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- EMDB-21453: SthK P300A cyclic nucleotide-gated potassium channel in the close... -

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Basic information

Entry
Database: EMDB / ID: EMD-21453
TitleSthK P300A cyclic nucleotide-gated potassium channel in the closed state, in complex with cAMP
Map datafinal map after 3D refinement
Sample
  • Complex: SthK P300A bound to cAMP
    • Protein or peptide: SthK
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


intracellularly cyclic nucleotide-activated monoatomic cation channel activity / protein-containing complex binding / membrane
Similarity search - Function
: / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Ion channel / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain ...: / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Ion channel / 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
Similarity search - Domain/homology
Transcriptional regulator, Crp/Fnr family
Similarity search - Component
Biological speciesSpirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsSchmidpeter PAM / Rheinberger J / Nimigean CM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124451 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088352 United States
American Heart Association18POST33960309 United States
CitationJournal: Nat Commun / Year: 2020
Title: Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel.
Authors: Philipp A M Schmidpeter / Jan Rheinberger / Crina M Nimigean /
Abstract: SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the ...SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.
History
DepositionFeb 25, 2020-
Header (metadata) releaseApr 1, 2020-
Map releaseNov 11, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vxz
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21453.png

Downloads & links

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Map

FileDownload / File: emd_21453.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map after 3D refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 256 pix.
= 280.602 Å		1.1 Å/pix.
x 256 pix.
= 280.602 Å		1.1 Å/pix.
x 256 pix.
= 280.602 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0961 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.025
Minimum - Maximum-0.14475423 - 0.24986358
Average (Standard dev.)0.00000087247923 (±0.0058345357)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 280.6016 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.09610156251.09610156251.0961015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z280.602280.602280.602
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1450.2500.000

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Supplemental data

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Mask #1

Fileemd_21453_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: final map after 3D refinement

Fileemd_21453_additional_1.map
Annotationfinal map after 3D refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_21453_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: final map after 3D refinement

Fileemd_21453_half_map_2.map
Annotationfinal map after 3D refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SthK P300A bound to cAMP

EntireName: SthK P300A bound to cAMP
Components
  • Complex: SthK P300A bound to cAMP
    • Protein or peptide: SthK
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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Supramolecule #1: SthK P300A bound to cAMP

SupramoleculeName: SthK P300A bound to cAMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: SthK P300A was reconstituted into nanodiscs.
Source (natural)Organism: Spirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)

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Macromolecule #1: SthK

MacromoleculeName: SthK / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Spirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)
Strain: ATCC 700085 / DSM 6578 / Z-1203
Molecular weightTheoretical: 51.092539 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKDIGINSD PNSSSVDKLM KSSGVSNPTY TLVWKVWILA VTLYYAIRIP LTLVFPSLFS PLLPLDILAS LALIADIPLD LAFESRRTS GRKPTLLAPS RLPDLLAALP LDLLVFALHL PSPLSLLSLV RLLKLISVQR SATRILSYRI NPALLRLLSL V GFILLAAH ...String:
MAKDIGINSD PNSSSVDKLM KSSGVSNPTY TLVWKVWILA VTLYYAIRIP LTLVFPSLFS PLLPLDILAS LALIADIPLD LAFESRRTS GRKPTLLAPS RLPDLLAALP LDLLVFALHL PSPLSLLSLV RLLKLISVQR SATRILSYRI NPALLRLLSL V GFILLAAH GIACGWMSLQ PPSENPAGTR YLSAFYWTIT TLTTIGYGDI TPSTPTQTVY TIVIELLGAA MYGLVIGNIA SL VSKLDAA KLLHRERVER VTAFLSYKRI SPELQRRIIE YFDYLWETRR GYEEREVLKE LPHPLRLAVA MEIHGDVIEK VAL FKGAGE EFIRDIILHL EPVIYGPGEY IIRAGEMGSD VYFINRGSVE VLSADEKTRY AILSEGQFFG EMALILRAPR TATV RARAF CDLYRLDKET FDRILSRYPE IAAQIQELAV RRKELESSGL VPRGSVKHHH H

UniProtKB: Transcriptional regulator, Crp/Fnr family

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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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Macromolecule #3: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

MacromoleculeName: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
type: ligand / ID: 3 / Number of copies: 24 / Formula: PGW
Molecular weightTheoretical: 749.007 Da

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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

Concentration7.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
10.0 mMHEPES
100.0 mMpotassium chlorideKCl
3.0 mMcAMP
3.0 mMFos-Choline-8, Fluorinated
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2494 / Average exposure time: 10.0 sec. / Average electron dose: 71.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 45620 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 657943
Startup modelType of model: EMDB MAP
EMDB ID:

7484

Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta) / Number images used: 61275
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0-beta)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0-beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6vxz:
SthK P300A cyclic nucleotide-gated potassium channel in the closed state, in complex with cAMP

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