+Open data
-Basic information
Entry | Database: PDB / ID: 7z85 | |||||||||
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Title | CRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-B5 nanobody complex | |||||||||
Components |
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Keywords | ANTIVIRAL PROTEIN / Complex / Spike glycoprotein / nanobody H11-B5 | |||||||||
Function / homology | Function and homology information virion component / 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 ...virion component / 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 | |||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 Escherichia virus T4 Lama glama (llama) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Weckener, M. / Naismith, J.H. | |||||||||
Funding support | United Kingdom, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7z85.cif.gz | 789.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7z85.ent.gz | 557.7 KB | Display | PDB format |
PDBx/mmJSON format | 7z85.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7z85_validation.pdf.gz | 2.6 MB | Display | wwPDB validaton report |
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Full document | 7z85_full_validation.pdf.gz | 2.6 MB | Display | |
Data in XML | 7z85_validation.xml.gz | 98.3 KB | Display | |
Data in CIF | 7z85_validation.cif.gz | 150.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/z8/7z85 ftp://data.pdbj.org/pub/pdb/validation_reports/z8/7z85 | HTTPS FTP |
-Related structure data
Related structure data | 14543MC 7z1aC 7z1bC 7z1cC 7z1dC 7z1eC 7z6vC 7z7xC 7z86C 7z9qC 7z9rC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: 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 | Mass: 14960.648 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 Source method: isolated from a genetically manipulated source #4: Sugar | ChemComp-NAG / Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 8 | ||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Grid material: COPPER/RHODIUM / Grid type: Homemade | ||||||||||||||||||||||||
Vitrification | Instrument: SPOTITON / Cryogen name: ETHANE 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 190 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 190 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 83% relative humidity and ambient temperature. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS / Details: EPU auto-function coma free correction |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3 sec. / Electron dose: 45 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 optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
Image scans | Width: 5760 / Height: 4092 |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 672533 | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 318816 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: RECIPROCAL | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 137.14 Å2 | ||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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