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- EMDB-73489: Computationally Designed Tetramer of Apo-HC4 (C1 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-73489
TitleComputationally Designed Tetramer of Apo-HC4 (C1 symmetry)
Map dataComputationally Designed Tetramer of Apo-HC4 (C1 symmetry)
Sample
  • Complex: Tetrameric HC4 construct
    • Protein or peptide: Designed tetrameric HC4 protein.
KeywordsTetramer / computationally designed protein / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.34 Å
AuthorsEng VH / Narehood SM / Tezcan FA
Funding support United States, 3 items
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, 2025-
Header (metadata) releaseFeb 25, 2026-
Map releaseFeb 25, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73489.png

Downloads & links

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Map

FileDownload / File: emd_73489.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComputationally Designed Tetramer of Apo-HC4 (C1 symmetry)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.72 Å/pix.
x 128 pix.
= 220.16 Å		1.72 Å/pix.
x 128 pix.
= 220.16 Å		1.72 Å/pix.
x 128 pix.
= 220.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.201
Minimum - Maximum-0.6824584 - 1.2254548
Average (Standard dev.)0.0008056739 (±0.030728243)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 220.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_73489_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_73489_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrameric HC4 construct

EntireName: Tetrameric HC4 construct
Components
  • Complex: Tetrameric HC4 construct
    • Protein or peptide: Designed tetrameric HC4 protein.

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Supramolecule #1: Tetrameric HC4 construct

SupramoleculeName: Tetrameric HC4 construct / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Designed tetrameric HC4 protein.

MacromoleculeName: Designed tetrameric HC4 protein. / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.233727 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SKTDKLLELA KLELILNFFL LASNANITKE SFLIAHKAWH KALNKLYEID KELAKEYFYY MHSFSIPYYE KLGFKDVAEW HKVMVEYFK VGDKDEALEL HNEGHKALRA GDEEKFAALL KKARELIEKA KSAENLYFQ

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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

Concentration5 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC7H15NO4SMOPS
150.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Details: 9030 micrographs were collected with an untilted stage, and 6556 micrographs were collected at a 30 degree tilt.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1246974
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.0) / Number images used: 108599
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

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

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