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- EMDB-26062: Camel nanobodies 7A3 and 8A2 broadly neutralize SARS-CoV-2 variants -

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Basic information

Entry
Database: EMDB / ID: EMD-26062
TitleCamel nanobodies 7A3 and 8A2 broadly neutralize SARS-CoV-2 variants
Map dataConsensus map refined with C1 symmetry and post-processed using DeepEMhancer
Sample
  • Complex: Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan strain) and camel nanobodies 7A3 and 8A2.
    • Complex: SARS-CoV-2 Spike glycoprotein
      • Protein or peptide: Spike glycoproteinSpike protein
    • Complex: Camel nanobody 8A2
      • Protein or peptide: Nanobody 8A2
    • Complex: Camel nanobody 7A3
      • Protein or peptide: Nanobody 7A3
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / plasma membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Camelus dromedarius (Arabian camel)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsButay KJ / Zhu J / Dandey VP / Hong J / Kwon HJ / Chen CZ / Duan Z / Li D / Ren H / Liang T ...Butay KJ / Zhu J / Dandey VP / Hong J / Kwon HJ / Chen CZ / Duan Z / Li D / Ren H / Liang T / Martin N / Esposito D / Ortega-Rodriguez U / Xu M / Xie H / Ho M / Cachau R / Borgnia MJ
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIA ES 103341 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 011943 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC ES 10326 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIC BC 011891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)Z01 BC010891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC010891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)75N91019D00024 United States
CitationJournal: bioRxiv / Year: 2021
Title: Camel nanobodies broadly neutralize SARS-CoV-2 variants.
Authors: Jessica Hong / Hyung Joon Kwon / Raul Cachau / Catherine Z Chen / Kevin John Butay / Zhijian Duan / Dan Li / Hua Ren / Tianyuzhou Liang / Jianghai Zhu / Venkata P Dandey / Negin Martin / ...Authors: Jessica Hong / Hyung Joon Kwon / Raul Cachau / Catherine Z Chen / Kevin John Butay / Zhijian Duan / Dan Li / Hua Ren / Tianyuzhou Liang / Jianghai Zhu / Venkata P Dandey / Negin Martin / Dominic Esposito / Uriel Ortega-Rodriguez / Miao Xu / Mario J Borgnia / Hang Xie / Mitchell Ho /
Abstract: With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H nanobodies (7A3 and 8A2) from dromedary camels by phage display, ...With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants.
ONE-SENTENCE SUMMARY: Dromedary camel ( ) V H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.
History
DepositionJan 25, 2022-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateApr 20, 2022-
Current statusApr 20, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26062.png

Downloads & links

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Map

FileDownload / File: emd_26062.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus map refined with C1 symmetry and post-processed using DeepEMhancer
Voxel sizeX=Y=Z: 0.932 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.0022977015 - 1.6769795
Average (Standard dev.)0.0019239914 (±0.029808192)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 298.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Consensus map refined with C1 symmetry. Half map A.

Fileemd_26062_half_map_1.map
AnnotationConsensus map refined with C1 symmetry. Half map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus map refined with C1 symmetry. Half map B.

Fileemd_26062_half_map_2.map
AnnotationConsensus map refined with C1 symmetry. Half 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 : Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan str...

EntireName: Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan strain) and camel nanobodies 7A3 and 8A2.
Components
  • Complex: Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan strain) and camel nanobodies 7A3 and 8A2.
    • Complex: SARS-CoV-2 Spike glycoprotein
      • Protein or peptide: Spike glycoproteinSpike protein
    • Complex: Camel nanobody 8A2
      • Protein or peptide: Nanobody 8A2
    • Complex: Camel nanobody 7A3
      • Protein or peptide: Nanobody 7A3

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Supramolecule #1: Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan str...

SupramoleculeName: Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan strain) and camel nanobodies 7A3 and 8A2.
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 17 KDa

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Supramolecule #2: SARS-CoV-2 Spike glycoprotein

SupramoleculeName: SARS-CoV-2 Spike glycoprotein / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Expi293F

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Supramolecule #3: Camel nanobody 8A2

SupramoleculeName: Camel nanobody 8A2 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Camelus dromedarius (Arabian camel)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: HB2151

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Supramolecule #4: Camel nanobody 7A3

SupramoleculeName: Camel nanobody 7A3 / type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Camelus dromedarius (Arabian camel)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: HB2151

