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- PDB-9ivk: cryo-EM structure of a tmFAP -

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Basic information

Entry
Database: PDB / ID: 9ivk
Titlecryo-EM structure of a tmFAP
Components
  • HBC599 membrane protein binder
  • Heavy chain, Fab fragment
  • Light Chain, Fab fragment
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / de novo protein design / transmembrane protein / ligand binding / fluorogenic / membrane / fluorescent protein. / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homologyChem-KY6
Function and homology information
Biological speciesartificial sequences (others)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsSun, K. / Zhu, J.Y. / Liang, M.F. / Lu, P.L.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0909200 China
CitationJournal: Nature / Year: 2025
Title: De novo design of transmembrane fluorescence-activating proteins.
Authors: Jingyi Zhu / Mingfu Liang / Ke Sun / Yu Wei / Ruiying Guo / Lijing Zhang / Junhui Shi / Dan Ma / Qi Hu / Gaoxingyu Huang / Peilong Lu /
Abstract: The recognition of ligands by transmembrane proteins is essential for the exchange of materials, energy and information across biological membranes. Progress has been made in the de novo design of ...The recognition of ligands by transmembrane proteins is essential for the exchange of materials, energy and information across biological membranes. Progress has been made in the de novo design of transmembrane proteins, as well as in designing water-soluble proteins to bind small molecules, but de novo design of transmembrane proteins that tightly and specifically bind to small molecules remains an outstanding challenge. Here we present the accurate design of ligand-binding transmembrane proteins by integrating deep learning and energy-based methods. We designed pre-organized ligand-binding pockets in high-quality four-helix backbones for a fluorogenic ligand, and generated a transmembrane span using gradient-guided hallucination. The designer transmembrane proteins specifically activated fluorescence of the target fluorophore with mid-nanomolar affinity, exhibiting higher brightness and quantum yield compared to those of enhanced green fluorescent protein. These proteins were highly active in the membrane fraction of live bacterial and eukaryotic cells following expression. The crystal and cryogenic electron microscopy structures of the designer protein-ligand complexes were very close to the structures of the design models. We showed that the interactions between ligands and transmembrane proteins within the membrane can be accurately designed. Our work paves the way for the creation of new functional transmembrane proteins, with a wide range of applications including imaging, ligand sensing and membrane transport.
History
DepositionJul 23, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HBC599 membrane protein binder
H: Heavy chain, Fab fragment
L: Light Chain, Fab fragment
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,0724
Polymers80,7133
Non-polymers3591
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein HBC599 membrane protein binder


Mass: 33037.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) artificial sequences (others) / Production host: Escherichia coli (E. coli)
#2: Antibody Heavy chain, Fab fragment


Mass: 24321.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Light Chain, Fab fragment


Mass: 23353.947 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-KY6 / 4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothiophen-2-yl}ethenyl]benzonitrile


Mass: 359.444 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H17N3OS
Has ligand of interestY
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

ComponentName: De novo design of HBC599 membrane protein binder / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21artificial sequences (others)81077
31Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 7.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 453223 / Symmetry type: POINT

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