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- PDB-7nmn: Rabbit HCN4 stabilised in amphipol A8-35 -

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Basic information

Entry
Database: PDB / ID: 7nmn
TitleRabbit HCN4 stabilised in amphipol A8-35
ComponentsPotassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4
KeywordsMEMBRANE PROTEIN / cryo-EM / ion channels
Function / homology
Function and homology information


HCN channel complex / intracellularly cAMP-activated cation channel activity / regulation of membrane depolarization / sodium channel activity / AMP binding / voltage-gated potassium channel activity / potassium channel activity / cAMP binding / cellular response to cAMP / potassium ion transmembrane transport ...HCN channel complex / intracellularly cAMP-activated cation channel activity / regulation of membrane depolarization / sodium channel activity / AMP binding / voltage-gated potassium channel activity / potassium channel activity / cAMP binding / cellular response to cAMP / potassium ion transmembrane transport / sodium ion transmembrane transport / regulation of heart rate / axon / dendrite / identical protein binding / plasma membrane
Similarity search - Function
Ion transport N-terminal / Ion transport protein N-terminal / : / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain ...Ion transport N-terminal / Ion transport protein N-terminal / : / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 1. / 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
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsChaves-Sanjuan, A.
CitationJournal: Mol Cell / Year: 2021
Title: Gating movements and ion permeation in HCN4 pacemaker channels.
Authors: Andrea Saponaro / Daniel Bauer / M Hunter Giese / Paolo Swuec / Alessandro Porro / Federica Gasparri / Atiyeh Sadat Sharifzadeh / Antonio Chaves-Sanjuan / Laura Alberio / Giacomo Parisi / ...Authors: Andrea Saponaro / Daniel Bauer / M Hunter Giese / Paolo Swuec / Alessandro Porro / Federica Gasparri / Atiyeh Sadat Sharifzadeh / Antonio Chaves-Sanjuan / Laura Alberio / Giacomo Parisi / Gabriele Cerutti / Oliver B Clarke / Kay Hamacher / Henry M Colecraft / Filippo Mancia / Wayne A Hendrickson / Steven A Siegelbaum / Dario DiFrancesco / Martino Bolognesi / Gerhard Thiel / Bina Santoro / Anna Moroni /
Abstract: The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current I/I that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy ...The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current I/I that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K/Na permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.
History
DepositionFeb 23, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 7, 2021Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 28, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jul 10, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4
B: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4
C: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4
D: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4


Theoretical massNumber of molelcules
Total (without water)392,8344
Polymers392,8344
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy, homology
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C"
d_2ens_1chain "B"
d_3ens_1chain "A"
d_4ens_1chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLNTHRA1 - 469
d_21ens_1GLNTHRB1 - 469
d_31ens_1GLNTHRC1 - 469
d_41ens_1GLNTHRD1 - 469

NCS oper:
IDCodeMatrixVector
1given(0.000128753922697, 0.99999985162, 0.000529322246215), (-0.999999904989, 0.000128533448977, 0.00041653370992), (0.000416465612501, -0.000529375826273, 0.999999773159)-0.0383374029261, 128.002927815, 0.0200202576956
2given(-0.999998179989, -0.00158152127345, -0.0010671500169), (0.00158213435408, -0.999998583742, -0.000573903351046), (-0.00106624086518, -0.000575590681238, 0.999999265913)128.178981249, 127.957750987, 0.107735696322
3given(-0.000928953404324, -0.999999504452, 0.000357967556631), (0.999999568483, -0.000928956545089, -8.60771407964E-6), (8.94024611877E-6, 0.000357959405997, 0.999999935893)128.048936331, 0.032523043619, -0.00834336729944

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Components

#1: Protein
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4 / Hyperpolarization-activated cation channel 4 / HAC-4


Mass: 98208.383 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: HCN4, HAC4 / Production host: Homo sapiens (human) / References: UniProt: Q9TV66

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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: Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK-293
Buffer solutionpH: 7
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 62 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1571

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_3751refinement
PHENIXdev_3751refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPU2image acquisition
4CTFFIND4.1.10CTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 100130
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11406 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.9 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009815812
ELECTRON MICROSCOPYf_angle_d1.154821356
ELECTRON MICROSCOPYf_chiral_restr0.22792328
ELECTRON MICROSCOPYf_plane_restr0.00792684
ELECTRON MICROSCOPYf_dihedral_angle_d20.68295864
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.00070773272005
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.000498358740117
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.00070806156801

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