EMDB-25685: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in...

Basic information

Entry

Database: EMDB / ID: EMD-25685

• Title: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with neutralizing fabs 2C12, 7I13 and 13H11
• Map data: Composite map generated by combining focused refined maps. Map used for model refinements

Sample

• Complex: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to fabs of human neutralizing antibodies 2C12, 7I13 and 13H11
  • Protein or peptide: x 11 types
• Ligand: x 1 types

Function / homology

Function:

host cell endosome membrane / host cell Golgi apparatus / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / fusion of virus membrane with host plasma membrane / viral envelope / host cell plasma membrane / virion membrane / membrane

Domain/homology:

Herpesvirus UL130, cytomegalovirus / HCMV glycoprotein pUL130 / Cytomegalovirus glycoprotein L / Cytomegalovirus glycoprotein L / Herpesvirus glycoprotein H main domain / Herpesvirus glycoprotein H / Herpesvirus glycoprotein H, C-terminal / Herpesvirus glycoprotein H, C-terminal domain superfamily / Herpesvirus glycoprotein H C-terminal domain

Component:

Envelope glycoprotein H / UL128 / UL130 / Envelope glycoprotein L / UL131A

• Biological species: Human betaherpesvirus 5 / Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 2.9 Å
• Authors: Kschonsak M / Johnson MC / Schelling R / Green EM / Rouge L / Ho H / Patel N / Kilic C / Kraft E / Arthur CP / Rohou AL / Comps-Agrar L / Martinez-Martin N / Perez L / Payandeh J / Ciferri C

Funding support

1 items

Organization	Grant number	Country
Not funded

• Citation: Journal: Sci Adv / Year: 2022; Title: Structural basis for HCMV Pentamer receptor recognition and antibody neutralization.; Authors: Marc Kschonsak / Matthew C Johnson / Rachel Schelling / Evan M Green / Lionel Rougé / Hoangdung Ho / Nidhi Patel / Cem Kilic / Edward Kraft / Christopher P Arthur / Alexis L Rohou / Laetitia Comps-Agrar / Nadia Martinez-Martin / Laurent Perez / Jian Payandeh / Claudio Ciferri / ; Abstract: Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.

History

Deposition	Dec 10, 2021	-
Header (metadata) release	Mar 23, 2022	-
Map release	Mar 23, 2022	-
Update	Mar 23, 2022	-
Current status	Mar 23, 2022	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_25685.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Composite map generated by combining focused refined maps. Map used for model refinements
• Voxel size: X=Y=Z: 1.1948 Å

Density

Contour Level	By AUTHOR: 4.9
Minimum - Maximum	-15.368733 - 30.294058
Average (Standard dev.)	-0.025309997 (±0.59128356)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 382.336 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Half map 2 of focused refinement on 2C12, 7I13, UL region.

• File: emd_25685_additional_1.map
• Annotation: Half map 2 of focused refinement on 2C12, 7I13, UL region.

Additional map: Sharpened map of focused refinement on gH and...

• File: emd_25685_additional_10.map
• Annotation: Sharpened map of focused refinement on gH and 13H11 region. Used for composite map generation.

Additional map: Non-sharpened map of focused refinement on gH and 13H11 region.

• File: emd_25685_additional_11.map
• Annotation: Non-sharpened map of focused refinement on gH and 13H11 region.

Additional map: Half map 2 of focused refinement on gH and 13H11 region.

• File: emd_25685_additional_12.map
• Annotation: Half map 2 of focused refinement on gH and 13H11 region.

Additional map: Half map 1 of focused refinement on gH and 13H11 region.

• File: emd_25685_additional_13.map
• Annotation: Half map 1 of focused refinement on gH and 13H11 region.

Additional map: Sharpened map of focused refinement on gL region....

• File: emd_25685_additional_2.map
• Annotation: Sharpened map of focused refinement on gL region. Used for composite map generation.

Additional map: Half map 2 of focused refinement on gL region.

