EMDB-25686: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in...

Basic information

Entry

Database: EMDB / ID: EMD-25686

• Title: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with THBD and neutralizing fabs MSL-109 and 13H11
• Map data: Composite map obtained by combining focused, sharpened maps. Map used for model building and refinements.

Sample

• Complex: Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to 1x THBD and 2x fabs of human neutralizing antibodies MSL-109 and 13H11
  • Protein or peptide: x 10 types
• Ligand: x 1 types

Function / homology

Function:

blood coagulation, common pathway / negative regulation of blood coagulation / apicolateral plasma membrane / serine-type endopeptidase complex / zymogen activation / vacuolar membrane / negative regulation of platelet activation / response to X-ray / negative regulation of fibrinolysis / Common Pathway of Fibrin Clot Formation / response to cAMP / host cell endosome membrane / female pregnancy / Cell surface interactions at the vascular wall / transmembrane signaling receptor activity / blood coagulation / signaling receptor activity / host cell Golgi apparatus / response to lipopolysaccharide / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / fusion of virus membrane with host plasma membrane / external side of plasma membrane / viral envelope / calcium ion binding / host cell plasma membrane / virion membrane / cell surface / proteolysis / extracellular space / membrane / plasma membrane

Domain/homology:

Thrombomodulin-like, EGF-like / Thrombomodulin like fifth domain, EGF-like / 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 / Complement Clr-like EGF domain / Complement Clr-like EGF-like / Calcium-binding EGF domain / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / Coagulation Factor Xa inhibitory site / C-type lectin-like/link domain superfamily / EGF-type aspartate/asparagine hydroxylation site / C-type lectin fold / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 2. / EGF-like domain

Component:

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

• Biological species: Homo sapiens (human) / Human betaherpesvirus 5
• Method: single particle reconstruction / cryo EM / Resolution: 3.3 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_25686.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Composite map obtained by combining focused, sharpened maps. Map used for model building and refinements.
• Voxel size: X=Y=Z: 1.0726 Å

Density

Contour Level	By AUTHOR: 0.15
Minimum - Maximum	-0.47399116 - 3.5848255
Average (Standard dev.)	0.0028422892 (±0.03619096)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 400 400 400 Spacing 400 400 400
Cell	A=B=C: 429.04 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Non-sharpened, full map of overall map (not focused).

• File: emd_25686_additional_1.map
• Annotation: Non-sharpened, full map of overall map (not focused).

Additional map: Half map 1 of focused gH-gL (A) region.

• File: emd_25686_additional_10.map
• Annotation: Half map 1 of focused gH-gL (A) region.

Additional map: Half map 2 of focused gH-gL (A) region.

• File: emd_25686_additional_11.map
• Annotation: Half map 2 of focused gH-gL (A) region.

Additional map: Density modified map of focused gH-gL (A) map...

• File: emd_25686_additional_12.map
• Annotation: Density modified map of focused gH-gL (A) map used for generation of composite map.

Additional map: Non-sharpened, full map of focused gH-gL (B) region.

• File: emd_25686_additional_13.map
• Annotation: Non-sharpened, full map of focused gH-gL (B) region.

Additional map: Half map 1 of focused gH-gL (B) region.

• File: emd_25686_additional_14.map
• Annotation: Half map 1 of focused gH-gL (B) region.

Additional map: Half map 2 of focused gH-gL (B) region.

• File: emd_25686_additional_15.map
• Annotation: Half map 2 of focused gH-gL (B) region.

Additional map: Density modified map of overall map (not focused)...

• File: emd_25686_additional_16.map
• Annotation: Density modified map of overall map (not focused) used for generation of composite map.

Additional map: Half map 1 of overall map (not focused).

• File: emd_25686_additional_2.map
• Annotation: Half map 1 of overall map (not focused).

Additional map: Density modified map of focused THBD-ULs map used...

• File: emd_25686_additional_3.map
• Annotation: Density modified map of focused THBD-ULs map used for generation of composite map.

Additional map: Half map 2 of overall map (not focused).

• File: emd_25686_additional_4.map
• Annotation: Half map 2 of overall map (not focused).

Additional map: Non-sharpened, full map of focused THBD-ULs region.

• File: emd_25686_additional_5.map
• Annotation: Non-sharpened, full map of focused THBD-ULs region.

Additional map: Half map 1 of focused THBD-ULs region.

• File: emd_25686_additional_6.map
• Annotation: Half map 1 of focused THBD-ULs region.

Additional map: Half map 2 of focused THBD-ULs region.

• File: emd_25686_additional_7.map
• Annotation: Half map 2 of focused THBD-ULs region.

Additional map: Density modified map of focused gH-gL (B) map...

• File: emd_25686_additional_8.map
• Annotation: Density modified map of focused gH-gL (B) map used for generation of composite map.

Additional map: Non-sharpened, full map of focused gH-gL (A) region.

• File: emd_25686_additional_9.map
• Annotation: Non-sharpened, full map of focused gH-gL (A) region.

Sample components

Entire : Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to...

