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- PDB-6zh9: Ternary complex CR3022 H11-H4 and RBD (SARS-CoV-2) -

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Basic information

Entry
Database: PDB / ID: 6zh9
TitleTernary complex CR3022 H11-H4 and RBD (SARS-CoV-2)
Components
  • CR3022 Light chain
  • CR3022 heavy
  • Nanobody H11-H4
  • Spike glycoproteinSpike protein
KeywordsANTIVIRAL PROTEIN / Covid19 / nanobody / SARS-CoV-2
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 speciesHomo sapiens (human)
Severe acute respiratory syndrome coronavirus 2
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.31 Å
AuthorsNaismith, J.H. / Mikolajek, H. / Le Bas, A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust100209/Z/12/Z). United Kingdom
Engineering and Physical Sciences Research CouncilRFI 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
DepositionJun 21, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
HHH: CR3022 heavy
LLL: CR3022 Light chain
EEE: Spike glycoprotein
FFF: Nanobody H11-H4


Theoretical massNumber of molelcules
Total (without water)84,2234
Polymers84,2234
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Purified before xtals
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6510 Å2
ΔGint-35 kcal/mol
Surface area33960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)156.411, 156.411, 116.205
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number94
Space group name H-MP42212

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Components

#1: Protein CR3022 heavy


Mass: 22874.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Antibody CR3022 Light chain


Mass: 24186.740 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Protein Spike glycoprotein / Spike protein / S glycoprotein / E2 / Peplomer protein


Mass: 22115.834 Da / Num. of mol.: 1
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
#4: Antibody Nanobody H11-H4


Mass: 15045.752 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.22 Å3/Da / Density % sol: 70.85 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: mixing 0.2 uL of the 18 mg/mL complex with 0.1 uL of the crystallization buffer containing 0.2 M Sodium acetate trihydrate, 0.1 M MES pH 6.0, 20 % w/v PEG 8000.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.98 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jun 18, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 3.31→80.253 Å / Num. obs: 22105 / % possible obs: 100 % / Redundancy: 25.6 % / CC1/2: 1 / Rmerge(I) obs: 0.091 / Net I/av σ(I): 21.9 / Net I/σ(I): 0.018
Reflection shellResolution: 3.31→3.4 Å / Rmerge(I) obs: 3.9 / Mean I/σ(I) obs: 0.5 / Num. unique obs: 1538 / CC1/2: 0.3 / Rpim(I) all: 0.77

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
pointlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6ZBY

6zby


Resolution: 3.31→80.243 Å / Cor.coef. Fo:Fc: 0.83 / Cor.coef. Fo:Fc free: 0.9 / SU B: 109.814 / SU ML: 0.696 / Cross valid method: FREE R-VALUE / ESU R Free: 0.545
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.3052 1088 4.931 %
Rwork0.2609 20977 -
all0.263 --
obs-22065 99.964 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 134 Å2
Baniso -1Baniso -2Baniso -3
1--1.696 Å2-0 Å2-0 Å2
2---1.696 Å2-0 Å2
3---3.392 Å2
Refinement stepCycle: LAST / Resolution: 3.31→80.243 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5856 0 0 0 5856
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0136072
X-RAY DIFFRACTIONr_bond_other_d0.0010.0175372
X-RAY DIFFRACTIONr_angle_refined_deg1.6261.6468275
X-RAY DIFFRACTIONr_angle_other_deg1.2281.57112533
X-RAY DIFFRACTIONr_dihedral_angle_1_deg21.5435.351797
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.9722.982275
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.09915939
X-RAY DIFFRACTIONr_dihedral_angle_other_3_deg20.671152
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.9911524
X-RAY DIFFRACTIONr_chiral_restr0.0590.2788
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.027576
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021299
X-RAY DIFFRACTIONr_nbd_refined0.1850.2920
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1880.25024
X-RAY DIFFRACTIONr_nbtor_refined0.1740.22807
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0770.22885
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1430.2120
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1790.219
X-RAY DIFFRACTIONr_nbd_other0.2540.255
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.2660.25
X-RAY DIFFRACTIONr_xyhbond_nbd_other0.0740.21
X-RAY DIFFRACTIONr_mcbond_it7.79111.6813051
X-RAY DIFFRACTIONr_mcbond_other7.78711.6823050
X-RAY DIFFRACTIONr_mcangle_it12.28917.513812
X-RAY DIFFRACTIONr_mcangle_other12.28817.5093813
X-RAY DIFFRACTIONr_scbond_it7.2511.9323020
X-RAY DIFFRACTIONr_scbond_other7.24911.9313021
X-RAY DIFFRACTIONr_scangle_it11.80917.7594455
X-RAY DIFFRACTIONr_scangle_other11.80817.7594456
X-RAY DIFFRACTIONr_lrange_it19.804221.69224022
X-RAY DIFFRACTIONr_lrange_other19.804221.68824023
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.31-3.3960.416890.4051494X-RAY DIFFRACTION99.6224
3.396-3.4890.395870.3781470X-RAY DIFFRACTION100
3.489-3.590.321860.3391437X-RAY DIFFRACTION100
3.59-3.70.374730.3191389X-RAY DIFFRACTION100
3.7-3.8220.356700.3171386X-RAY DIFFRACTION100
3.822-3.9560.355820.2971305X-RAY DIFFRACTION100
3.956-4.1050.353570.2981270X-RAY DIFFRACTION100
4.105-4.2720.343570.31246X-RAY DIFFRACTION100
4.272-4.4620.319630.261187X-RAY DIFFRACTION100
4.462-4.6790.24470.2341130X-RAY DIFFRACTION100
4.679-4.9320.315510.2291093X-RAY DIFFRACTION100
4.932-5.230.358540.2461023X-RAY DIFFRACTION100
5.23-5.5910.296490.246983X-RAY DIFFRACTION100
5.591-6.0370.303520.244892X-RAY DIFFRACTION100
6.037-6.6120.362350.259843X-RAY DIFFRACTION100
6.612-7.3890.29360.229785X-RAY DIFFRACTION100
7.389-8.5270.232270.211689X-RAY DIFFRACTION100
8.527-10.4290.251300.181596X-RAY DIFFRACTION100
10.429-14.690.284220.198474X-RAY DIFFRACTION100
14.69-80.2430.277210.504285X-RAY DIFFRACTION99.3506
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.6597-0.4119-2.07672.4891.98292.93760.48420.03810.6667-0.0637-0.06980.4182-0.5798-0.3661-0.41440.80450.42080.37930.80550.50130.644821.24-32.608-52.836
24.835-1.7169-2.75471.08171.88064.14660.0385-0.1652-0.15410.3322-0.06450.27150.3703-0.20090.0260.7090.11250.43360.57650.25370.491819.386-45.311-39.516
33.76651.64130.43446.14221.76193.2516-0.36340.8443-0.0714-0.23890.7744-0.1956-0.0859-0.3469-0.41110.3471-0.045-0.02310.8091-0.11150.104940.243-60.063-86.095
41.4255-1.74930.67972.2119-0.80650.35690.64560.63570.2229-1.1106-0.5932-0.44750.26910.2289-0.05242.26-0.72340.25712.9487-0.52780.683944.791-67.016-122.069
Refinement TLS group
IDRefine-IDRefine TLS-IDSelectionAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1ALLHHH0 - 219
2X-RAY DIFFRACTION2ALLLLL1 - 219
3X-RAY DIFFRACTION3ALLEEE334 - 528
4X-RAY DIFFRACTION4ALLFFF1 - 128

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