• File: emd_25685_additional_3.map
• Annotation: Half map 2 of focused refinement on gL region.

Additional map: Sharpened map of focused refinement on 2C12, 7I13,...

• File: emd_25685_additional_4.map
• Annotation: Sharpened map of focused refinement on 2C12, 7I13, UL region. Used for composite map generation.

Additional map: Non-sharpened map of focused refinement on gL region.

• File: emd_25685_additional_5.map
• Annotation: Non-sharpened map of focused refinement on gL region.

Additional map: Non-sharpened map of overall (non-focused) refinement.

• File: emd_25685_additional_6.map
• Annotation: Non-sharpened map of overall (non-focused) refinement.

Additional map: Half map 1 of focused refinement on gL region.

• File: emd_25685_additional_7.map
• Annotation: Half map 1 of focused refinement on gL region.

Additional map: Non-sharpened map of focused refinement on 2C12, 7I13, UL region.

• File: emd_25685_additional_8.map
• Annotation: Non-sharpened map of focused refinement on 2C12, 7I13, UL region.

Additional map: Half map 1 of focused refinement on 2C12, 7I13, UL region.

• File: emd_25685_additional_9.map
• Annotation: Half map 1 of focused refinement on 2C12, 7I13, UL region.

Sample components

Entire : Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A ...

• Entire: Name: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to fabs of human neutralizing antibodies 2C12, 7I13 and 13H11

Components

• Complex: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to fabs of human neutralizing antibodies 2C12, 7I13 and 13H11
  • Protein or peptide: Envelope glycoprotein H
  • Protein or peptide: Envelope glycoprotein L
  • Protein or peptide: Envelope protein UL128
  • Protein or peptide: Envelope glycoprotein UL130
  • Protein or peptide: Envelope protein UL131A
  • Protein or peptide: Fab 2C12 heavy chain
  • Protein or peptide: Fab 2C12 light chain
  • Protein or peptide: Fab 7I13 light chain
  • Protein or peptide: Fab 7I13 heavy chain
  • Protein or peptide: Fab 13H11 heavy chain
  • Protein or peptide: Fab 13H11 light chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

Supramolecule #1: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A ...

• Supramolecule: Name: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to fabs of human neutralizing antibodies 2C12, 7I13 and 13H11; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#11
• Molecular weight: Theoretical: 320 KDa

Macromolecule #1: Envelope glycoprotein H

• Macromolecule: Name: Envelope glycoprotein H / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 87.311273 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MRPGLPSYLI ILAVCLFSHL LSSRYGAEAV SEPLDKAFHL LLNTYGRPIR FLRENTTQCT YNSSLRNSTV VRENAISFNF FQSYNQYYV FHMPRCLFAG PLAEQFLNQV DLTETLERYQ QRLNTYALVS KDLASYRSFS QQLKAQDSLG EQPTTVPPPI D LSIPHVWM PPQTTPHGWT ESHTTSGLHR PHFNQTCILF DGHDLLFSTV TPCLHQGFYL IDELRYVKIT LTEDFFVVTV SI DDDTPML LIFGHLPRVL FKAPYQRDNF ILRQTEKHEL LVLVKKDQLN RHSYLKDPDF LDAALDFNYL DLSALLRNSF HRY AVDVLK SGRCQMLDRR TVEMAFAYAL ALFAAARQEE AGAQVSVPRA LDRQAALLQI QEFMITCLSQ TPPRTTLLLY PTAV DLAKR ALWTPNQITD ITSLVRLVYI LSKQNQQHLI PQWALRQIAD FALKLHKTHL ASFLSAFARQ ELYLMGSLVH SMLVH TTER REIFIVETGL CSLAELSHFT QLLAHPHHEY LSDLYTPCSS SGRRDHSLER LTRLFPDATV PATVPAALSI LSTMQP STL ETFPDLFCLP LGESFSALTV SEHVSYIVTN QYLIKGISYP VSTTVVGQSL IITQTDSQTK CELTRNMHTT HSITVAL NI SLENCAFCQS ALLEYDDTQG VINIMYMHDS DDVLFALDPY NEVVVSSPRT HYLMLLKNGT VLEVTDVVVD ATDGTKLG P EQKLISEEDL NSAVDGSGLN DIFEAQKIEW HENLYFQGHH HHHHHH

