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- PDB-7z1e: Nanobody H11-H4 Q98R H100E bound to RBD -

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Basic information

Entry
Database: PDB / ID: 7z1e
TitleNanobody H11-H4 Q98R H100E bound to RBD
Components
  • H11-H4 Q98R H100E
  • Spike protein S1
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 / 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 / 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
NITRATE ION / Spike glycoprotein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.59 Å
AuthorsMikolajek, H. / Naismith, J.H.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust100209/Z/12/Z) United Kingdom
Engineering and Physical Sciences Research CouncilEP/S025243/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.
Authors: Halina Mikolajek / Miriam Weckener / Z Faidon Brotzakis / Jiandong Huo / Evmorfia V Dalietou / Audrey Le Bas / Pietro Sormanni / Peter J Harrison / Philip N Ward / Steven Truong / Lucile ...Authors: Halina Mikolajek / Miriam Weckener / Z Faidon Brotzakis / Jiandong Huo / Evmorfia V Dalietou / Audrey Le Bas / Pietro Sormanni / Peter J Harrison / Philip N Ward / Steven Truong / Lucile Moynie / Daniel K Clare / Maud Dumoux / Joshua Dormon / Chelsea Norman / Naveed Hussain / Vinod Vogirala / Raymond J Owens / Michele Vendruscolo / James H Naismith /
Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. ...Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
History
DepositionFeb 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 23, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Derived calculations / Category: atom_type / citation / citation_author
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
EEE: Spike protein S1
FFF: H11-H4 Q98R H100E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,4048
Polymers38,7822
Non-polymers6226
Water3,711206
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)78.243, 78.243, 126.812
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Components on special symmetry positions
IDModelComponents
11EEE-832-

HOH

21EEE-839-

HOH

31EEE-841-

HOH

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Components

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Protein / Antibody / Sugars , 3 types, 3 molecules EEEFFF

#1: Protein Spike protein S1


Mass: 23716.580 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
#2: Antibody H11-H4 Q98R H100E


Mass: 15065.783 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)
#3: 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.: 1 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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

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Non-polymers , 3 types, 211 molecules

#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-NO3 / NITRATE ION / Nitrate


Mass: 62.005 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: NO3
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 206 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.89 Å3/Da / Density % sol: 57.43 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 0.2M Ammonium nitrate, 20 % Peg 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jul 14, 2021
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.59→67.761 Å / Num. obs: 61104 / % possible obs: 100 % / Redundancy: 18.8 % / CC1/2: 0.999 / Rmerge(I) obs: 0.046 / Net I/σ(I): 24.9
Reflection shellResolution: 1.59→1.62 Å / Redundancy: 20.2 % / Rmerge(I) obs: 4.156 / Mean I/σ(I) obs: 0.4 / Num. unique obs: 3027 / CC1/2: 0.361 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
xia2data reduction
xia2data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6ZBP

6zbp


Resolution: 1.59→67.761 Å / Cor.coef. Fo:Fc: 0.976 / Cor.coef. Fo:Fc free: 0.967 / SU B: 4.816 / SU ML: 0.067 / Cross valid method: THROUGHOUT / ESU R: 0.077 / ESU R Free: 0.072
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2023 3095 5.071 %
Rwork0.161 57940 -
all0.163 --
obs-61035 99.972 %
Solvent computationIon probe radii: 0.7 Å / Shrinkage radii: 0.7 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 38.799 Å2
Baniso -1Baniso -2Baniso -3
1--0.473 Å2-0.237 Å2-0 Å2
2---0.473 Å20 Å2
3---1.535 Å2
Refinement stepCycle: LAST / Resolution: 1.59→67.761 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2520 0 40 206 2766
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.0122688
X-RAY DIFFRACTIONr_bond_other_d0.0010.0152400
X-RAY DIFFRACTIONr_angle_refined_deg1.3061.6563666
X-RAY DIFFRACTIONr_angle_other_deg1.2671.5835521
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9945338
X-RAY DIFFRACTIONr_dihedral_angle_2_deg29.59721.597144
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.69115399
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.0321517
X-RAY DIFFRACTIONr_chiral_restr0.0610.2337
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.023115
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02701
X-RAY DIFFRACTIONr_nbd_refined0.1860.2416
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1780.22172
X-RAY DIFFRACTIONr_nbtor_refined0.1710.21266
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0750.21249
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1230.2169
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.2660.228
X-RAY DIFFRACTIONr_nbd_other0.2150.250
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1470.218
X-RAY DIFFRACTIONr_mcbond_it2.3073.8661298
X-RAY DIFFRACTIONr_mcbond_other2.2933.8641297
X-RAY DIFFRACTIONr_mcangle_it3.055.8011624
X-RAY DIFFRACTIONr_mcangle_other3.0555.8041625
X-RAY DIFFRACTIONr_scbond_it2.6214.2311390
X-RAY DIFFRACTIONr_scbond_other2.5274.2251385
X-RAY DIFFRACTIONr_scangle_it3.2876.2112032
X-RAY DIFFRACTIONr_scangle_other3.2376.2062027
X-RAY DIFFRACTIONr_lrange_it4.12844.4712949
X-RAY DIFFRACTIONr_lrange_other4.13144.4842950
X-RAY DIFFRACTIONr_rigid_bond_restr1.3635088
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.59-1.6310.5112240.45742280.4644690.440.44199.61960.447
1.631-1.6760.4222260.43841090.43743350.5920.5931000.431
1.676-1.7250.3742220.36939910.36942130.6570.71000.364
1.725-1.7780.3331990.31539390.31641380.8060.8121000.31
1.778-1.8360.2452020.23637690.23639710.8980.91000.219
1.836-1.90.222090.17836560.1838650.930.9371000.151
1.9-1.9720.1882080.1435440.14337520.9460.9571000.112
1.972-2.0520.1861640.13134270.13435910.9590.9651000.105
2.052-2.1440.1621570.12132560.12334130.9680.9721000.098
2.144-2.2480.1861810.12631370.1333180.9550.971000.104
2.248-2.3690.1871690.11929970.12331660.9590.9721000.099
2.369-2.5130.1791320.12828520.1329840.9610.9721000.106
2.513-2.6860.1771430.12726600.1328030.9660.9731000.111
2.686-2.9010.2051600.13524720.13926320.9530.9721000.122
2.901-3.1770.1871230.15123040.15324270.960.9691000.142
3.177-3.5520.1951180.1621050.16222230.9610.9711000.157
3.552-4.0990.183920.16218560.16319480.970.9711000.168
4.099-5.0160.156690.13416090.13516780.9780.981000.15
5.016-7.0740.251600.18512730.18813330.9630.971000.205
7.074-67.7610.259370.2247560.2267930.9450.9471000.256

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