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- EMDB-47340: De novo calcium channel hexamer, CalC6_3 with DHR extensions -

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Basic information

Entry
Database: EMDB / ID: EMD-47340
TitleDe novo calcium channel hexamer, CalC6_3 with DHR extensions
Map dataNon Uniform Refinement, DeepEMhancer sharpened map. C6 symmetry. Main map used for structure refinement. 3.75 Angstrom
Sample
  • Complex: CalC6_3 with DHR extension
    • Protein or peptide: CalC6_3 with DHR extension
KeywordsCalcium Channel / CalC6_3 / De novo / Membrane protein / CalC6_3 with DHR extensions / hexamer / pore / De novo protein / Ca
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsWeidle C / Liu Y / Borst AJ
Funding support United States, China, France, 4 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, China)DGE-2140004 China
Human Frontier Science Program (HFSP)LT000880/2019 France
Defense Advanced Research Projects Agency (DARPA)HR001120S0052 United States
CitationJournal: Nature / Year: 2025
Title: Bottom-up design of Ca channels from defined selectivity filter geometry.
Authors: Yulai Liu / Connor Weidle / Ljubica Mihaljević / Joseph L Watson / Zhe Li / Le Tracy Yu / Sagardip Majumder / Andrew J Borst / Kenneth D Carr / Ryan D Kibler / Tamer M Gamal El-Din / ...Authors: Yulai Liu / Connor Weidle / Ljubica Mihaljević / Joseph L Watson / Zhe Li / Le Tracy Yu / Sagardip Majumder / Andrew J Borst / Kenneth D Carr / Ryan D Kibler / Tamer M Gamal El-Din / William A Catterall / David Baker /
Abstract: Native ion channels play key roles in biological systems, and engineered versions are widely used as chemogenetic tools and in sensing devices. Protein design has been harnessed to generate pore- ...Native ion channels play key roles in biological systems, and engineered versions are widely used as chemogenetic tools and in sensing devices. Protein design has been harnessed to generate pore-containing transmembrane proteins, but the design of selectivity filters with precise arrangements of amino acid side chains specific for a target ion, a crucial feature of native ion channels, has been constrained by the lack of methods for placing the metal-coordinating residues with atomic-level precision. Here we describe a bottom-up RFdiffusion-based approach to construct Ca channels from defined selectivity filter residue geometries, and use this approach to design symmetric oligomeric channels with Ca selectivity filters having different coordination numbers and different geometries at the entrance of a wider pore buttressed by multiple transmembrane helices. The designed channel proteins assemble into homogeneous pore-containing particles and, for both tetrameric and hexameric ion-coordinating configurations, patch-clamp experiments show that the designed channels have higher conductances for Ca than for Na and other divalent ions (Sr and Mg) that are eliminated after mutation of selectivity filter residues. Cryogenic electron microscopy indicates that the design method has high accuracy: the structure of the hexameric Ca channel is nearly identical to that of the design model. Our bottom-up design approach now enables the testing of hypotheses relating filter geometry to ion selectivity by direct construction, and provides a roadmap for creating selective ion channels for a wide range of applications.
History
DepositionOct 17, 2024-
Header (metadata) releaseOct 1, 2025-
Map releaseOct 1, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47340.png

Downloads & links

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Map

FileDownload / File: emd_47340.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNon Uniform Refinement, DeepEMhancer sharpened map. C6 symmetry. Main map used for structure refinement. 3.75 Angstrom
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 340 pix.
= 285.6 Å		0.84 Å/pix.
x 340 pix.
= 285.6 Å		0.84 Å/pix.
x 340 pix.
= 285.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0463
Minimum - Maximum-0.0018169921 - 1.8135867
Average (Standard dev.)0.0007987067 (±0.020220935)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 285.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Non Uniform Refinement, un-sharpened map. C6 symmetry. 3.75 Angstrom

Fileemd_47340_additional_1.map
AnnotationNon Uniform Refinement, un-sharpened map. C6 symmetry. 3.75 Angstrom
Projections & Slices
AxesZYX

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Additional map: Non Uniform Refinement, auto sharpened map. C6 symmetry....

Fileemd_47340_additional_2.map
AnnotationNon Uniform Refinement, auto sharpened map. C6 symmetry. 3.75 Angstrom
Projections & Slices
AxesZYX

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Additional map: Non Uniform Refinement, DeepEMhancer sharpened map. C1 symmetry....

Fileemd_47340_additional_3.map
AnnotationNon Uniform Refinement, DeepEMhancer sharpened map. C1 symmetry. 3.98 Angstrom
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AxesZYX

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Additional map: Non Uniform Refinement, auto sharpened map. C1 symmetry....

