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- PDB-8w6e: De novo design of HBC599 binder -

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Basic information

Entry
Database: PDB / ID: 8w6e
TitleDe novo design of HBC599 binder
ComponentsHBC599 in complex with wFAP1.1
KeywordsDE NOVO PROTEIN / de novo protein design / ligand binding / fluorogenic / fluorescent protein
Function / homologyChem-KY6
Function and homology information
Biological speciesartificial sequences (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsLiang, M.F. / Zhu, J.Y. / 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
DepositionAug 28, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 26, 2025Group: 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.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Mar 5, 2025Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Apr 16, 2025Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HBC599 in complex with wFAP1.1
B: HBC599 in complex with wFAP1.1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,6414
Polymers42,9222
Non-polymers7192
Water55831
1
A: HBC599 in complex with wFAP1.1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,8202
Polymers21,4611
Non-polymers3591
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: HBC599 in complex with wFAP1.1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,8202
Polymers21,4611
Non-polymers3591
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)40.557, 91.912, 52.198
Angle α, β, γ (deg.)90.000, 107.791, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein HBC599 in complex with wFAP1.1


Mass: 21460.938 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) artificial sequences (others) / Production host: Escherichia coli (E. coli)
#2: 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.: 2 / Source method: obtained synthetically / Formula: C21H17N3OS / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 31 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2 Å3/Da / Density % sol: 43.01 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop / pH: 4.2
Details: 0.2M Lithium sulfate, 20% W/V PEG1000, 0.1M Sodium phosphate citrate PH4.2

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: 100K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL02U1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jan 7, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.1→30 Å / Num. obs: 20877 / % possible obs: 99.5 % / Redundancy: 11.4 % / Biso Wilson estimate: 37.5 Å2 / CC1/2: 1 / Net I/σ(I): 26
Reflection shellResolution: 2.1→2.14 Å / Redundancy: 5.5 % / Rmerge(I) obs: 0.824 / Mean I/σ(I) obs: 1.33 / Num. unique obs: 1864 / CC1/2: 0.797 / CC star: 0.942 / Χ2: 0.415 / % possible all: 95.1

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Processing

Software
NameVersionClassification
PHENIX1.19.1_4122refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1→29.57 Å / SU ML: 0.263 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 29.6492
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2441 989 4.74 %
Rwork0.2176 19888 -
obs0.2188 20877 98.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 51.96 Å2
Refinement stepCycle: LAST / Resolution: 2.1→29.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2755 0 52 31 2838
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01072821
X-RAY DIFFRACTIONf_angle_d1.48533749
X-RAY DIFFRACTIONf_chiral_restr0.0894418
X-RAY DIFFRACTIONf_plane_restr0.0045480
X-RAY DIFFRACTIONf_dihedral_angle_d19.05361222
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.220.32251250.27462629X-RAY DIFFRACTION91.4
2.22-2.350.28421190.24392877X-RAY DIFFRACTION99.47
2.35-2.540.23981320.22822884X-RAY DIFFRACTION99.93
2.54-2.790.26941760.22882841X-RAY DIFFRACTION99.93
2.79-3.190.25631330.222875X-RAY DIFFRACTION99.97
3.2-4.020.21771510.19312885X-RAY DIFFRACTION100
4.02-29.570.23521530.21742897X-RAY DIFFRACTION99.54

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