Macromolecule #2: Envelope glycoprotein L

• Macromolecule: Name: Envelope glycoprotein L / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 30.846492 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MCRRPDCGFS FSPGPVILLW CCLLLPIVSS AAVSVAPTAA EKVPAECPEL TRRCLLGEVF EGDKYESWLR PLVNVTGRDG PLSQLIRYR PVTPEAANSV LLDEAFLDTL ALLYNNPDQL RALLTLLSSD TAPRWMTVMR GYSECGDGSP AVYTCVDDLC R GYDLTRLS YGRSIFTEHV LGFELVPPSL FNVVVAIRNE ATRTNRAVRL PVSTAAAPEG ITLFYGLYNA VKEFCLRHQL DP PLLRHLD KYYAGLPPEL KQTRVNLPAH SRYGPQAVDA R

Macromolecule #3: Envelope protein UL128

• Macromolecule: Name: Envelope protein UL128 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 19.746023 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MSPKNLTPFL TALWLLLGHS RVPRVRAEEC CEFINVNHPP ERCYDFKMCN RFTVALRCPD GEVCYSPEKT AEIRGIVTTM THSLTRQVV HNKLTSCNYN PLYLEADGRI RCGKVNDKAQ YLLGAAGSVP YRWINLEYDK ITRIVGLDQY LESVKKHKRL D VCRAKMGY MLQ

Macromolecule #4: Envelope glycoprotein UL130

• Macromolecule: Name: Envelope glycoprotein UL130 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 28.866811 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MLRLLLRHHF HCLLLCAVWA TPCLASPWFT LTANQNPSPP WSKLTYPKPH DAATFYCPFL YPSPPRSPSQ FSGFQRVSTG PECRNETLY LLYNREGQTL VERSSTWVKK VIWYLSGRNQ TILQRMPRTA SKPSDGNVQI SVEDAKIFGA HMVPKQTKLL R FVVNDGTR YQMCVMKLES WAHVFRDYSV SFQVRLTFTE ANNQTYTFCT HPNLIVGSEN LYFQGSAWSH PQFEKGGGSG GG SGGGSAW SHPQFEK

Macromolecule #5: Envelope protein UL131A

• Macromolecule: Name: Envelope protein UL131A / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 15.011043 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MRLCRVWLSV CLCAVVLGQC QRETAEKNDY YRVPHYWDAC SRALPDQTRY KYVEQLVDLT LNYHYDASHG LDNFDVLKRI NVTEVSLLI SDFRRQNRRG GTNKRTTFNA AGSLAPHARS LEFSVRLFAN

Macromolecule #6: Fab 2C12 heavy chain

• Macromolecule: Name: Fab 2C12 heavy chain / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 27.00177 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAQITLRES GPTLVKPTQT LTLTCTFSGF SLNTNGVGVG WIRQPPGKAL EWLALIYWNG NEGYSPSLK SRLTITKDTS KNQVVLTMTN MDPVDTATYY CVHWPQGLTT VTRLAFDIWG QGTMVTVSSA STKGPSVFPL A PSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NT KVDKKVE PKSCD

Macromolecule #7: Fab 2C12 light chain

• Macromolecule: Name: Fab 2C12 light chain / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.594654 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAQSALTQP RSVSGSPGQS VTISCTGTTS DVGRYNFVSW YQQHPGKAPK LLMYDVSQRP SGVPSRFSG SKSGNTASLT ISGLQAEDEA VFYCCSYAGG NFFSYVFGTG TKVTVLGQPK AAPSVTLFPP SSEELQANKA T LVCLISDF YPGAVTVAWK ADSSPVKAGV ETTTPSKQSN NKYAASSYLS LTPEQWKSHR SYSCQVTHEG STVEKTVAPT EC

