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- EMDB-32081: Cryo-EM structure of the SARS-CoV-2 spike protein (2-up RBD) boun... -

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Entry
Database: EMDB / ID: EMD-32081
TitleCryo-EM structure of the SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobodies P17
Map data
Sample
  • Complex: SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobodies P17
    • Complex: SARS-CoV-2 spike protein (2-up RBD)
      • Protein or peptide: Spike glycoproteinSpike protein
    • Complex: neutralizing nanobodies P17
      • Protein or peptide: Neutralizing nanobody P17
Keywordsspike / nanobody / VHH / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsMaeda R / Fujita J / Konishi Y / Kazuma Y / Yamazaki H / Anzai I / Yamaguchi K / Kasai K / Nagata K / Yamaoka Y ...Maeda R / Fujita J / Konishi Y / Kazuma Y / Yamazaki H / Anzai I / Yamaguchi K / Kasai K / Nagata K / Yamaoka Y / Miyakawa K / Ryo A / Shirakawa K / Makino F / Matsuura Y / Inoue T / Imura A / Namba K / Takaori-Kondo A
Funding support Japan, 8 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP20fk0108268 Japan
Japan Agency for Medical Research and Development (AMED)JP20fk018517 Japan
Japan Agency for Medical Research and Development (AMED)JP20fk018413 Japan
Japan Society for the Promotion of Science (JSPS)JP20K22630 Japan
Japan Society for the Promotion of Science (JSPS)JP25000013 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101117 Japan
Japan Agency for Medical Research and Development (AMED)JP17pc0101020 Japan
Japan Science and TechnologyJPMJOP1861 Japan
CitationJournal: Commun Biol / Year: 2022
Title: A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron.
Authors: Ryota Maeda / Junso Fujita / Yoshinobu Konishi / Yasuhiro Kazuma / Hiroyuki Yamazaki / Itsuki Anzai / Tokiko Watanabe / Keishi Yamaguchi / Kazuki Kasai / Kayoko Nagata / Yutaro Yamaoka / Kei ...Authors: Ryota Maeda / Junso Fujita / Yoshinobu Konishi / Yasuhiro Kazuma / Hiroyuki Yamazaki / Itsuki Anzai / Tokiko Watanabe / Keishi Yamaguchi / Kazuki Kasai / Kayoko Nagata / Yutaro Yamaoka / Kei Miyakawa / Akihide Ryo / Kotaro Shirakawa / Kei Sato / Fumiaki Makino / Yoshiharu Matsuura / Tsuyoshi Inoue / Akihiro Imura / Keiichi Namba / Akifumi Takaori-Kondo /
Abstract: We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, ...We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.
History
DepositionOct 18, 2021-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32081.png

Downloads & links

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Map

FileDownload / File: emd_32081.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.874 Å
Density
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-1.5179745 - 2.4713376
Average (Standard dev.)-0.001566974 (±0.059003342)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 314.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32081_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_32081_half_map_1.map
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Half map: #2

Fileemd_32081_half_map_2.map
Projections & Slices
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Sample components

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Entire : SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobod...

EntireName: SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobodies P17
Components
  • Complex: SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobodies P17
    • Complex: SARS-CoV-2 spike protein (2-up RBD)
      • Protein or peptide: Spike glycoproteinSpike protein
    • Complex: neutralizing nanobodies P17
      • Protein or peptide: Neutralizing nanobody P17

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Supramolecule #1: SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobod...

SupramoleculeName: SARS-CoV-2 spike protein (2-up RBD) bound to neutralizing nanobodies P17
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Supramolecule #2: SARS-CoV-2 spike protein (2-up RBD)

SupramoleculeName: SARS-CoV-2 spike protein (2-up RBD) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Vicugna pacos (alpaca)

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Supramolecule #3: neutralizing nanobodies P17

SupramoleculeName: neutralizing nanobodies P17 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQGVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PGSASSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDPPEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGSH HHHHHHH

GENBANK: GENBANK: QHD43416.1

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Macromolecule #2: Neutralizing nanobody P17

MacromoleculeName: Neutralizing nanobody P17 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
QVQLQLQESG GGLVQAGGSL RLSCAASGRT SSVYNMAWFR QTPGKEREFV AAITGNGGTT LYADSVKGRL TISRGNAKNT VSLQMNVLKP DDTAVYYCAA GGWGKERNYA YWGQGTQVTV SSHHHHHH

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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

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormula
20.0 mMHEPES
150.0 mMNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: GRAPHENE
Details: The graphene grid was chemically oxidized and modified.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsMixed with 5 times molar excess of P86.

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2

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Image processing

Particle selectionNumber selected: 766652
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 48715
FSC plot (resolution estimation)

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