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Details: Hexa-Pro construct mutations / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 126.821453 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: CVNLTTRTQL PPAYTNSFTR GVYYPDKVFR SSVLHSTQDL FLPFFSNVTW FHAIHVSGTN GTKRFDNPVL PFNDGVYFAS TEKSNIIRG WIFGTTLDSK TQSLLIVNNA TNVVIKVCEF QFCNDPFLGV YYHKNNKSWM ESEFRVYSSA NNCTFEYVSQ P FLMDLEGK ...String:
CVNLTTRTQL PPAYTNSFTR GVYYPDKVFR SSVLHSTQDL FLPFFSNVTW FHAIHVSGTN GTKRFDNPVL PFNDGVYFAS TEKSNIIRG WIFGTTLDSK TQSLLIVNNA TNVVIKVCEF QFCNDPFLGV YYHKNNKSWM ESEFRVYSSA NNCTFEYVSQ P FLMDLEGK QGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLL ALHRSYLTPG DS SSGWTAG AAAYYVGYLQ PRTFLLKYNE NGTITDAVDC ALDPLSETKC TLKSFTVEKG IYQTSNFRVQ PTESIVRFPN ITN LCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQ TGKIA DYNYKLPDDF TGCVIAWNSN NLDSKVGGNY NYLYRLFRKS NLKPFERDIS TEIYQAGSTP CNGVEGFNCY FPLQS YGFQ PTNGVGYQPY RVVVLSFELL HAPATVCGPK KSTNLVKNKC VNFNFNGLTG TGVLTESNKK FLPFQQFGRD IADTTD AVR DPQTLEILDI TPCSFGGVSV ITPGTNTSNQ VAVLYQDVNC TEVPVAIHAD QLTPTWRVYS TGSNVFQTRA GCLIGAE HV NNSYECDIPI GAGICASYQT QTNSPGSASS VASQSIIAYT MSLGAENSVA YSNNSIAIPT NFTISVTTEI LPVSMTKT S VDCTMYICGD STECSNLLLQ YGSFCTQLNR ALTGIAVEQD KNTQEVFAQV KQIYKTPPIK DFGGFNFSQI LPDPSKPSK RSPIEDLLFN KVTLADAGFI KQYGDCLGDI AARDLICAQK FNGLTVLPPL LTDEMIAQYT SALLAGTITS GWTFGAGPAL QIPFPMQMA YRFNGIGVTQ NVLYENQKLI ANQFNSAIGK IQDSLSSTPS ALGKLQDVVN QNAQALNTLV KQLSSNFGAI S SVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASA NLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQ SAPHGVV FLHVTYVPAQ EKNFTTAPAI CHDGKAHFPR EGVFVSNGTH WFVTQRNFYE PQIITTDNTF VSGNCDVVIG IVN NTVYDP LQPELDSFKE ELDKYFKNH

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Macromolecule #2: Nanobody 8A2

MacromoleculeName: Nanobody 8A2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Camelus dromedarius (Arabian camel)
Molecular weightTheoretical: 13.993344 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AVQLVDSGGG SVQAGGSLRL SCAASGYTYS ICTMGWYRQA PGEGLEWVSG INADGSNTHY TDSVKGRFTI SRDNAKKTLY LQMNSLKPE DTAIYYCAAH GTYDKYAPCG GFAGTYTYWG QGTQVTVSSS GQA

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Macromolecule #3: Nanobody 7A3

MacromoleculeName: Nanobody 7A3 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Camelus dromedarius (Arabian camel)
Molecular weightTheoretical: 13.755188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG SVQPGGSLRL SCVVSGYTSS SRYMGWFRQV PGKGLEWVSG IKRDGTNTYY ADSVKGRFTI SQDNAKNTVY LQMNSLKPE DTAMYYCAAG SWYNQWGYSM DYWGKGTQVT VSSSGQ

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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

Concentration1.93 mg/mL
BufferpH: 7.5 / Details: PBS
GridModel: Homemade / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 30.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
DetailsComplexes of spike with 7A3 and/or 8A2 VHHs were prepared by mixing the components at a spike trimer to nanobody molar ratio of 1:6. The final concentration of spike trimer was 3 uM in PBS at pH 7 with the addition of 5 mM imidazole. Because the 8A2 stock was too diluted, complexes involving this nanobody were prepared at 0.5 uM spike trimer followed by a 6-fold concentration using a 10 kDa cutoff centrifugal filter (Amicon Ultra). All complexes were incubated on ice for at least 5 min prior to grid preparation.

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Electron microscopy #1

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 60.0 µm / Calibrated magnification: 53648 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Microscopy ID1
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Electron microscopy #1~

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 47348 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID1
Image recordingImage recording ID: 2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.000001 µm / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1539054
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 580888
Image recording ID2

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