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- PDB-5v4s: CryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel -

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Basic information

Entry
Database: PDB / ID: 5v4s
TitleCryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel
ComponentsTransporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
KeywordsTRANSPORT PROTEIN / Ion channel / cyclic nucleotide / allostery / vision / olfaction
Function / homology
Function and homology information


HCN channel complex / regulation of membrane depolarization / voltage-gated potassium channel activity / sodium ion transmembrane transport
Similarity search - Function
: / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold ...: / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
Similarity search - Component
Biological speciesLeptospira licerasiae serovar Varillal str. VAR 010 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsJames, Z.M. / Borst, A.J. / Haitin, Y. / Frenz, B. / DiMaio, F. / Zagotta, W.N. / Veesler, D.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R01EY010329 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01MH102378 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008268 United States
American Heart Association14CSA20380095 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel.
Authors: Zachary M James / Andrew J Borst / Yoni Haitin / Brandon Frenz / Frank DiMaio / William N Zagotta / David Veesler /
Abstract: Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. ...Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from -which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.
History
DepositionMar 10, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 12, 2017Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2017Group: Database references
Revision 1.2May 3, 2017Group: Other
Revision 1.3May 10, 2017Group: Database references
Revision 1.4Sep 27, 2017Group: Author supporting evidence / Data collection / Experimental preparation
Category: em_image_scans / em_sample_support ...em_image_scans / em_sample_support / em_software / pdbx_audit_support
Item: _em_sample_support.grid_type / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.5Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
B: Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
C: Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
D: Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein


Theoretical massNumber of molelcules
Total (without water)213,8594
Polymers213,8594
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein


Mass: 53464.785 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Leptospira licerasiae serovar Varillal str. VAR 010 (bacteria)
Gene: LEP1GSC185_1946 / Production host: Escherichia coli (E. coli) / References: UniProt: I0XVQ9

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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: LliK / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Leptospira licerasiae serovar Varillal str. VAR 010 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMPotassium ChlorideKCl1
30.05 mMLMNG1
40.005 mMCholesterol Hemi-Succinate1
50.02 mMcAMP1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 1.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1FindEMparticle selection
2Leginon3.2image acquisition
4GctfCTF correction
7Rosettamodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13Rosettamodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 379000
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18737 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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