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- EMDB-47678: Cu-bound tetrameric copper storage protein 1 with anti-rhodopsin ... -

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Basic information

Entry
Database: EMDB / ID: EMD-47678
TitleCu-bound tetrameric copper storage protein 1 with anti-rhodopsin 1D4 epitope
Map dataCu-bound tetrameric copper storage protein 1 with anti-rhodopsin 1D4 epitope
Sample
  • Complex: Cu-bound tetrameric copper storage protein 1
    • Protein or peptide: Four-helix bundle copper-binding protein
Keywordstetrameric copper storage protein 1 / METAL BINDING PROTEIN
Function / homologyTwin-arginine translocation signal, Cys-rich four helix bundle protein / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / metal ion binding / identical protein binding / Four-helix bundle copper-binding protein
Function and homology information
Biological speciesMethylosinus trichosporium OB3b (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsYao Y / Oken AC / Farrens DL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R01 EY029343 United States
CitationJournal: Biophys J / Year: 2025
Title: A novel "bio-tag" for cryo-EM studies based on the small, electron-dense protein Csp1.
Authors: Weekie Yao / Adam C Oken / David L Farrens
Abstract: Small proteins can be challenging to study by single-particle cryogenic electron microscopy (cryo-EM) techniques because they have low signal-to-noise ratios, making them difficult to identify and ...Small proteins can be challenging to study by single-particle cryogenic electron microscopy (cryo-EM) techniques because they have low signal-to-noise ratios, making them difficult to identify and analyze. Here we investigated the use of Csp1, a small (∼50 kDa) tetrameric metal-binding protein, to act as a "bio-tag" to help overcome this problem. We find Csp1 is compact, stable, and exhibits enhanced electron scattering and excellent particle contrast in cryo-EM micrographs. As a result, we could determine the structure of Csp1 to 2.98-Å resolution using standard cryo-EM approaches. We also tested if Csp1 could be used as a tag or fiducial to help determine the structure of a protein bound to it. Specifically, we analyzed an epitope-tagged Csp1 bound to a ∼40 kDa Fab fragment from the antibody 1D4. Data from these complexes yielded medium-resolution structures of the complex (5.70 Å) and the bound 1D4 Fab (5.40 Å). These results suggest that, with further optimization, electron-rich Csp1 is a promising bio-tag for use in cryo-EM studies.
History
DepositionNov 1, 2024-
Header (metadata) releaseMay 7, 2025-
Map releaseMay 7, 2025-
UpdateJul 23, 2025-
Current statusJul 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47678.png

Downloads & links

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Map

FileDownload / File: emd_47678.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCu-bound tetrameric copper storage protein 1 with anti-rhodopsin 1D4 epitope
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 400 pix.
= 259.32 Å		0.65 Å/pix.
x 400 pix.
= 259.32 Å		0.65 Å/pix.
x 400 pix.
= 259.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.6483 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.235
Minimum - Maximum-0.96076196 - 1.3023218
Average (Standard dev.)0.000087459506 (±0.020585122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 259.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47678_msk_1.map
Projections & Slices
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Half map: Half Map A

Fileemd_47678_half_map_1.map
AnnotationHalf Map A
Projections & Slices
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Histogram (log scale)

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Half map: Half Map B

Fileemd_47678_half_map_2.map
AnnotationHalf Map B
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Sample components

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Entire : Cu-bound tetrameric copper storage protein 1

EntireName: Cu-bound tetrameric copper storage protein 1
Components
  • Complex: Cu-bound tetrameric copper storage protein 1
    • Protein or peptide: Four-helix bundle copper-binding protein

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Supramolecule #1: Cu-bound tetrameric copper storage protein 1

SupramoleculeName: Cu-bound tetrameric copper storage protein 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Methylosinus trichosporium OB3b (bacteria)
Molecular weightTheoretical: 54 KDa

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Macromolecule #1: Four-helix bundle copper-binding protein

MacromoleculeName: Four-helix bundle copper-binding protein / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Methylosinus trichosporium OB3b (bacteria)
Molecular weightTheoretical: 11.321135 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GAKYKALLDS SSHCVAVGED CLRHCFEMLA MNDASMGACT KATYDLVAAC GALAKLAGTN SAFTPAFAKV VADVCAACKK ECDKFPSIA ECKACGEACQ ACAEECHKVA A

UniProtKB: Four-helix bundle copper-binding protein

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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

BufferpH: 7.5 / Component - Concentration: 20.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7020 / Average exposure time: 1.0 sec. / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6383264
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3) / Software - details: patch CTF correction / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5fje

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 125008
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5fje


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9e7j:
Cu-bound tetrameric copper storage protein 1 with anti-rhodopsin 1D4 epitope

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