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- EMDB-43522: SthK R120A R124A in the presence of PIP2 and cAMP -

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Basic information

Entry
Database: EMDB / ID: EMD-43522
TitleSthK R120A R124A in the presence of PIP2 and cAMP
Map dataunsharpened map after 3D-refinement
Sample
  • Complex: tetrameric SthK R120A R124A protein
    • Protein or peptide: Transcriptional regulator, Crp/Fnr family
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
Keywordscyclic-nucleotide binding / potassium channel / lipid modulation / TRANSPORT PROTEIN
Function / homology
Function and homology information


monoatomic ion transmembrane transport / 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 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsSchmidpeter PAM / Thon O / Nimigean CM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124451 United States
CitationJournal: Nat Commun / Year: 2024
Title: PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK.
Authors: Oliver Thon / Zhihan Wang / Philipp A M Schmidpeter / Crina M Nimigean /
Abstract: The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the ...The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.
History
DepositionJan 26, 2024-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43522.png

Downloads & links

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Map

FileDownload / File: emd_43522.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map after 3D-refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 330 pix.
= 297. Å		0.9 Å/pix.
x 330 pix.
= 297. Å		0.9 Å/pix.
x 330 pix.
= 297. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00755
Minimum - Maximum-0.023772309 - 0.058377955
Average (Standard dev.)-0.0000021994458 (±0.0011503081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 297.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half-map 1

Fileemd_43522_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: half-map 2

Fileemd_43522_half_map_2.map
Annotationhalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : tetrameric SthK R120A R124A protein

EntireName: tetrameric SthK R120A R124A protein
Components
  • Complex: tetrameric SthK R120A R124A protein
    • Protein or peptide: Transcriptional regulator, Crp/Fnr family
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: tetrameric SthK R120A R124A protein

SupramoleculeName: tetrameric SthK R120A R124A protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: SthK R120A R124A in complex with cAMP reconstituted into MSP1E3 nanodiscs containing PIP2
Source (natural)Organism: Spirochaeta thermophila (bacteria)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Transcriptional regulator, Crp/Fnr family

MacromoleculeName: Transcriptional regulator, Crp/Fnr family / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Spirochaeta thermophila (bacteria)
Molecular weightTheoretical: 50.588918 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKDIGINSD PNSSSVMKSS GVSNPTYTLV WKVWILAVTL YYAIRIPLTL VFPSLFSPLL PLDILASLAL IADIPLDLAF ESRRTSGRK PTLLAPSRLP DLLAALPLDL LVFALHLPSP LSLLSLVRLL KLISVQASAT AILSYRINPA LLRLLSLVGF I LLAAHGIA ...String:
MAKDIGINSD PNSSSVMKSS GVSNPTYTLV WKVWILAVTL YYAIRIPLTL VFPSLFSPLL PLDILASLAL IADIPLDLAF ESRRTSGRK PTLLAPSRLP DLLAALPLDL LVFALHLPSP LSLLSLVRLL KLISVQASAT AILSYRINPA LLRLLSLVGF I LLAAHGIA CGWMSLQPPS ENPAGTRYLS AFYWTITTLT TIGYGDITPS TPTQTVYTIV IELLGAAMYG LVIGNIASLV SK LDAAKLL HRERVERVTA FLSYKRISPE LQRRIIEYFD YLWETRRGYE EREVLKELPH PLRLAVAMEI HGDVIEKVPL FKG AGEEFI RDIILHLEPV IYGPGEYIIR AGEMGSDVYF INRGSVEVLS ADEKTRYAIL SEGQFFGEMA LILRAPRTAT VRAR AFCDL YRLDKETFDR ILSRYPEIAA QIQELAVRRK ELESSGLVPR GSVKHHHH

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: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 24 / Formula: PCW
Molecular weightTheoretical: 787.121 Da
Chemical component information

ChemComp-PCW:
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DOPC, phospholipid*YM

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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:
ConcentrationName
10.0 mMHepes
100.0 mMKCl
3.0 mMcAMP
3.0 mMf-Foscholine 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 30 s incubation 2 s blotting blot force -4.
Detailsnanodisc reconstitution was carried out in the presence of DOPC, POPG, and PIP2

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 56.64 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1799891
Startup modelType of model: OTHER / Details: ab-initio model from dataset
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 431535
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6cjq


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8vtb:
SthK R120A R124A in the presence of PIP2 and cAMP

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