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- PDB-9e0h: De novo calcium channel heptamer, CalC6_3 with DHR extensions. Of... -

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Basic information

Entry
Database: PDB / ID: 9e0h
TitleDe novo calcium channel heptamer, CalC6_3 with DHR extensions. Off target multimerization state
ComponentsCalC6_3 with DHR extension
KeywordsDE NOVO PROTEIN / Calcium Channel / CalC6_3 / De novo / Membrane protein / CalC6_3 with DHR extensions / heptamer / Ca
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.62 Å
AuthorsWeidle, C. / Liu, Y. / Borst, A.J.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CalC6_3 with DHR extension
B: CalC6_3 with DHR extension
C: CalC6_3 with DHR extension
D: CalC6_3 with DHR extension
E: CalC6_3 with DHR extension
F: CalC6_3 with DHR extension
G: CalC6_3 with DHR extension


Theoretical massNumber of molelcules
Total (without water)253,1477
Polymers253,1477
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CalC6_3 with DHR extension


Mass: 36163.828 Da / Num. of mol.: 7
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: CalC6_3 with DHR extensions, hepatmer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.25276902 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.006% GDN
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane / Hydrogen ChlorideTris/HCl1
2150 mMSodium ChlorideNaCl1
30.006 %Glyco-DiosgeninGDN1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: membrane protein in purified detergent micelle
Specimen supportDetails: 5 mA current / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4 sec. / Electron dose: 47.32 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6893
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13UCSF ChimeraXmodel refinement
14ISOLDEmodel refinement
15Cootmodel refinement
16PHENIXmodel refinement
17PyMOLmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 886110
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 4.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45077 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: For 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 ...Details: For 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.
Atomic model buildingDetails: Design protein, see paper / Source name: Other / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0016118
ELECTRON MICROSCOPYf_angle_d0.3558442
ELECTRON MICROSCOPYf_dihedral_angle_d2.8881099
ELECTRON MICROSCOPYf_chiral_restr0.0281092
ELECTRON MICROSCOPYf_plane_restr0.0021162

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