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- PDB-7z86: CRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H4 Q98R H100E nanobod... -

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Basic information

Entry
Database: PDB / ID: 7z86
TitleCRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H4 Q98R H100E nanobody complex in 1Up2Down conformation
Components
  • Nanobody H11-H4 Q98R H100E
  • Spike glycoprotein,Fibritin
KeywordsANTIVIRAL PROTEIN / Complex / Spike glycoprotein / nanobody H11-H4 Q98R H100E
Function / homology
Function and homology information


virion component / Maturation of spike protein / viral translation / Translation of Structural Proteins / host cell surface / Virion Assembly and Release / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion ...virion component / Maturation of spike protein / viral translation / Translation of Structural Proteins / host cell surface / Virion Assembly and Release / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / membrane fusion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / 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
Fibritin C-terminal / Fibritin C-terminal region / Spike glycoprotein, N-terminal domain / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. ...Fibritin C-terminal / Fibritin C-terminal region / Spike glycoprotein, N-terminal domain / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / 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 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Jelly Rolls / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Spike glycoprotein / Fibritin
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Escherichia virus T4
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWeckener, M. / Naismith, J.H.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research CouncilP2019-0006 United Kingdom
Wellcome Trust100209/Z/12/Z 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
DepositionMar 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._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 1.2Nov 6, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein,Fibritin
B: Spike glycoprotein,Fibritin
C: Spike glycoprotein,Fibritin
X: Nanobody H11-H4 Q98R H100E
Y: Nanobody H11-H4 Q98R H100E
Z: Nanobody H11-H4 Q98R H100E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)476,19652
Polymers461,9576
Non-polymers14,23946
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47340 Å2
ΔGint40 kcal/mol
Surface area148900 Å2

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Components

#1: Protein Spike glycoprotein,Fibritin / S glycoprotein / E2 / Peplomer protein / Collar protein / Whisker antigen control 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. exp.) Escherichia virus T4
Gene: S, 2, wac / Cell line (production host): HEK 293T Lenti-X / Production host: Homo sapiens (human) / References: UniProt: P0DTC2, UniProt: P10104
#2: Antibody Nanobody H11-H4 Q98R H100E


Mass: 14107.723 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) / Strain (production host): WK6
#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.: 20
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.: 26 / 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
Has protein modificationY

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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
IDNameTypeEntity IDParent-IDSource
1Complex between Spike and nanobody H11-H4 Q98R H100ECOMPLEX#1-#20MULTIPLE SOURCES
2Spike glycoprotein trimer with fibritin tagCOMPLEX#11RECOMBINANT
3Nanobody H11-H4 Q98R H100ECOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.52 MDaNO
210.142 MDaNO
310.015 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Severe acute respiratory syndrome coronavirus 22697049
32Escherichia virus T410665
43Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Homo sapiens (human)9606HEK 293T
32Homo sapiens (human)9606HEK 293T
43Escherichia coli (E. coli)562WK6
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESHEPES1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged twice, 60 s each time. / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.85 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Details: The images were collected as 50 frame movies
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1RELION3.08particle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10REFMACmodel refinement
11RELION3.08initial Euler assignment
12RELION3.08final Euler assignment
13RELION3.08classification
14RELION3.083D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 909790
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 396910 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL
Atomic model building

3D fitting-ID: 1 / Accession code: 6ZHD / Initial refinement model-ID: 1 / Pdb chain residue range: 27-1147 / PDB-ID: 6ZHD

6zhd

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3C

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