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- EMDB-31328: The cryo-EM map of the MR3-Spike complex -

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Basic information

Entry
Database: EMDB / ID: EMD-31328
TitleThe cryo-EM map of the MR3-Spike complex
Map dataThe cryo-EM map of MR3-Spike complex.Two 'up' RBDs (orange) and one 'down' RBD (red) are appropriately labled.
Sample
  • Complex: The cryo-EM map of the MR3-Spike complex.
    • Complex: Spike protein
      • Other: SARS-CoV-2 Spike protein S1
    • Complex: MR3
      • Other: MR3 nanobody
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 / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / 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 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.25 Å
AuthorsHan W / Liu CX / Wang YF / Cong Y
Funding support China, 1 items
OrganizationGrant numberCountry
Chinese Academy of Sciences China
CitationJournal: Nat Commun / Year: 2021
Title: A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.
Authors: Tingting Li / Hongmin Cai / Hebang Yao / Bingjie Zhou / Ning Zhang / Martje Fentener van Vlissingen / Thijs Kuiken / Wenyu Han / Corine H GeurtsvanKessel / Yuhuan Gong / Yapei Zhao / Quan ...Authors: Tingting Li / Hongmin Cai / Hebang Yao / Bingjie Zhou / Ning Zhang / Martje Fentener van Vlissingen / Thijs Kuiken / Wenyu Han / Corine H GeurtsvanKessel / Yuhuan Gong / Yapei Zhao / Quan Shen / Wenming Qin / Xiao-Xu Tian / Chao Peng / Yanling Lai / Yanxing Wang / Cedric A J Hutter / Shu-Ming Kuo / Juan Bao / Caixuan Liu / Yifan Wang / Audrey S Richard / Hervé Raoul / Jiaming Lan / Markus A Seeger / Yao Cong / Barry Rockx / Gary Wong / Yuhai Bi / Dimitri Lavillette / Dianfan Li /
Abstract: SARS-CoV-2, the causative agent of COVID-19, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein. Neutralizing antibodies that block RBD-ACE2 interaction are candidates ...SARS-CoV-2, the causative agent of COVID-19, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (K = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC = 0.42 μg mL). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.
History
DepositionMay 21, 2021-
Header (metadata) releaseJul 21, 2021-
Map releaseJul 21, 2021-
UpdateNov 30, 2022-
Current statusNov 30, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31328.png

Downloads & links

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Map

FileDownload / File: emd_31328.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe cryo-EM map of MR3-Spike complex.Two 'up' RBDs (orange) and one 'down' RBD (red) are appropriately labled.
Voxel sizeX=Y=Z: 1.02 Å
Density
Contour LevelBy AUTHOR: 0.0027 / Movie #1: 0.005
Minimum - Maximum-0.0112968385 - 0.024727488
Average (Standard dev.)-5.2456636e-07 (±0.0011167357)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 408.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.021.021.02
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z408.000408.000408.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0110.025-0.000

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Supplemental data

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Sample components

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Entire : The cryo-EM map of the MR3-Spike complex.

EntireName: The cryo-EM map of the MR3-Spike complex.
Components
  • Complex: The cryo-EM map of the MR3-Spike complex.
    • Complex: Spike protein
      • Other: SARS-CoV-2 Spike protein S1
    • Complex: MR3
      • Other: MR3 nanobody

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Supramolecule #1: The cryo-EM map of the MR3-Spike complex.

SupramoleculeName: The cryo-EM map of the MR3-Spike complex. / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Spike protein

SupramoleculeName: Spike protein / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Supramolecule #3: MR3

SupramoleculeName: MR3 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: SARS-CoV-2 Spike protein S1

MacromoleculeName: SARS-CoV-2 Spike protein S1 / type: other / ID: 1 / Classification: other
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 LYQDVNCTEV 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 VLLSTFLENL YFQGDYKDDD DKHHHHHHHH H

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Macromolecule #2: MR3 nanobody

MacromoleculeName: MR3 nanobody / type: other / ID: 2 / Classification: other
SequenceString:
GSSSQVQLVE SGGGLVQAGG SLRLSCAASG FPVNAHFMYW YRQAPGKERE WVAAIYSYGR TLYADSVKGR FTISRDNAKN TVYLQMNSLK PEDTAVYYCN VKDYGAASWE YDYWGQGTQV TVSAGRAGEQ KLISEEDLNS AVDHHHHHH

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 49.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 34243

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