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- EMDB-11068: Cryo-EM Structure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-11068
TitleCryo-EM Structure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex
Map dataStructure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex
Sample
  • Complex: Spike glycoprotein in complex with H11-D4 nanobodiesSpike protein
    • Complex: Spike glycoproteinSpike protein
      • Protein or peptide: Spike glycoproteinSpike protein
    • Complex: NanobodySingle-domain antibody
      • Protein or peptide: NanobodySingle-domain antibody
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / viral life cycle / receptor-mediated endocytosis of virus by host cell / membrane fusion / positive regulation of viral entry into host cell ...Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / viral life cycle / receptor-mediated endocytosis of virus by host cell / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host endosome membrane / viral envelope / viral entry into host cell / endoplasmic reticulum lumen / host cell plasma membrane / virion membrane / integral component of membrane / membrane / identical protein binding
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. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 2 ...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. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Coronavirus spike glycoprotein S2 / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRuza RR / Duyvesteyn HME / Shah P / Carrique L / Ren J / Malinauskas T / Zhou D / Stuart DI / Naismith JH
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research CouncilEP/S025243/1 United Kingdom
Cancer Research UKC20724/A14414 United Kingdom
Wellcome Trust100209/Z/12/Z United Kingdom
Wellcome Trust101122/Z/13/Z United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.
Authors: Jiandong Huo / Audrey Le Bas / Reinis R Ruza / Helen M E Duyvesteyn / Halina Mikolajek / Tomas Malinauskas / Tiong Kit Tan / Pramila Rijal / Maud Dumoux / Philip N Ward / Jingshan Ren / ...Authors: Jiandong Huo / Audrey Le Bas / Reinis R Ruza / Helen M E Duyvesteyn / Halina Mikolajek / Tomas Malinauskas / Tiong Kit Tan / Pramila Rijal / Maud Dumoux / Philip N Ward / Jingshan Ren / Daming Zhou / Peter J Harrison / Miriam Weckener / Daniel K Clare / Vinod K Vogirala / Julika Radecke / Lucile Moynié / Yuguang Zhao / Javier Gilbert-Jaramillo / Michael L Knight / Julia A Tree / Karen R Buttigieg / Naomi Coombes / Michael J Elmore / Miles W Carroll / Loic Carrique / Pranav N M Shah / William James / Alain R Townsend / David I Stuart / Raymond J Owens / James H Naismith /
Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the ...The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
History
DepositionMay 22, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z43
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11068.map.gz / Format: CCP4 / Size: 561.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 528 pix.
= 438.24 Å
0.83 Å/pix.
x 528 pix.
= 438.24 Å
0.83 Å/pix.
x 528 pix.
= 438.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.10291079 - 0.17046817
Average (Standard dev.)0.0000813213 (±0.002073685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions528528528
Spacing528528528
CellA=B=C: 438.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z528528528
origin x/y/z0.0000.0000.000
length x/y/z438.240438.240438.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS528528528
D min/max/mean-0.1030.1700.000

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Supplemental data

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Sample components

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Entire : Spike glycoprotein in complex with H11-D4 nanobodies

EntireName: Spike glycoprotein in complex with H11-D4 nanobodiesSpike protein
Components
  • Complex: Spike glycoprotein in complex with H11-D4 nanobodiesSpike protein
    • Complex: Spike glycoproteinSpike protein
      • Protein or peptide: Spike glycoproteinSpike protein
    • Complex: NanobodySingle-domain antibody
      • Protein or peptide: NanobodySingle-domain antibody
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Spike glycoprotein in complex with H11-D4 nanobodies

SupramoleculeName: Spike glycoprotein in complex with H11-D4 nanobodies / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 480 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
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI- / Recombinant plasmid: pHR-CMV-TetO2

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Supramolecule #3: Nanobody

SupramoleculeName: Nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 142.399375 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV Y SSANNCTF ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV Y SSANNCTF EYVSQPFLMD LEGKQGNFKN LREFVFKNID GYFKIYSKHT PINLVRDLPQ GFSALEPLVD LPIGINITRF QT LLALHRS YLTPGDSSSG WTAGAAAYYV GYLQPRTFLL KYNENGTITD AVDCALDPLS ETKCTLKSFT VEKGIYQTSN FRV QPTESI VRFPNITNLC PFGEVFNATR FASVYAWNRK RISNCVADYS VLYNSASFST FKCYGVSPTK LNDLCFTNVY ADSF VIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPC NGVE GFNCYFPLQS YGFQPTNGVG YQPYRVVVLS FELLHAPATV CGPKKSTNLV KNKCVNFNFN GLTGTGVLTE SNKKFL PFQ QFGRDIADTT DAVRDPQTLE ILDITPCSFG GVSVITPGTN TSNQVAVLYQ DVNCTEVPVA IHADQLTPTW RVYSTGS NV FQTRAGCLIG AEHVNNSYEC DIPIGAGICA SYQTQTNSPG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTI S VTTEILPVSM TKTSVDCTMY ICGDSTECSN LLLQYGSFCT QLNRALTGIA VEQDKNTQEV FAQVKQIYKT PPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFG AGAALQIPFA MQMAYRFNGI GVTQNVLYEN QKLIANQFNS AIGKIQDSLS STASALGKLQ DVVNQNAQAL N TLVKQLSS NFGAISSVLN DILSRLDPPE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RV DFCGKGY HLMSFPQSAP HGVVFLHVTY VPAQEKNFTT APAICHDGKA HFPREGVFVS NGTHWFVTQR NFYEPQIITT DNT FVSGNC DVVIGIVNNT VYDPLQPELD SFKEELDKYF KNHTSPDVDL GDISGINASV VNIQKEIDRL NEVAKNLNES LIDL QELGK YEQGSGYIPE APRDGQAYVR KDGEWVLLST FLGRSLEVLF QGPGHHHHHH HHGSAWSHPQ FEKGGGSGGG SGGSA WSHP QFEK

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Macromolecule #2: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 16.679453 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LMQAGGSLRL SCAVSGRTFS TAAMGWFRQA PGKEREFVAA IRWSGGSAYY ADSVKGRFTI SRDKAKNTVY LQMNSLKYE DTAVYYCART ENVRSLLSDY ATWPYDYWGQ GTQVTVSSGP GGQHHHHHHG AEQKLISEED LS

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 26 / 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

Concentration0.25 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
200.0 mMNaClSodium chlorideSodium Chloride
0.02 %NaN3Sodium Azide
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.65 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 6 seconds with a blotting force of -1.

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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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 42.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 305513

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6z43:
Cryo-EM Structure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex

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