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- PDB-7oan: Nanobody C5 bound to Spike -

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Basic information

Entry
Database: PDB / ID: 7oan
TitleNanobody C5 bound to Spike
Components
  • Spike glycoprotein
  • immunoglobulin mu heavy chain
KeywordsANTIVIRAL PROTEIN / Spike / nanobody / high affinity
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 / entry receptor-mediated virion attachment to host cell ...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 / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / membrane fusion / Attachment and Entry / 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 / identical protein binding / membrane / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsNaismith, J.H. / Weckener, M.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust100209/Z/12/Z) United Kingdom
Engineering and Physical Sciences Research CouncilEP/S025243/1 United Kingdom
Citation
Journal: Nat Commun / Year: 2021
Title: A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19.
Authors: Jiandong Huo / Halina Mikolajek / Audrey Le Bas / Jordan J Clark / Parul Sharma / Anja Kipar / Joshua Dormon / Chelsea Norman / Miriam Weckener / Daniel K Clare / Peter J Harrison / Julia A ...Authors: Jiandong Huo / Halina Mikolajek / Audrey Le Bas / Jordan J Clark / Parul Sharma / Anja Kipar / Joshua Dormon / Chelsea Norman / Miriam Weckener / Daniel K Clare / Peter J Harrison / Julia A Tree / Karen R Buttigieg / Francisco J Salguero / Robert Watson / Daniel Knott / Oliver Carnell / Didier Ngabo / Michael J Elmore / Susan Fotheringham / Adam Harding / Lucile Moynié / Philip N Ward / Maud Dumoux / Tessa Prince / Yper Hall / Julian A Hiscox / Andrew Owen / William James / Miles W Carroll / James P Stewart / James H Naismith / Raymond J Owens /
Abstract: SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain ...SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.
#1: Journal: Res Sq / Year: 2021
Title: A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19
Authors: Huo, J. / Mikolajek, H. / Le Bas, A. / Clark, J. / Sharma, P. / Kipar, A. / Dormon, J. / Norman, C. / Weckener, M. / Clare, D. / Harrison, P. / Tree, J. / Buttigieg, K. / Salguero, F. / ...Authors: Huo, J. / Mikolajek, H. / Le Bas, A. / Clark, J. / Sharma, P. / Kipar, A. / Dormon, J. / Norman, C. / Weckener, M. / Clare, D. / Harrison, P. / Tree, J. / Buttigieg, K. / Salguero, F. / Watson, R. / Knott, D. / Carnell, O. / Ngabo, D. / Elmore, M. / Fotheringham, S. / Harding, A. / Ward, P. / Moynie, L. / Dumoux, M. / Hall, Y. / Hiscox, J. / Owen, A. / James, W. / Carroll, M. / Stewart, J. / Naismith, J. / Owens, R.
History
DepositionApr 19, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
F: immunoglobulin mu heavy chain
G: immunoglobulin mu heavy chain
H: immunoglobulin mu heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)475,68657
Polymers461,3566
Non-polymers14,33051
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47330 Å2
ΔGint31 kcal/mol
Surface area152070 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C
14F
24G
15F
25H
16G
26H

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A14 - 1147
2010B14 - 1147
1020A14 - 1147
2020C14 - 1147
1030B14 - 1147
2030C14 - 1147
1040F1 - 121
2040G1 - 121
1050F1 - 121
2050H1 - 121
1060G1 - 121
2060H1 - 121

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 139877.906 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Antibody immunoglobulin mu heavy chain


Mass: 13907.522 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Gene: LOC102538064 / Production host: Escherichia coli (E. coli)
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 15
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 36 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: nanobody complex / Type: COMPLEX / Details: proteins produced incubated and run on grids / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Buffer solutionpH: 7 / Details: Standard buffer
Buffer componentConc.: 100 mM / Name: Tris
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample well dispersed
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
Image processingDetails: high pass filter
CTF correctionType: NONE
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 227898 / Symmetry type: POINT
RefinementResolution: 3→198.22 Å / Cor.coef. Fo:Fc: 0.909 / SU B: 15.017 / SU ML: 0.268 / ESU R: 0.392
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34545 --
obs0.34545 309862 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 119.93 Å2
Baniso -1Baniso -2Baniso -3
1-0.2 Å2-0.01 Å2-0.02 Å2
2--0.19 Å2-0.01 Å2
3----0.39 Å2
Refinement stepCycle: 1 / Total: 28203
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01328926
ELECTRON MICROSCOPYr_bond_other_d00.01726532
ELECTRON MICROSCOPYr_angle_refined_deg1.1751.69439480
ELECTRON MICROSCOPYr_angle_other_deg1.4611.61461158
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.78953483
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.69723.2551401
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.723154410
ELECTRON MICROSCOPYr_dihedral_angle_4_deg14.50715126
ELECTRON MICROSCOPYr_chiral_restr0.0580.24032
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0232757
ELECTRON MICROSCOPYr_gen_planes_other0.0020.026801
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.69612.81814013
ELECTRON MICROSCOPYr_mcbond_other2.69612.81714012
ELECTRON MICROSCOPYr_mcangle_it5.07219.20117469
ELECTRON MICROSCOPYr_mcangle_other5.07219.20317470
ELECTRON MICROSCOPYr_scbond_it2.12713.21314913
ELECTRON MICROSCOPYr_scbond_other2.12713.21314913
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other4.14819.71922012
ELECTRON MICROSCOPYr_long_range_B_refined13.636114536
ELECTRON MICROSCOPYr_long_range_B_other13.636114535
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A672620.01
12B672620.01
21A672380.01
22C672380.01
31B672440.01
32C672440.01
41F75860
42G75860
51F75680.01
52H75680.01
61G75660.01
62H75660.01
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.984 22773 -
obs--100 %

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