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- PDB-6zhd: H11-H4 bound to Spike -

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Basic information

Entry
Database: PDB / ID: 6zhd
TitleH11-H4 bound to Spike
Components
  • Nanobody H11-H4
  • Spike glycoprotein,Fibritin
KeywordsANTIVIRAL PROTEIN / SARS-CoV-2 / nanobody / llama / neutralisation / spike
Function / homology
Function and homology information


host cell endoplasmic reticulum-Golgi intermediate compartment membrane / host cell surface receptor binding / virion / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / pathogenesis / host cell plasma membrane / virion membrane / integral component of membrane
Fibritin C-terminal / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike receptor binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus
Spike glycoprotein / Fibritin
Biological speciesSevere acute respiratory syndrome coronavirus 2
Enterobacteria phage T4 (bacteriophage)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsClare, D.K. / Naismith, J.H. / Weckener, M. / Vogirala, V.K.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust100209/Z/12/Z United Kingdom
Engineering and Physical Sciences Research CouncilRosalind Franklin United Kingdom
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2020
Title: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.
Authors: Jiangdong 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: Jiangdong 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 22, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Spike glycoprotein,Fibritin
B: Spike glycoprotein,Fibritin
C: Spike glycoprotein,Fibritin
D: Nanobody H11-H4
E: Nanobody H11-H4
F: Nanobody H11-H4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)486,57552
Polymers472,3356
Non-polymers14,23946
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44890 Å2
ΔGint24 kcal/mol
Surface area150010 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A27 - 1147
2010B27 - 1147
1020A27 - 1147
2020C27 - 1147
1030B27 - 1147
2030C27 - 1147

NCS ensembles:
ID
1
2
3

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Components

#1: Protein Spike glycoprotein,Fibritin / S glycoprotein / E2 / Peplomer protein / Collar protein / Whisker antigen control protein


Mass: 142399.375 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2, (gene. exp.) Enterobacteria phage T4 (bacteriophage)
Gene: S, 2, wac / Production host: Homo sapiens (human) / References: UniProt: P0DTC2, UniProt: P10104
#2: Antibody Nanobody H11-H4


Mass: 15045.752 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


GlcNAc

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GlcNAc

Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 20
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 26
Source method: isolated from a genetically manipulated source
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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex between Spike and nanobody H11-H4COMPLEXNanobody complexed to Spike protein1,20RECOMBINANT
2SpikeCOMPLEX11RECOMBINANT
3nanobody H11-H4COMPLEX21RECOMBINANT
Molecular weightValue: 0.52 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Enterobacteria phage T4 (bacteriophage)10665
22Severe acute respiratory syndrome coronavirus 22697049
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
Buffer solutionpH: 7 / Details: 50 mM Tris, pH 7, 150 mM NaCl
SpecimenConc.: 1 mg/ml / Details: Complex mixed together / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Plasma Cleaner PDC-002-CE, Harrick Plasma / Grid type: Homemade
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 81 %
Details: SPT Labtech prototype 300 mesh 1.2/2.0 nanowire grids with a highly reproduceable rectangular bar cross-section were used. The grids were glow-discharged on low for 90 s (Plasma Cleaner PDC- ...Details: SPT Labtech prototype 300 mesh 1.2/2.0 nanowire grids with a highly reproduceable rectangular bar cross-section were used. The grids were glow-discharged on low for 90 s (Plasma Cleaner PDC-002-CE, Harrick Plasma) to activate the nanowires. Approximately 6 nL of the complex were applied to the grids using a Chameleon EP system (SPT Labtech) at 81 % relative humidity and ambient temperature.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS / Details: EPU auto-function coma free correction
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 47170 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 46 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10000 / Details: The images were collected as 50 frame movies
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: REFMAC / Version: 5.8.0258 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLOparticle selection
2EPU2.6.1image acquisitionAFIS enabled version
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELION3.08initial Euler assignment
10RELION3.08final Euler assignment
11RELIONclassification
12RELION3.083D reconstruction
13REFMAC5model refinement
Image processingDetails: Images were aligned and reconstructed in Releion
CTF correctionDetails: Relion / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 786392
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126938 / Algorithm: BACK PROJECTION / Details: Relion / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Correlation / Details: REFMAC was used via ccpem GUI
Atomic model buildingPDB-ID: 6Z43
RefinementResolution: 3.7→216.24 Å / Cor.coef. Fo:Fc: 0.929 / SU B: 27.809 / SU ML: 0.385 / ESU R: 0.801
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.28488 --
Obs0.28488 154374 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 183.518 Å2
Baniso -1Baniso -2Baniso -3
1-0.33 Å20.96 Å2-0.02 Å2
2--2.08 Å2-0.24 Å2
3----2.41 Å2
Refinement stepCycle: 1 / Total: 26927
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01227629
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.2641.68837727
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.96853329
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.57523.0931306
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.385154147
ELECTRON MICROSCOPYr_dihedral_angle_4_deg17.03215116
ELECTRON MICROSCOPYr_chiral_restr0.0850.23869
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0220927
ELECTRON MICROSCOPYr_gen_planes_other
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_it10.22317.16513388
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it17.66125.72716684
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it14.58719.42514241
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined40.206110735
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

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

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A597160.1
12B597160.1
21A599820.1
22C599820.1
31B609540.09
32C609540.09
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.934 11466 -
Obs--100 %

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