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- EMDB-60929: cryo-EM structure of a tmFAP -

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Basic information

Entry
Database: EMDB / ID: EMD-60929
Titlecryo-EM structure of a tmFAP
Map data
Sample
  • Complex: De novo design of HBC599 membrane protein binder
    • Protein or peptide: HBC599 membrane protein binder
    • Protein or peptide: Heavy chain, Fab fragment
    • Protein or peptide: Light Chain, Fab fragment
  • Ligand: 4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothiophen-2-yl}ethenyl]benzonitrile
Keywordsde novo protein design / transmembrane protein / ligand binding / fluorogenic / membrane / fluorescent protein. / MEMBRANE PROTEIN/IMMUNE SYSTEM / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Biological speciesartificial sequences (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsSun K / Zhu JY / Liang MF / Lu PL
Funding support China, 1 items
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, 2024-
Header (metadata) releaseNov 20, 2024-
Map releaseNov 20, 2024-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_60929.png

Downloads & links

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Map

FileDownload / File: emd_60929.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 192 pix.
= 218.88 Å		1.14 Å/pix.
x 192 pix.
= 218.88 Å		1.14 Å/pix.
x 192 pix.
= 218.88 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.136
Minimum - Maximum-1.1038141 - 1.8270135
Average (Standard dev.)0.00028416121 (±0.031624958)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 218.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_60929_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60929_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : De novo design of HBC599 membrane protein binder

EntireName: De novo design of HBC599 membrane protein binder
Components
  • Complex: De novo design of HBC599 membrane protein binder
    • Protein or peptide: HBC599 membrane protein binder
    • Protein or peptide: Heavy chain, Fab fragment
    • Protein or peptide: Light Chain, Fab fragment
  • Ligand: 4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothiophen-2-yl}ethenyl]benzonitrile

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Supramolecule #1: De novo design of HBC599 membrane protein binder

SupramoleculeName: De novo design of HBC599 membrane protein binder / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: artificial sequences (others)

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Macromolecule #1: HBC599 membrane protein binder

MacromoleculeName: HBC599 membrane protein binder / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: artificial sequences (others)
Molecular weightTheoretical: 33.037648 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DEERLKEILF FLLLIIIFVV FLLIVDYKFL EEFKEKNVTD KEEFNEVIKI DEAVMLISAF LLAIAAALLK ELEELIRKRF EEWEKEEET LKKLEDNWET LNDNLKVIEK ADNAAQVKDA LTKMRAAALD AQKATPPKLE DKSPDSPEMK DFRHGFDILV G QIDDALKL ...String:
DEERLKEILF FLLLIIIFVV FLLIVDYKFL EEFKEKNVTD KEEFNEVIKI DEAVMLISAF LLAIAAALLK ELEELIRKRF EEWEKEEET LKKLEDNWET LNDNLKVIEK ADNAAQVKDA LTKMRAAALD AQKATPPKLE DKSPDSPEMK DFRHGFDILV G QIDDALKL ANEGKVKEAQ AAAEQLKTTR NAYIQKYLEK AKETMEKRKE IRRELEKILG VLAGLYVGAA FLMVIAKFLT KK IKEENTT DKEKLNEWYE FVYVLLIFAF FIILAIAVVL LKLLEILG

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Macromolecule #2: Heavy chain, Fab fragment

MacromoleculeName: Heavy chain, Fab fragment / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.321084 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK S

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Macromolecule #3: Light Chain, Fab fragment

MacromoleculeName: Light Chain, Fab fragment / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.353947 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRG

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Macromolecule #4: 4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothioph...

MacromoleculeName: 4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothiophen-2-yl}ethenyl]benzonitrile
type: ligand / ID: 4 / Number of copies: 1 / Formula: KY6
Molecular weightTheoretical: 359.444 Da
Chemical component information

ChemComp-KY6:
4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothiophen-2-yl}ethenyl]benzonitrile

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7.6 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 453223
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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