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- EMDB-21454: SthK P300A cyclic nucleotide-gated potassium channel in a putativ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21454
TitleSthK P300A cyclic nucleotide-gated potassium channel in a putative active state, in complex with cAMP
Map datamap after refinement
Sample
  • Complex: SthK P300A bound to cAMP
    • Protein or peptide: SthK
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


monoatomic ion transmembrane transport / 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 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) / Spirochaeta thermophila DSM 6578 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.68 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: 0.0166
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vy0
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21454.png

Downloads & links

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Map

FileDownload / File: emd_21454.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap after refinement
Voxel sizeX=Y=Z: 1.0961 Å
Density
Contour LevelBy AUTHOR: 0.0166 / Movie #1: 0.0166
Minimum - Maximum-0.003229137 - 0.034275237
Average (Standard dev.)0.0003746998 (±0.0022572991)
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-0.0030.0340.000

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Supplemental data

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Mask #1

Fileemd_21454_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: map after postprocessing

Fileemd_21454_additional_1.map
Annotationmap after postprocessing
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered half map 2 from the refinement

Fileemd_21454_half_map_1.map
Annotationunfiltered half map 2 from the refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered half map 1 from the refinement

Fileemd_21454_half_map_2.map
Annotationunfiltered half map 1 from the 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

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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: all
Details: Low-populated state of SthK P300A reconstituted into nanodiscs. Due to the low resolution, side chains could not be assigned and all amino acids are modeled as alanine. The register is ...Details: Low-populated state of SthK P300A reconstituted into nanodiscs. Due to the low resolution, side chains could not be assigned and all amino acids are modeled as alanine. The register is maintained from the high-resolution structure of the closed state.
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 DSM 6578 (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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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 45620 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
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

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0-beta)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0-beta)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta) / Number images used: 19918
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6vy0:
SthK P300A cyclic nucleotide-gated potassium channel in a putative active state, in complex with cAMP

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