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- EMDB-12513: cAMP-bound rabbit HCN4 stabilized in LMNG-CHS detergent mixture -

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Basic information

Entry
Database: EMDB / ID: EMD-12513
TitlecAMP-bound rabbit HCN4 stabilized in LMNG-CHS detergent mixture
Map datasharped map
Sample
  • Complex: HCN4
    • Protein or peptide: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
KeywordsHCN channels / cAMP / ion transport / MEMBRANE PROTEIN
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 / sodium ion transmembrane transport / cAMP binding / cellular response to cAMP ...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 / sodium ion transmembrane transport / cAMP binding / cellular response to cAMP / potassium 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGiese H / Saponaro A
Funding support United States, Italy, European Union, 6 items
OrganizationGrant numberCountry
Leducq FoundationTNE19CVD03 United States
Fondazione CARIPLO2018-0231 Italy
European Research Council (ERC)ERC Advanced grant noMAGIC 695078European Union
National Institutes of Health/National Center for Research Resources (NIH/NCRR)NIH grants R01-NS109366 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)P41-GM116799 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01-GM107462 via Type a message United States
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 26, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7np4
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12513.png

Downloads & links

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Map

FileDownload / File: emd_12513.map.gz / Format: CCP4 / Size: 76.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharped map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 272 pix.
= 228.48 Å		0.84 Å/pix.
x 272 pix.
= 228.48 Å		0.84 Å/pix.
x 272 pix.
= 228.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.70107806 - 1.4001751
Average (Standard dev.)0.002262026 (±0.04830512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions272272272
Spacing272272272
CellA=B=C: 228.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z272272272
origin x/y/z0.0000.0000.000
length x/y/z228.480228.480228.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS272272272
D min/max/mean-0.7011.4000.002

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Supplemental data

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Mask #1

Fileemd_12513_msk_1.map
Projections & Slices
AxesZYX

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Additional map: unsharped map

Fileemd_12513_additional_1.map
Annotationunsharped map
Projections & Slices
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Half map: half 1

Fileemd_12513_half_map_1.map
Annotationhalf 1
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Half map: half 2

Fileemd_12513_half_map_2.map
Annotationhalf 2
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Sample components

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Entire : HCN4

EntireName: HCN4
Components
  • Complex: HCN4
    • Protein or peptide: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

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Supramolecule #1: HCN4

SupramoleculeName: HCN4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: HCN4
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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

MacromoleculeName: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 98.522727 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 SNL

UniProtKB: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4, Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4

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Macromolecule #2: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

MacromoleculeName: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: CMP
Molecular weightTheoretical: 329.206 Da
Chemical component information

ChemComp-CMP:
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

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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

Concentration0.5 mg/mL
BufferpH: 7 / Component - Concentration: 200.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride
GridModel: Quantifoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 302 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 71.85 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5u6o

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 54828
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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