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

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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/homologyPotassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding, conserved site / voltage-gated potassium channel activity / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding-like / Ion transport domain / Cyclic nucleotide-binding domain / Ion transport protein ...Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding, conserved site / voltage-gated potassium channel activity / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding-like / Ion transport domain / Cyclic nucleotide-binding domain / Ion transport protein / RmlC-like jelly roll fold / integral component of membrane / Transporter, cation channel family / cyclic nucleotide-binding domain multi-domain protein
Function and homology information
Specimen sourceLeptospira licerasiae serovar varillal str. var 010 / bacteria
MethodElectron microscopy (4.2 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsJames, Z.M. / Borst, A.J. / Haitin, Y. / Frenz, B. / DiMaio, F. / Zagotta, W.N. / Veesler, D.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 10, 2017 / Release: Apr 12, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 12, 2017Structure modelrepositoryInitial release
1.1Apr 26, 2017Structure modelDatabase references
1.2May 3, 2017Structure modelOther
1.3May 10, 2017Structure modelDatabase references
1.4Sep 27, 2017Structure modelAuthor supporting evidence / Data collection / Experimental preparationem_image_scans / em_sample_support / em_software / pdbx_audit_support_em_sample_support.grid_type / _em_software.name / _pdbx_audit_support.funding_organization

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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
Polyers213,8594
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 53464.785 Da / Num. of mol.: 4
Source: (gene. exp.) Leptospira licerasiae serovar varillal str. var 010 / bacteria
Gene: LEP1GSC185_1946 / Production host: Escherichia coli / References: UniProt:I0XVQ9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: LliK / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.2 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Leptospira licerasiae serovar Varillal str. VAR 010
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 7.9
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mMTris1
2150mMPotassium ChlorideKCl1
30.05mMLMNG1
40.005mMCholesterol Hemi-Succinate1
50.02mMcAMP1
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 / 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
MicroscopyMicroscope model: 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
9RELION2.0INITIAL EULER ASSIGNMENT
10RELION2.0FINAL EULER ASSIGNMENT
11RELION2.0CLASSIFICATION
12RELION2.0RECONSTRUCTION
13RosettaMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 379000
SymmetryPoint symmetry: C4
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 18737 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL

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