480 pix.
= 399.936 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-1.4809011 - 2.0780818
Average (Standard dev.)0.00019169471 (±0.017862068)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 399.93597 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43671_msk_1.map
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Additional map: unsharpened

Fileemd_43671_additional_1.map
Annotationunsharpened
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Half map: half A

Fileemd_43671_half_map_1.map
Annotationhalf A
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Half map: half B

Fileemd_43671_half_map_2.map
Annotationhalf B
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Sample components

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Entire : Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B...

EntireName: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3
Components
  • Complex: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3
    • Complex: human cytomegalovirus (HCMV) glycoprotein B (gB) ectodomain
    • Complex: Fab 1G2
    • Complex: Fab 7H3

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Supramolecule #1: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B...

SupramoleculeName: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: human cytomegalovirus (HCMV) glycoprotein B (gB) ectodomain

SupramoleculeName: human cytomegalovirus (HCMV) glycoprotein B (gB) ectodomain
type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Human betaherpesvirus 5

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Supramolecule #3: Fab 1G2

SupramoleculeName: Fab 1G2 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Fab 7H3

SupramoleculeName: Fab 7H3 / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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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

Concentration4 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
2.0 mMTris
200.0 mMsodium chlorideNaCl
0.02 percent (w/v)sodium azide
0.12 percent (w/v)CHAPS
0.01 percent (w/v)amphipol A8-35
3.0 percent (v/v)glycerol

Details: 2 mM Tris pH 8, 200 mM NaCl, 0.02% w/v sodium azide, 3% (v/v) glycerol, 0.12% (w/v) CHAPS, 0.01% (w/v) amphipol A8-35
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSoluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the stabilized in a prefusion-like conformation, 1G2 Fab, and 7H3 Fab purified separately, mixed, and incubated for 30 minutes on ice prior to grid preparation

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 12524 / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.0) / Number images used: 165982
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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