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- EMDB-11980: SARS-CoV-spike RBD bound to two neutralising nanobodies. -

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Basic information

Entry
Database: EMDB / ID: EMD-11980
TitleSARS-CoV-spike RBD bound to two neutralising nanobodies.
Map dataResolve Cryo-EM in the PHENIX suite used on the half-maps from localised reconstruction.
Sample
  • Complex: Structure of SARS-CoV-2 spike RBD in complex with SARS-CoV-2 neutralizing biparatopic nanobody VE
    • Complex: Spike glycoproteinSpike protein
      • Protein or peptide: Spike protein S1
    • Complex: biparatopic nanobody VE
      • Protein or peptide: SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from Lama glama,SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from Lama glama
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / Vicugna pacos (alpaca) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.01 Å
AuthorsHallberg BM / Das H
CitationJournal: Science / Year: 2021
Title: Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.
Authors: Paul-Albert Koenig / Hrishikesh Das / Hejun Liu / Beate M Kümmerer / Florian N Gohr / Lea-Marie Jenster / Lisa D J Schiffelers / Yonas M Tesfamariam / Miki Uchima / Jennifer D Wuerth / Karl ...Authors: Paul-Albert Koenig / Hrishikesh Das / Hejun Liu / Beate M Kümmerer / Florian N Gohr / Lea-Marie Jenster / Lisa D J Schiffelers / Yonas M Tesfamariam / Miki Uchima / Jennifer D Wuerth / Karl Gatterdam / Natalia Ruetalo / Maria H Christensen / Caroline I Fandrey / Sabine Normann / Jan M P Tödtmann / Steffen Pritzl / Leo Hanke / Jannik Boos / Meng Yuan / Xueyong Zhu / Jonathan L Schmid-Burgk / Hiroki Kato / Michael Schindler / Ian A Wilson / Matthias Geyer / Kerstin U Ludwig / B Martin Hällberg / Nicholas C Wu / Florian I Schmidt /
Abstract: The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost ...The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.
History
DepositionNov 23, 2020-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateDec 21, 2022-
Current statusDec 21, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.472
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.472
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7b17
  • Surface level: 0.472
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7b17
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11980.png

Downloads & links

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Map

FileDownload / File: emd_11980.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationResolve Cryo-EM in the PHENIX suite used on the half-maps from localised reconstruction.
Voxel sizeX=Y=Z: 1.02 Å
Density
Contour LevelBy AUTHOR: 0.472 / Movie #1: 0.472
Minimum - Maximum-3.3222024 - 13.063079
Average (Standard dev.)-3.9585986e-12 (±0.30428848)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin275255264
Dimensions629071
Spacing716290
CellA: 72.42 Å / B: 63.239998 Å / C: 91.799995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.021.021.02
M x/y/z716290
origin x/y/z0.0000.0000.000
length x/y/z72.42063.24091.800
α/β/γ90.00090.00090.000
start NX/NY/NZ264275255
NX/NY/NZ716290
MAP C/R/S321
start NC/NR/NS255275264
NC/NR/NS906271
D min/max/mean-3.32213.063-0.000

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Supplemental data

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Half map: Half map 1 from the localised reconstruction.

Fileemd_11980_half_map_1.map
AnnotationHalf map 1 from the localised reconstruction.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from the localised reconstruction.

Fileemd_11980_half_map_2.map
AnnotationHalf map 1 from the localised reconstruction.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of SARS-CoV-2 spike RBD in complex with SARS-CoV-2 neut...

EntireName: Structure of SARS-CoV-2 spike RBD in complex with SARS-CoV-2 neutralizing biparatopic nanobody VE
Components
  • Complex: Structure of SARS-CoV-2 spike RBD in complex with SARS-CoV-2 neutralizing biparatopic nanobody VE
    • Complex: Spike glycoproteinSpike protein
      • Protein or peptide: Spike protein S1
    • Complex: biparatopic nanobody VE
      • Protein or peptide: SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from Lama glama,SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from Lama glama
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Structure of SARS-CoV-2 spike RBD in complex with SARS-CoV-2 neut...

