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- EMDB-49883: Apo WT SthK in 3:1 DOPC:POPE nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-49883
TitleApo WT SthK in 3:1 DOPC:POPE nanodiscs
Map dataunsharpened full map
Sample
  • Complex: tetrameric SthK
    • Protein or peptide: Putative transcriptional regulator, Crp/Fnr family
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate
Keywordstrans-membrane protein ion channel cyclic nucleotide-gated channel / MEMBRANE 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 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsSchmidpeter PA / Newton AJ
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Protein Sci / Year: 2025
Title: Membrane-forming phospholipids allosterically modulate native-state prolyl isomerization in a CNG channel.
Authors: Ashley J Newton / Robert D Latvala / Adefoluke E Kuforiji / Philipp A M Schmidpeter /
Abstract: Ion channel activity is intricately linked to the surrounding lipid environment, yet the molecular effects of lipid-mediated regulation remain largely understudied. Here, we show that membrane- ...Ion channel activity is intricately linked to the surrounding lipid environment, yet the molecular effects of lipid-mediated regulation remain largely understudied. Here, we show that membrane-forming phospholipids, which are known to modulate the activity of the cyclic nucleotide-gated channel SthK from Spirochaeta thermophila, exhibit effects that extend well beyond the membrane boundary. Using stopped-flow flux assays, we demonstrate that anionic lipids, which are known to promote channel opening, also affect the fast-to-slow activation ratio and the cAMP potency in SthK. Enzymatic catalysis studies confirm that this occurs by altering the cis/trans equilibrium at Pro300 in the apo state. Additionally, cryogenic electron microscopy structures of SthK reveal lipid-dependent conformational changes that propagate from the bundle crossing into the cytosolic domains. All observed effects correlate with the electronegativity of the lipid headgroup, indicating a common underlying mechanism. Our results highlight membrane-forming phospholipids as allosteric regulators of SthK, controlling multiple functional characteristics of the channel.
History
DepositionMar 23, 2025-
Header (metadata) releaseNov 12, 2025-
Map releaseNov 12, 2025-
UpdateDec 3, 2025-
Current statusDec 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49883.png

Downloads & links

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Map

FileDownload / File: emd_49883.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened full map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.8 Å/pix.
x 330 pix.
= 264.191 Å		0.8 Å/pix.
x 330 pix.
= 264.191 Å		0.8 Å/pix.
x 330 pix.
= 264.191 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.80058 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00471
Minimum - Maximum-0.011176955 - 0.029612368
Average (Standard dev.)0.000010238757 (±0.000819843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 264.1914 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_49883_msk_1.map
Projections & Slices
AxesZYX

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Half map: unfiltered half map 2

Fileemd_49883_half_map_1.map
Annotationunfiltered half map 2
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: unfiltered half map 1

Fileemd_49883_half_map_2.map
Annotationunfiltered half map 1
Projections & Slices
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Sample components

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Entire : tetrameric SthK

EntireName: tetrameric SthK
Components
  • Complex: tetrameric SthK
    • Protein or peptide: Putative transcriptional regulator, Crp/Fnr family
  • Ligand: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate

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Supramolecule #1: tetrameric SthK

SupramoleculeName: tetrameric SthK / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: tetrameric SthK protein reconstituted into MSP1E3 nanodiscs composed of 3:1 DOPC:POPE
Source (natural)Organism: Spirochaeta thermophila (bacteria) / Strain: DSM 6578
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Putative transcriptional regulator, Crp/Fnr family

MacromoleculeName: Putative transcriptional regulator, Crp/Fnr family / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Spirochaeta thermophila (bacteria) / Strain: DSM 6578
Molecular weightTheoretical: 51.118574 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 VPL 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: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyl...

MacromoleculeName: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate
type: ligand / ID: 2 / Number of copies: 20 / Formula: 6OU
Molecular weightTheoretical: 717.996 Da
Chemical component information

ChemComp-6OU:
[(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate / 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:
ConcentrationNameFormula
10.0 mMHepes
100.0 mMPotassium chlorideKCl
3.0 mMfluorinated Fos-choline 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK IV
Detailsapo SthK in 3:1 DOPC:POPE nanodiscs

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 9686 / Average exposure time: 1.2 sec. / Average electron dose: 52.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1798810
CTF correctionSoftware - Name: CTFFIND / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Details: initial model generated in Relion from 2D-sorted particles
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 224867
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
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: RIGID BODY FIT
Output model

PDB-9nwj:
Apo WT SthK in 3:1 DOPC:POPE nanodiscs

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