+Open data
-Basic information
Entry | Database: PDB / ID: 7r9d | ||||||
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Title | Crystal structure of Nb_0 in complex with Fab_8D3 | ||||||
Components |
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Keywords | PROTEIN BINDING / scaffold / protein binder | ||||||
Biological species | Mus musculus (house mouse) Vicugna pacos (alpaca) | ||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.83 Å | ||||||
Authors | Wu, X.D. / Rapoport, T.A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies). Authors: Xudong Wu / Tom A Rapoport / Abstract: We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding ...We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7r9d.cif.gz | 320 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7r9d.ent.gz | 261.6 KB | Display | PDB format |
PDBx/mmJSON format | 7r9d.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/r9/7r9d ftp://data.pdbj.org/pub/pdb/validation_reports/r9/7r9d | HTTPS FTP |
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-Related structure data
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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-Components
#1: Antibody | Mass: 24095.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293 freestyle / Production host: Homo sapiens (human) |
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#2: Antibody | Mass: 13153.450 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 |
#3: Antibody | Mass: 24322.207 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293 freesytle / Production host: Homo sapiens (human) |
#4: Water | ChemComp-HOH / |
-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 2.3 Å3/Da / Density % sol: 46.57 % |
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Crystal grow | Temperature: 277 K / Method: vapor diffusion, hanging drop / Details: 0.2 M Ammonium formate, 20-22 % w/v PEG 3350 |
-Data collection
Diffraction | Mean temperature: 100 K / Serial crystal experiment: N |
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Diffraction source | Source: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.979 Å |
Detector | Type: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Feb 4, 2021 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 0.979 Å / Relative weight: 1 |
Reflection | Resolution: 1.83→30 Å / Num. obs: 49251 / % possible obs: 99.35 % / Redundancy: 7.6 % / Rmerge(I) obs: 0.068 / Net I/σ(I): 16.46 |
Reflection shell | Resolution: 1.83→1.87 Å / Rmerge(I) obs: 0.55 / Num. unique obs: 3189 |
-Processing
Software |
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Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: Fab with similar sequence Resolution: 1.83→28.45 Å / SU ML: 0.17 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 25.15 / Stereochemistry target values: ML
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 115.59 Å2 / Biso mean: 39.5196 Å2 / Biso min: 19.59 Å2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: final / Resolution: 1.83→28.45 Å
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LS refinement shell | Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14
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Refinement TLS params. | Method: refined / Origin x: -16.3594 Å / Origin y: 5.6634 Å / Origin z: -12.5449 Å
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Refinement TLS group |
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