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- EMDB-22788: Cryo-EM structure of the HCMV pentamer bound by Fabs 2-18 and 8I21 -

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Basic information

Entry
Database: EMDB / ID: EMD-22788
TitleCryo-EM structure of the HCMV pentamer bound by Fabs 2-18 and 8I21
Map dataLocally refined map sharpened using DeepEMhancer
Sample
  • Complex: HCMV pentamer bound by antibodies 2-18 and 8I21
    • Complex: HCMV pentamer (refinement focused on three of the subunits)
      • Protein or peptide: Envelope protein UL128
      • Protein or peptide: Envelope glycoprotein UL130
      • Protein or peptide: UL131A
    • Complex: Fab 2-18
      • Protein or peptide: 2-18 Fab Heavy Chain
      • Protein or peptide: 2-18 Fab Light Chain
    • Complex: Fab 8I21
      • Protein or peptide: 8I21 Fab Heavy Chain
      • Protein or peptide: 8I21 Fab Light Chain
KeywordsHCMV pentamer / Fab / complex / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homologyHerpesvirus UL130, cytomegalovirus / HCMV glycoprotein pUL130 / viral envelope / Envelope glycoprotein UL130 / UL128 / UL131A
Function and homology information
Biological speciesHuman betaherpesvirus 5 / Homo sapiens (human) / Human cytomegalovirus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsWrapp D / McLellan JS
CitationJournal: Sci Adv / Year: 2022
Title: Structural basis for HCMV Pentamer recognition by neuropilin 2 and neutralizing antibodies.
Authors: Daniel Wrapp / Xiaohua Ye / Zhiqiang Ku / Hang Su / Harrison G Jones / Nianshuang Wang / Akaash K Mishra / Daniel C Freed / Fengsheng Li / Aimin Tang / Leike Li / Dabbu Kumar Jaijyan / Hua ...Authors: Daniel Wrapp / Xiaohua Ye / Zhiqiang Ku / Hang Su / Harrison G Jones / Nianshuang Wang / Akaash K Mishra / Daniel C Freed / Fengsheng Li / Aimin Tang / Leike Li / Dabbu Kumar Jaijyan / Hua Zhu / Dai Wang / Tong-Ming Fu / Ningyan Zhang / Zhiqiang An / Jason S McLellan /
Abstract: Human cytomegalovirus (HCMV) encodes multiple surface glycoprotein complexes to infect a variety of cell types. The HCMV Pentamer, composed of gH, gL, UL128, UL130, and UL131A, enhances entry into ...Human cytomegalovirus (HCMV) encodes multiple surface glycoprotein complexes to infect a variety of cell types. The HCMV Pentamer, composed of gH, gL, UL128, UL130, and UL131A, enhances entry into epithelial, endothelial, and myeloid cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here, we describe the cryo-EM structure of NRP2 bound to Pentamer. The high-affinity interaction between these proteins is calcium dependent and differs from the canonical carboxyl-terminal arginine (CendR) binding that NRP2 typically uses. We also determine the structures of four neutralizing human antibodies bound to the HCMV Pentamer to define susceptible epitopes. Two of these antibodies compete with NRP2 binding, but the two most potent antibodies recognize a previously unidentified epitope that does not overlap the NRP2-binding site. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization.
History
DepositionOct 1, 2020-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kbb
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kbb
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22788.png

Downloads & links

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Map

FileDownload / File: emd_22788.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally refined map sharpened using DeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 384 pix.
= 402.048 Å		1.05 Å/pix.
x 384 pix.
= 402.048 Å		1.05 Å/pix.
x 384 pix.
= 402.048 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.047 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.0018058335 - 2.1686828
Average (Standard dev.)0.000373216 (±0.014037825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 402.04803 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z402.048402.048402.048
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0022.1690.000

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Supplemental data

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Additional map: Locally refined map unsharpened

Fileemd_22788_additional_1.map
AnnotationLocally refined map unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map sharpened with DeepEMhancer

Fileemd_22788_additional_2.map
AnnotationFull map sharpened with DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_22788_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_22788_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HCMV pentamer bound by antibodies 2-18 and 8I21

EntireName: HCMV pentamer bound by antibodies 2-18 and 8I21
Components
  • Complex: HCMV pentamer bound by antibodies 2-18 and 8I21
    • Complex: HCMV pentamer (refinement focused on three of the subunits)
      • Protein or peptide: Envelope protein UL128
      • Protein or peptide: Envelope glycoprotein UL130
      • Protein or peptide: UL131A
    • Complex: Fab 2-18
      • Protein or peptide: 2-18 Fab Heavy Chain
      • Protein or peptide: 2-18 Fab Light Chain
    • Complex: Fab 8I21
      • Protein or peptide: 8I21 Fab Heavy Chain
      • Protein or peptide: 8I21 Fab Light Chain

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Supramolecule #1: HCMV pentamer bound by antibodies 2-18 and 8I21

SupramoleculeName: HCMV pentamer bound by antibodies 2-18 and 8I21 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 280 KDa

