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- EMDB-47356: De novo calcium channel heptamer, CalC6_3 with DHR extensions. Of... -

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Basic information

Entry
Database: EMDB / ID: EMD-47356
TitleDe novo calcium channel heptamer, CalC6_3 with DHR extensions. Off target multimerization state
Map dataNon uniform refinement in C7, sharpened with DeepEMhancer, Main map for structure building. 4.62 Angstrom
Sample
  • Complex: CalC6_3 with DHR extensions, hepatmer
    • Protein or peptide: CalC6_3 with DHR extension
KeywordsCalcium Channel / CalC6_3 / De novo / Membrane protein / CalC6_3 with DHR extensions / heptamer / De novo protein / Ca
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.62 Å
AuthorsWeidle C / Liu Y / Borst AJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) 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_47356.png

Downloads & links

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Map

FileDownload / File: emd_47356.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNon uniform refinement in C7, sharpened with DeepEMhancer, Main map for structure building. 4.62 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)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.0018723768 - 2.0103915
Average (Standard dev.)-0.00074011995 (±0.027550498)
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 in C7, un-sharpened. 4.62 Angstrom

Fileemd_47356_additional_1.map
AnnotationNon uniform refinement in C7, un-sharpened. 4.62 Angstrom
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Additional map: Non uniform refinement in C7, auto sharpened. 4.62 Angstrom

Fileemd_47356_additional_2.map
AnnotationNon uniform refinement in C7, auto sharpened. 4.62 Angstrom
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Additional map: Non uniform refinement in C1, un-sharpened. 5.13 Angstrom

Fileemd_47356_additional_3.map
AnnotationNon uniform refinement in C1, un-sharpened. 5.13 Angstrom
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Additional map: Non uniform refinement in C1, half map A.

Fileemd_47356_additional_4.map
AnnotationNon uniform refinement in C1, half map A.
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Additional map: Non uniform refinement in C1, sharpened. 5.13 Angstrom

Fileemd_47356_additional_5.map
AnnotationNon uniform refinement in C1, sharpened. 5.13 Angstrom
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Additional map: Non uniform refinement in C1, half map B

Fileemd_47356_additional_6.map
AnnotationNon uniform refinement in C1, half map B
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Half map: Non uniform refinement in C7, half map A.

Fileemd_47356_half_map_1.map
AnnotationNon uniform refinement in C7, half map A.
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Half map: Non uniform refinement in C7, half map B.

Fileemd_47356_half_map_2.map
AnnotationNon uniform refinement in C7, half map B.
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Sample components

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Entire : CalC6_3 with DHR extensions, hepatmer

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

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Supramolecule #1: CalC6_3 with DHR extensions, hepatmer

SupramoleculeName: CalC6_3 with DHR extensions, hepatmer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 252.76902 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: 7 / 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 mMTris/HCltris(hydroxymethyl)aminomethane / Hydrogen Chloride
150.0 mMNaClSodium Chloride
0.006 %GDNGlyco-Diosgenin

Details: 20 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.006% GDN
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 - Atmosphere: AIR / Pretreatment - Pressure: 39.0 kPa / Details: 5 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
Detailsmembrane protein in purified detergent micelle

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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 correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: Ab Initio
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 45077
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Avg.num./class: 10470 / Software - Name: cryoSPARC / Details: Ab Initio, C7
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 paper
DetailsFor the heptamer, CalC6_3 with DHR extension hexamer design model was used as a starting model. A single chain for the heptamer was isolated in PyMOL, and 7 copies of this chain were fitted to the map in UCSF Chimera to reform the channel. The heptamer 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: FLEXIBLE FIT
Output model

PDB-9e0h:
De novo calcium channel heptamer, CalC6_3 with DHR extensions. Off target multimerization state

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