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- EMDB-12466: Rabbit HCN4 stabilised in amphipol A8-35 -

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Basic information

Entry
Database: EMDB / ID: EMD-12466
TitleRabbit HCN4 stabilised in amphipol A8-35
Map data
Sample
  • Complex: Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35
    • Protein or peptide: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4
Function / homology
Function and homology information


intracellularly cAMP-activated cation channel activity / sodium channel activity / monoatomic ion channel complex / voltage-gated potassium channel activity / cAMP binding / cellular response to cAMP / potassium ion transmembrane transport / regulation of heart rate / 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 / Cyclic nucleotide-binding domain superfamily ...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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSwuec P
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, 2021-
Header (metadata) releaseJun 30, 2021-
Map releaseJun 30, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nmn
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12466.png

Downloads & links

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Map

FileDownload / File: emd_12466.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.889 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.1351381 - 0.20094103
Average (Standard dev.)1.5620984e-05 (±0.0068359766)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 263.144 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8890.8890.889
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z263.144263.144263.144
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-0.1350.2010.000

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Supplemental data

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Additional map: #1

Fileemd_12466_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35

EntireName: Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35
Components
  • Complex: Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35
    • Protein or peptide: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4

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Supramolecule #1: Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35

SupramoleculeName: Rabbit HCN4 cAMP-unbound stabilised in amphipol A8-35 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK-293

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Macromolecule #1: Potassium/sodium hyperpolarization-activated cyclic nucleotide-ga...

MacromoleculeName: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Rabbit HCN4
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 98.208383 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDKLPPSMRK RLYSLPQQVG AKAWIMDEEE DAEEEGAGGR QDPRRRSIRL RPLPSPSPSP SAAAAAAGGA ESRGAALGGA ADGEGPARG AAKSSTNGDC RRFRGSLASL GSRGGGGGGG STGGGSHGHL HDSAEERRLI AEGDASPGED RTPPGLAAEP E RPGAPAPP ...String:
MDKLPPSMRK RLYSLPQQVG AKAWIMDEEE DAEEEGAGGR QDPRRRSIRL RPLPSPSPSP SAAAAAAGGA ESRGAALGGA ADGEGPARG AAKSSTNGDC RRFRGSLASL GSRGGGGGGG STGGGSHGHL HDSAEERRLI AEGDASPGED RTPPGLAAEP E RPGAPAPP AASPPQVPSS CGEQRPADAA VKVEGGAAAG DQILPEAEAR LGQAGFMQRQ FGAMLQPGVN KFSLRMFGSQ KA VEREQER VKSAGFWIIH PYSDFRFYWD LTMLLLMVGN LIIIPVGITF FKDENTTPWI VFNVVSDTFF LIDLVLNFRT GIV VEDNTD IILDPRRIKM KYLKSWFVVD FVSSIPVDYI FLIVETRIDS EVYKTARALR IVRFTKILSL LRLLRLSRLI RYIH QWEEI FHMTYDLASA VVRIVNLIGM MLLLCHWDGC LQFLVPMLQD FPDDCWVSLN NMVNNSWGKQ YSYALFKAMS HMLCI GYGR QAPMGMSDVW LTMLSMIVGA TCYAMFIGHA TALIQSLDSS RRQYQEKYKQ VEQYMSFHKL PPDTRQRIHD YYEHRY QGK MFDEESILGE LSEPLREEII NFNCRKLVAS MPLFANADPN FVTSMLTKLR FEVFQPGDYI IREGTIGKKM YFIQHGV VS VLTKGNKETK LADGSYFGEI CLLTRGRRTA SVRADTYCRL YSLSVDNFNE VLEEYPMMRR AFETVALDRL DRIGKKNS I HKVQHDLSSG VSNYQENAIV QRIVQHDREM AHCARRAQAT TPVAPAIWTP LIQAPLQAAA QDLKLISASQ PALPQDGAQ TLRRASPHSS SGESVAALPP AGGPFPRAPG RPPGAGPGQH VTLTLPRKAS SGSLPPPLSL FGPRAAPRLT AAPQREPGAK SEPVRSKLP

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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

Concentration0.3 mg/mL
BufferpH: 7
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1571 / Average exposure time: 62.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 100130
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 11406
FSC plot (resolution estimation)

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