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- EMDB-43672: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B... -

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Basic information

Entry
Database: EMDB / ID: EMD-43672
TitleSoluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3, composite map (global and local) and model
Map dataComposite Map
Sample
  • Complex: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) stabilized in a prefusion-like conformation in complex with 1G2 and 7H3
    • Protein or peptide: Envelope glycoprotein B
    • Protein or peptide: 7H3 Fab Heavy Chain
    • Protein or peptide: 7H3 Fab Light Chain
    • Protein or peptide: 1G2 Fab Heavy Chain
    • Protein or peptide: 1G2 Fab Light Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsorthoherpesvirus / betaherpesvirus / cytomegalovirus / human betaherpesvirus 5 / human cytomegalovirus / HCMV / glycoprotein B / gB / HCMV gB / prefusion / prefusion-stabilized / disulfide / VIRAL PROTEIN / 7H2 / 1G2 / 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 speciesHuman betaherpesvirus 5 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 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-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43672.png

Downloads & links

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Map

FileDownload / File: emd_43672.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-1.3997841 - 1.8847591
Average (Standard dev.)-0.000049661045 (±0.030048987)
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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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
    • Protein or peptide: Envelope glycoprotein B
    • Protein or peptide: 7H3 Fab Heavy Chain
    • Protein or peptide: 7H3 Fab Light Chain
    • Protein or peptide: 1G2 Fab Heavy Chain
    • Protein or peptide: 1G2 Fab Light Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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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 / Macromolecule list: #1-#5

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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: Human betaherpesvirus 5
Molecular weightTheoretical: 88.918586 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKY PYRVCSMAQG LDLIRFERNI VCTSMKPINE DLDEGIMVVY KRNICAHTFK VRVYQKVLTF RRSYAYIHTT Y LLGSNTEY ...String:
MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKY PYRVCSMAQG LDLIRFERNI VCTSMKPINE DLDEGIMVVY KRNICAHTFK VRVYQKVLTF RRSYAYIHTT Y LLGSNTEY VAPPMWEIHH INSHSQCYSS YSRVIAGTVF VAYHRDSYEN KTMQLMPDDY SNTCSTRYVT VKDQWHSRGS TW LYRETSN LNCMVTITTA RSKYPYHFFI TSTGDVVDIS PFYNGTNRNA SYFGENADKF FIFPNYTIVS DFGRPNSALE THR LVAFLE RADSVISWDI QDEKNVTCQL TFWEASERTI RSEAEDSYHF SSAKMTATFL SKKQEVNMSD SALDCVRDEA INKL QQIFN TSYNQTYEKY GNVSVFETTG GLVVFWQGIK QKSLVELERL ANRSSLNLTH NSTKSSTDGN NATHLSNMES VHNLV YAQL QFTYDTLRGY INRALAQIAE AWCVDQRRTL EVFKELSKIN PSAILSAIYN KPIAARFMGD VLGLASCVTI NQTSVK VLR DMNVKESPGR CYSRPVVIFN FANSSYVQYG QLGEDNEILL GNHRTEECQL PSLKIFIAGN SAYEYVDYLF KRMIDLS SI STVDSMIALD CDPLCNTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV DPGSGYIPEA PRDGQAYV R KDGEWVLLST FLGAAASLEV LFQGPGHHHH HHHHSAWSHP QFEKGGASGG GGSGGSAWSH PQFEK

UniProtKB: Envelope glycoprotein B

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Macromolecule #2: 7H3 Fab Heavy Chain

MacromoleculeName: 7H3 Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.152064 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGAE VKNPGASVKV SCKASGYTFT DYYIHWVRQA PGQGLEWMGW FNPNSGGTNF VQNFQGRVTM TRDTSISTAY MELSRLRSD DTAMYYCAKD SAKTASAYYG LNFFYYGMDV WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT ...String:
QVQLVQSGAE VKNPGASVKV SCKASGYTFT DYYIHWVRQA PGQGLEWMGW FNPNSGGTNF VQNFQGRVTM TRDTSISTAY MELSRLRSD DTAMYYCAKD SAKTASAYYG LNFFYYGMDV WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCD

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Macromolecule #3: 7H3 Fab Light Chain

MacromoleculeName: 7H3 Fab Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.879361 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVLSQPPSA SGTPGQRVTI SCSGSSSNIG KNYVYWYQQV PGTAPKLLMF KNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYC SAWDGSLSRP LFGGGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP ...String:
QSVLSQPPSA SGTPGQRVTI SCSGSSSNIG KNYVYWYQQV PGTAPKLLMF KNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYC SAWDGSLSRP LFGGGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTECS

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Macromolecule #4: 1G2 Fab Heavy Chain

MacromoleculeName: 1G2 Fab Heavy Chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.206172 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QLQLQESGPG LVKPSETLSL TCTVSGASID RSTYYWGWIR QPPGKGLEWI ANIYYNGRAV YSPSLKSRVT ISVDTSKNQF SLKVRSLTA ADTAVYYCAT RWNYFFDFDY WGRGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QLQLQESGPG LVKPSETLSL TCTVSGASID RSTYYWGWIR QPPGKGLEWI ANIYYNGRAV YSPSLKSRVT ISVDTSKNQF SLKVRSLTA ADTAVYYCAT RWNYFFDFDY WGRGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCD

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Macromolecule #5: 1G2 Fab Light Chain

MacromoleculeName: 1G2 Fab Light Chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.853125 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVLTQPPSA SGTPGQRVTI SCSGSSSNIE TNYVSWYQQF PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEAEYYC GTWDDNSWVF GGGTKLTVLG QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA G VETTTPSK ...String:
QSVLTQPPSA SGTPGQRVTI SCSGSSSNIE TNYVSWYQQF PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEAEYYC GTWDDNSWVF GGGTKLTVLG QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA G VETTTPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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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

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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: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.0)
Details: A mask was created around gB structural domain I (DI) and 1G2 using ChimeraX and imported to cryoSPARC for local refinement. The resulting local map was combined with the global map using ...Details: A mask was created around gB structural domain I (DI) and 1G2 using ChimeraX and imported to cryoSPARC for local refinement. The resulting local map was combined with the global map using PHENIX combine_focused_maps
Number images used: 165982
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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