Macromolecule #8: Fab 7I13 light chain

• Macromolecule: Name: Fab 7I13 light chain / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.8449 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAEIVLTQS PGTLSLSPGE RATLSCRASQ SVSSDFLAWY QQKPGQAPRL LIYGASSRAT GIPDRFSGS GSGTDFTLTI SRLEPEDFAV YYCQQYAASP PFGQGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL N NFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Macromolecule #9: Fab 7I13 heavy chain

• Macromolecule: Name: Fab 7I13 heavy chain / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 27.046506 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAQVQLVES GGGVVQPGRS LRLSCAASGF TFSNYGMHWV RQGPGKGLEW VAVISYDASS KYYTDSVQG RFTISRDNSK NTLFLQMNSL RGEDTAVYYC AKALRYLDWF LSDPFDYWGQ GTLVTVSSAS TKGPSVFPLA P SSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TK VDKKVEP KSCD

Macromolecule #10: Fab 13H11 heavy chain

• Macromolecule: Name: Fab 13H11 heavy chain / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 26.600086 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAQVQLVQS GAEVKKPGAS VKVSCKASGY TFTNYYIHWV RQAPGQGLEW MGIIHPSSGG TSYAQKFQG RVTMTRDTST STVSMDLSSL RSEDTAVYYC GRAFRILGLS DVFVNDWGQG TVVTVSSAST KGPSVFPLAP S SKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KV DKKVEPK SCD

Macromolecule #11: Fab 13H11 light chain

• Macromolecule: Name: Fab 13H11 light chain / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.78002 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYADIQMTQS PSSLSASVGD RVTITCRASQ GINNYLAWYQ QKPGKVPKLL IYAASTLQSG VPSRFSGSG SGTAFTLTIL SLQPEDVATY YCQKYNSAPF TFGPGTKVDI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL N NFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Macromolecule #12: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 12 / Number of copies: 6 / Formula: NAG
• Molecular weight: Theoretical: 221.208 Da

Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1.77 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Formula	Name
300.0 mM	NaClSodium chloride	sodium chloride
25.0 mM	HEPES	HEPES

• Grid: Model: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 ; Details: The grid was incubated with a thiol reactive self-assembling reaction mixture of 4mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol, (SPT-0011P6, SensoPath Technologies, Inc., Bozeman, MT)[23]. Grids were incubated with this self-assembled, monolayer (SAM) solution for 24 hours. Prior to grid freezing, grids were removed from the SAM solution and rinsed with EtOH.
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP / Details: blot for 3.5s before plunging.
• Details: The sample was monodisperse.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 18326 / Average exposure time: 10.0 sec. / Average electron dose: 58.0 e/Ų; Details: Images were collected in movie-mode at 5 frames/second.
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 5128264 / Details: template-matching particle picking
• CTF correction: Software - Name: CTFFIND (ver. 4.1.13)
• Startup model: Type of model: OTHER; Details: ab-initio 3D model generation from select particles within cisTEM
• Initial angle assignment: Type: OTHER / Software - Name: cisTEM (ver. 1.02) / Details: Auto-refine in cisTEM
• Final 3D classification: Number classes: 100 / Software - Name: RELION (ver. 3.1)
• Final angle assignment: Type: OTHER / Software - Name: cisTEM (ver. 1.02); Details: Manual refine in cisTEM. Map was divided in 3 sections to refine map with focused refinements.
• Final reconstruction: Number classes used: 39 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.02); Details: A composite map was generated from the three individual focused 3D maps using phenix combine_focused_maps; Number images used: 4792984
• Details: Movie frames were corrected for motion and aligned. Images with a CTF fit resolution of 8.0 A or better were selected for particle picking.

Atomic model buiding 1

Initial model

PDB ID:

5vob

Output model

PDB-7t4q:
CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with neutralizing fabs 2C12, 7I13 and 13H11