• Entire: Name: Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to 1x THBD and 2x fabs of human neutralizing antibodies MSL-109 and 13H11

Components

• Complex: Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to 1x THBD and 2x fabs of human neutralizing antibodies MSL-109 and 13H11
  • Protein or peptide: Thrombomodulin
  • 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 13H11 heavy chain
  • Protein or peptide: Fab 13H11 light chain
  • Protein or peptide: Fab MSL-109 light chain
  • Protein or peptide: Fab MSL-109 heavy chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

Supramolecule #1: Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to...

• Supramolecule: Name: Complex of 2x HCMV Pentamer gH, gL, UL128, UL130, UL131A bound to 1x THBD and 2x fabs of human neutralizing antibodies MSL-109 and 13H11; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
• Molecular weight: Theoretical: 590 KDa

Macromolecule #1: Thrombomodulin

• Macromolecule: Name: Thrombomodulin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 55.128371 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MLGVLVLGAL ALAGLGFPAP AEPQPGGSQC VEHDCFALYP GPATFLNASQ ICDGLRGHLM TVRSSVAADV ISLLLNGDGG VGRRRLWIG LQLPPGCGDP KRLGPLRGFQ WVTGDNNTSY SRWARLDLNG APLCGPLCVA VSAAEATVPS EPIWEEQQCE V KADGFLCE FHFPATCRPL AVEPGAAAAA VSITYGTPFA ARGADFQALP VGSSAAVAPL GLQLMCTAPP GAVQGHWARE AP GAWDCSV ENGGCEHACN AIPGAPRCQC PAGAALQADG RSCTASATQS CNDLCEHFCV PNPDQPGSYS CMCETGYRLA ADQ HRCEDV DDCILEPSPC PQRCVNTQGG FECHCYPNYD LVDGECVEPV DPCFRANCEY QCQPLNQTSY LCVCAEGFAP IPHE PHRCQ MFCNQTACPA DCDPNTQASC ECPEGYILDD GFICTDIDEC ENGGFCSGVC HNLPGTFECI CGPDSALARH IGTDC DSGK VDGGDSGSGE PPPSPTPGST LTPPAVGLVH SGGNSDYKDD DDK

Macromolecule #2: Envelope glycoprotein H

• Macromolecule: Name: Envelope glycoprotein H / type: protein_or_peptide / ID: 2 / Number of copies: 2 / 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 #3: Envelope glycoprotein L

• Macromolecule: Name: Envelope glycoprotein L / type: protein_or_peptide / ID: 3 / Number of copies: 2 / 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 #4: Envelope protein UL128

• Macromolecule: Name: Envelope protein UL128 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / 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 #5: Envelope glycoprotein UL130

• Macromolecule: Name: Envelope glycoprotein UL130 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / 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 #6: Envelope protein UL131A

• Macromolecule: Name: Envelope protein UL131A / type: protein_or_peptide / ID: 6 / Number of copies: 2 / 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 #7: Fab 13H11 heavy chain

• Macromolecule: Name: Fab 13H11 heavy chain / type: protein_or_peptide / ID: 7 / Number of copies: 2 / 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 #8: Fab 13H11 light chain

• Macromolecule: Name: Fab 13H11 light chain / type: protein_or_peptide / ID: 8 / Number of copies: 2 / 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 #9: Fab MSL-109 light chain

• Macromolecule: Name: Fab MSL-109 light chain / type: protein_or_peptide / ID: 9 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 28.355809 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYADIVMTQS PLSLSVTPGE PASISCRSSQ SLLHTNGYNY LDWYVQKPGQ SPQLLIYLAS NRASGVPDR FSGSGSGTDF TLKISRVETE DVGVYYCMQA LQIPRTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS V VCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RG ECGLNDI FEAQKIEWHE

Macromolecule #10: Fab MSL-109 heavy chain

• Macromolecule: Name: Fab MSL-109 heavy chain / type: protein_or_peptide / ID: 10 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 27.547818 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAEEQVLES GGGLVKPGGS LRLSCAASGF TFSPYSVFWV RQAPGKGLEW VSSINSDSTY KYYADSVKG RFTISRDNAE NSIFLQMNSL RAEDTAVYYC ARDRSYYAFS SGSLSDYYYG LDVWGQGTLV TVSSASTKGP S VFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NH KPSNTKV DKKVEPKSCD

Macromolecule #11: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 11 / Number of copies: 14 / 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: 0.6 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.

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 / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10926 / Average electron dose: 64.0 e/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 10680163
• CTF correction: Software - Name: CTFFIND (ver. 4.1.13)
• Startup model: Type of model: OTHER / Details: ab-initio using C2 symmetry in cisTEM
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
• Final 3D classification: Number classes: 27 / Software - Name: RELION (ver. 3.1)
• Final angle assignment: Type: OTHER; Details: Manual refine in cisTEM. Map was divided in 3 sections to refine map with focused refinements.
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 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.; Number images used: 504492
• Details: Movie frames were corrected for motion and aligned. Images with a CTF fit resolution of 5.0 A or better were selected for particle picking.