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- PDB-8dt8: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex wi... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8dt8 | |||||||||
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Title | LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein (focused refinement) | |||||||||
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![]() | VIRAL PROTEIN / nanobody / bispecific nanobody / coronavirus / antibody engineering | |||||||||
Function / homology | ![]() 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 / identical protein binding / membrane / plasma membrane Similarity search - Function | |||||||||
Biological species | ![]() ![]() synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å | |||||||||
![]() | Ozorowski, G. / Turner, H.L. / Ward, A.B. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins. Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan ...Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan Verma / Jingjia Liu / Robyn L Stanfield / Xueyong Zhu / Hannah L Turner / Devin Sok / Po-Ssu Huang / Dennis R Burton / Andrew B Ward / Ian A Wilson / Joseph G Jardine / ![]() Abstract: Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple ...Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V and V domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 579.9 KB | Display | ![]() |
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PDB format | ![]() | 465.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.4 MB | Display | ![]() |
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Full document | ![]() | 1.4 MB | Display | |
Data in XML | ![]() | 84.7 KB | Display | |
Data in CIF | ![]() | 127.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 27692MC ![]() 8eloC ![]() 8elpC ![]() 8elqC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 141328.359 Da / Num. of mol.: 3 Mutation: R682G, R683S, R685S, V705C, F817P, T883C, A892P, A899P, A942P, K986P, V987P Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: S, 2 / Cell line (production host): HEK293F / Production host: ![]() #2: Antibody | | Mass: 14064.400 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Cell line (production host): HEK293F / Production host: ![]() #3: Antibody | | Mass: 13610.862 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: ![]() #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 |
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Sample preparation
Component | Name: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() | ||||||||||||||||||||
Buffer solution | pH: 7.4 / Details: Detergent added shortly to grid application | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.85 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: complex incubated for 100 minutes prior to freezing | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 9 sec. / Electron dose: 49.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4296 |
Image scans | Movie frames/image: 36 |
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Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50160 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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