Fileemd_47340_additional_4.map
AnnotationNon Uniform Refinement, auto sharpened map. C1 symmetry. 3.98 Angstrom
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Additional map: Non Uniform Refinement, half map B. C1 symmetry. 3.98 Angstrom

Fileemd_47340_additional_5.map
AnnotationNon Uniform Refinement, half map B. C1 symmetry. 3.98 Angstrom
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Additional map: Non Uniform Refinement, half map B. C1 symmetry. 3.98 Angstrom

Fileemd_47340_additional_6.map
AnnotationNon Uniform Refinement, half map B. C1 symmetry. 3.98 Angstrom
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Additional map: Non Uniform Refinement, un-sharpened map. C1 symmetry. 3.98 Angstrom

Fileemd_47340_additional_7.map
AnnotationNon Uniform Refinement, un-sharpened map. C1 symmetry. 3.98 Angstrom
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Non Uniform Refinement, half map A. C6 symmetry. 3.75 Angstrom

Fileemd_47340_half_map_1.map
AnnotationNon Uniform Refinement, half map A. C6 symmetry. 3.75 Angstrom
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Non Uniform Refinement, half map B. C6 symmetry. 3.75 Angstrom

Fileemd_47340_half_map_2.map
AnnotationNon Uniform Refinement, half map B. C6 symmetry. 3.75 Angstrom
Projections & Slices
AxesZYX

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Slices (1/2)
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Sample components

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Entire : CalC6_3 with DHR extension

EntireName: CalC6_3 with DHR extension
Components
  • Complex: CalC6_3 with DHR extension
    • Protein or peptide: CalC6_3 with DHR extension

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Supramolecule #1: CalC6_3 with DHR extension

SupramoleculeName: CalC6_3 with DHR extension / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: 6 monomers assemble to form a calcium channel
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 216.65916 KDa

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Macromolecule #1: CalC6_3 with DHR extension

MacromoleculeName: CalC6_3 with DHR extension / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 36.163828 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAELRERLLR AARWILLLGL LVLVGFVVLA YLERSPLIRA FVLSAGVVLV ALFAAALAWL YLAAALLGRS PLLALVALAL GLITLAAAS AAMAATFAHL LLEAPPEYRE AMLYVFGIAV LIVGLLLLGL VWLLEEALEA LLEEEKRREE EEKRRELVKR A EEALQKAQ ...String:
MAELRERLLR AARWILLLGL LVLVGFVVLA YLERSPLIRA FVLSAGVVLV ALFAAALAWL YLAAALLGRS PLLALVALAL GLITLAAAS AAMAATFAHL LLEAPPEYRE AMLYVFGIAV LIVGLLLLGL VWLLEEALEA LLEEEKRREE EEKRRELVKR A EEALQKAQ EAEKQGDVEK AVKAAQEAVR AAKESGDNDV LRRVAEQALQ IAKEAEKQGN VEVAVKAARV AVEAAKQAGD ND VLRKVAE QALRIAKEAE KQGNVEVAVK AARVAVEAAK QAGDQDVLRK VSEQAERISK EAKKQGNSEV SEEARKVADE AKK QTGGSG GSHHHHHH

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTRIStris(hydroxymethyl)aminomethane
150.0 mMNaClsodium chloride
0.006 %GDNglyco-diosgenin
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: GRAPHENE / Support film - #0 - topology: CONTINUOUS / Support film - #0 - Film thickness: 2 / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY / Support film - #1 - Film thickness: 40 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 16 sec. / Pretreatment - Pressure: 39.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
Details20 mM Tris/HCl pH 8, 150 mM NaCl, 0.006% GDN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6893 / Average exposure time: 4.0 sec. / Average electron dose: 47.32 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: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 886110
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: Ab Initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Non Uniform Refinement / Number images used: 67181
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 8 / Avg.num./class: 10470 / Details: Heterogeneous Refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: Design protein, see methods
DetailsThe design model was used as an initial reference for building the final cryo-EM structure. For the hexamer the design model of the CalC6_3 was used as an initial reference model fit with UCSF Chimera. Hexamer was refined using several rounds of relaxation and minimization, performed on the complete structures, which were manually inspected for errors each time using ISOLDE in UCSF ChimeraX, Coot, and PHENIX real-space refinement. The final model quality was analyzed using MolProbity.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9dzw:
De novo calcium channel hexamer, CalC6_3 with DHR extensions

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