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Supramolecule #2: HCMV pentamer (refinement focused on three of the subunits)

SupramoleculeName: HCMV pentamer (refinement focused on three of the subunits)
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3-#5
Source (natural)Organism: Human betaherpesvirus 5

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Supramolecule #3: Fab 2-18

SupramoleculeName: Fab 2-18 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Fab 8I21

SupramoleculeName: Fab 8I21 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6-#7
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: 2-18 Fab Heavy Chain

MacromoleculeName: 2-18 Fab Heavy Chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.640506 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVQSGGG LVQPGGSLRV SCAASGFSFS DHDMDWVRQA PGKGFEWVGR SRNKDYSSTT EYAASVRGRF TISRHTSEDL LYLELNTVK TEDTAVYFCA RGPHHSDRSG YYGGTFDIWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD Y FPEPVTVS ...String:
EVQLVQSGGG LVQPGGSLRV SCAASGFSFS DHDMDWVRQA PGKGFEWVGR SRNKDYSSTT EYAASVRGRF TISRHTSEDL LYLELNTVK TEDTAVYFCA RGPHHSDRSG YYGGTFDIWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD Y FPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKGLEVL FQ

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Macromolecule #2: 2-18 Fab Light Chain

MacromoleculeName: 2-18 Fab Light Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.414055 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPSS VSASVGDRVI ITCRASQGIS SWLAWYQQKP GRAPRLLIYD ASTLESGVPS RFSGRGSGTE FTLTINSLQP EDFATYYCQ QGNMFPLTFG GGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPSS VSASVGDRVI ITCRASQGIS SWLAWYQQKP GRAPRLLIYD ASTLESGVPS RFSGRGSGTE FTLTINSLQP EDFATYYCQ QGNMFPLTFG GGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #3: Envelope protein UL128

MacromoleculeName: Envelope protein UL128 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human cytomegalovirus
Molecular weightTheoretical: 16.684299 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EECCEFINVN HPPERCYDFK MCNRFTVALR CPDGEVCYSP EKTAEIRGIV TTMTHSLTRQ VVHNKLTSCN YNPLYLEADG RIRCGKVND KAQYLLGAAG SVPYRWINLE YDKITRIVGL DQYLESVKKH KRLDVCRAKM GYMLQ

UniProtKB: UL128

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Macromolecule #4: Envelope glycoprotein UL130

MacromoleculeName: Envelope glycoprotein UL130 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human cytomegalovirus
Molecular weightTheoretical: 21.761678 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
SPWSTLTANQ NPSPPWSKLT YSKPHDAATF YCPFLYPSPP RSPLQFSGFQ QVSTGPECRN ETLYLLYNRE GQTLVERSST WVKKVIWYL SGRNQTILQR MPQTASKPSD GNVQISVEDA KIFGAHMVPK QTKLLRFVVN DGTRYQMCVM KLESWAHVFR D YSVSFQVR LTFTEANNQT YTFCTHPNLI V

UniProtKB: Envelope glycoprotein UL130

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Macromolecule #5: UL131A

MacromoleculeName: UL131A / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human cytomegalovirus
Molecular weightTheoretical: 13.005457 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QCQRETAEKN DYYRVPHYWD ACSRALPDQT RYKYVEQLVD LTLNYHYDAS HGLDNFDVLK RINVTEVSLL ISDFRRQNRR GGTNKRTTF NAAGSLAPHA RSLEFSVRLF AN

UniProtKB: UL131A

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Macromolecule #6: 8I21 Fab Heavy Chain

MacromoleculeName: 8I21 Fab Heavy Chain / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.19634 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LVELVESGGG VVQPGRSLRL SCAASGFTFS SDGMHWVRQS PGRGLEWVAF ISSDGSTPYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAMYFCAKD WALFRWLRTF DHWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV T VSWNSGAL ...String:
LVELVESGGG VVQPGRSLRL SCAASGFTFS SDGMHWVRQS PGRGLEWVAF ISSDGSTPYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAMYFCAKD WALFRWLRTF DHWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV T VSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKG LEVLFQ

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Macromolecule #7: 8I21 Fab Light Chain

MacromoleculeName: 8I21 Fab Light Chain / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.459006 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ETVMTQSPAT LSVSPGGRAT LSCRASQSVG INLAWYQQKP GQAPRLLIYG ASTRASGFPA RFSGSGSGTE FTLTITSLQS EDFAVYYCQ QYNDWPPWTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
ETVMTQSPAT LSVSPGGRAT LSCRASQSVG INLAWYQQKP GQAPRLLIYG ASTRASGFPA RFSGSGSGTE FTLTITSLQS EDFAVYYCQ QYNDWPPWTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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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

Concentration0.25 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
2.0 mMC4H11NO3Tris
200.0 mMNaClsodium chloride
0.2 %NaN3sodium azide
0.01 %(C6.2H10.3O1.35N0.65Na0.35)72amphipol A8-35
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 36.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 249802
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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