SupramoleculeName: Structure of SARS-CoV-2 spike RBD in complex with SARS-CoV-2 neutralizing biparatopic nanobody VE
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Density from localised reconstruction from full spike - VE data.
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: Spike glycoprotein

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

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Supramolecule #3: biparatopic nanobody VE

SupramoleculeName: biparatopic nanobody VE / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Vicugna pacos (alpaca)

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Macromolecule #1: Spike protein S1

MacromoleculeName: Spike protein S1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 21.90157 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NLCPFGEVFN ATRFASVYAW NRKRISNCVA DYSVLYNSAS FSTFKCYGVS PTKLNDLCFT NVYADSFVIR GDEVRQIAPG QTGKIADYN YKLPDDFTGC VIAWNSNNLD SKVGGNYNYL YRLFRKSNLK PFERDISTEI YQAGSTPCNG VEGFNCYFPL Q SYGFQPTN ...String:
NLCPFGEVFN ATRFASVYAW NRKRISNCVA DYSVLYNSAS FSTFKCYGVS PTKLNDLCFT NVYADSFVIR GDEVRQIAPG QTGKIADYN YKLPDDFTGC VIAWNSNNLD SKVGGNYNYL YRLFRKSNLK PFERDISTEI YQAGSTPCNG VEGFNCYFPL Q SYGFQPTN GVGYQPYRVV VLSFELLHAP ATVCGPK

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Macromolecule #2: SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from L...

MacromoleculeName: SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from Lama glama,SARS-CoV-2 neutralizing biparatopic nanobody VE,nanobody E from Lama glama
type: protein_or_peptide / ID: 2
Details: Nanobody V from Vicugna pacos and nanobody E from Lama glama connected with a 15 residue linker,Nanobody V from Vicugna pacos and nanobody E from Lama glama connected with a 15 residue ...Details: Nanobody V from Vicugna pacos and nanobody E from Lama glama connected with a 15 residue linker,Nanobody V from Vicugna pacos and nanobody E from Lama glama connected with a 15 residue linker, 1-119 nanobody from Alpacka, 120-136: linker, 137-263: nanobody from Llama, 264-283: cloning and purification tag,Nanobody V from Vicugna pacos and nanobody E from Lama glama connected with a 15 residue linker,Nanobody V from Vicugna pacos and nanobody E from Lama glama connected with a 15 residue linker, 1-119 nanobody from Alpacka, 120-136: linker, 137-263: nanobody from Llama, 264-283: cloning and purification tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 30.102807 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLVETGGG LVQPGGSLRL SCAASGFTFS SYAMGWARQV PGKGLEWVSY IYSDGSTEYQ DSVKGRFTIS RDNAKSTVYL QMNSLKPED TAVYYCATEG SLGGWGRDFG SWGQGTQVTV SSGGGGSGGG GSGGGGSQVQ LVETGGGFVQ PGGSLRLSCA A SGVTLDYY ...String:
QVQLVETGGG LVQPGGSLRL SCAASGFTFS SYAMGWARQV PGKGLEWVSY IYSDGSTEYQ DSVKGRFTIS RDNAKSTVYL QMNSLKPED TAVYYCATEG SLGGWGRDFG SWGQGTQVTV SSGGGGSGGG GSGGGGSQVQ LVETGGGFVQ PGGSLRLSCA A SGVTLDYY AIGWFRQAPG KEREGVSCIG SSDGRTYYSD SVKGRFTISR DNAKNTVYLQ MNSLKPEDTA VYYCALTVGT YY SGNYHYT CSDDMDYWGK GTQVTVSSGG YPYDVPDYAG HHHHHH

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 1.5 sec. / Average electron dose: 48.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.15)
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 92938
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7b17:
SARS-CoV-spike RBD bound to two neutralising nanobodies.

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