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- PDB-9ytq: Computationally Designed Tetramer of Apo-HC4 (C1 symmetry) -

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Basic information

Entry
Database: PDB / ID: 9ytq
TitleComputationally Designed Tetramer of Apo-HC4 (C1 symmetry)
ComponentsDesigned tetrameric HC4 protein.
KeywordsDE NOVO PROTEIN / Tetramer / computationally designed protein
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.34 Å
AuthorsEng, V.H. / Narehood, S.M. / Tezcan, F.A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE-2038238 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R24GM154186 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM138884 United States
CitationJournal: J Am Chem Soc / Year: 2026
Title: Computational Design of a Highly Stable Dicopper Catechol Oxidase.
Authors: Vanessa H Eng / Sarah M Narehood / Yiying Li / Mauro Gascón / Alexander M Hoffnagle / Angela A Shiau / Manny Semonis / Michael T Green / R David Britt / F Akif Tezcan /
Abstract: Type 3 (T3) Cu proteins play essential roles in binding and activating molecular oxygen (O) and are prevalent across all domains of life. Despite sharing the same coordination motif, T3 Cu proteins ...Type 3 (T3) Cu proteins play essential roles in binding and activating molecular oxygen (O) and are prevalent across all domains of life. Despite sharing the same coordination motif, T3 Cu proteins display divergent functions: hemocyanin transports O, while tyrosinase catalyzes the hydroxylation of monophenols and the subsequent oxidation of diphenols and catechol oxidase oxidizes only diphenols. Here, we report the design and characterization of a di-Cu protein (Cu-HC4) inspired by the active sites of natural T3 Cu proteins to investigate the structural features that facilitate catalytic oxidase activity. Cu-HC4 is roughly 1/5th the size of the commercially available mushroom tyrosinase and shares only around 20% sequence identity with the T3 Cu protein templates. Notably, Cu-HC4 displays high thermostability and exhibits diphenol oxidation activity at ambient and elevated temperatures (≥60 °C). Cu-HC4 also initiates the formation of melanin polymers, mimicking melanin biosynthesis of natural tyrosinases. Mechanistic investigations demonstrate that Cu-HC4 utilizes both Cu centers cooperatively for diphenol oxidation and requires O for catalysis like natural Cu oxidases but follows a distinct catalytic pathway compared to those enzymes. Cryo-EM characterization of a tetrameric form of HC4 reveals slight deviations in the relative positions of the active site His residues that may account for differences in reactivity between Cu-HC4 and natural T3 Cu enzymes.
History
DepositionOct 21, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Designed tetrameric HC4 protein.
B: Designed tetrameric HC4 protein.
C: Designed tetrameric HC4 protein.
D: Designed tetrameric HC4 protein.


Theoretical massNumber of molelcules
Total (without water)64,9354
Polymers64,9354
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Designed tetrameric HC4 protein.


Mass: 16233.727 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
Has protein modificationN

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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: Tetrameric HC4 construct / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMOPSC7H15NO4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
Details: 9030 micrographs were collected with an untilted stage, and 6556 micrographs were collected at a 30 degree tilt.
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
2EPUimage acquisition
4cryoSPARC4.6.0CTF correction
9cryoSPARC4.6.0initial Euler assignment
10cryoSPARC4.6.0final Euler assignment
12cryoSPARC4.6.03D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1246974
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108599 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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