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- PDB-7tj5: SthK closed state, cAMP-bound in the presence of POPA -

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Basic information

Entry
Database: PDB / ID: 7tj5
TitleSthK closed state, cAMP-bound in the presence of POPA
ComponentsPutative transcriptional regulator, Crp/Fnr family
KeywordsTRANSPORT PROTEIN / cyclic nucleotide-gated channel / lipid modulation / pacemaker channel
Function / homology
Function and homology information


intracellular cyclic nucleotide activated cation channel complex / intracellularly cGMP-activated cation channel activity / intracellularly cAMP-activated cation channel activity / cGMP binding / protein-containing complex binding / plasma 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
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / Chem-D21 / Transcriptional regulator, Crp/Fnr family
Similarity search - Component
Biological speciesSpirochaeta thermophila DSM 6578 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.41 Å
AuthorsSchmidpeter, P.A. / Nimigean, C.M.
Funding support United States, 3items
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 Struct Mol Biol / Year: 2022
Title: Anionic lipids unlock the gates of select ion channels in the pacemaker family.
Authors: Philipp A M Schmidpeter / Di Wu / Jan Rheinberger / Paul M Riegelhaupt / Haiping Tang / Carol V Robinson / Crina M Nimigean /
Abstract: Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker ...Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.
History
DepositionJan 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative transcriptional regulator, Crp/Fnr family
B: Putative transcriptional regulator, Crp/Fnr family
C: Putative transcriptional regulator, Crp/Fnr family
D: Putative transcriptional regulator, Crp/Fnr family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)224,68936
Polymers204,4744
Non-polymers20,21532
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 51118.574 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spirochaeta thermophila DSM 6578 (bacteria)
Strain: ATCC 700085 / DSM 6578 / Z-1203 / Gene: Spith_0644 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: G0GA88
#2: Chemical
ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.206 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H12N5O6P
#3: Chemical...
ChemComp-D21 / (2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (9Z)-octadec-9-enoate


Mass: 674.929 Da / Num. of mol.: 28 / Source method: obtained synthetically / Formula: C37H71O8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: closed conformation of SthK bound to cAMP / Type: COMPLEX
Details: SthK was reconstituted into nanodiscs containing POPA lipids.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Spirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41(DE3)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2100 mMpotassium chlorideKCl1
33 mMcAMP1
43 mMFos-Choline-8, Fluorinated1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: WT SthK in MSP1E3 nanodiscs composed of 3:1 DOPC:POPA
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 50.812 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5684
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.8.0b41particle selection
2Leginonimage acquisition
4CTFFIND4.1CTF correction
9RELION3.1.3initial Euler assignment
10RELION3.1.3final Euler assignment
12RELION3.1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1561870
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 294383 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6CJQ

6cjq


Accession code: 6CJQ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413420
ELECTRON MICROSCOPYf_angle_d0.80818124
ELECTRON MICROSCOPYf_dihedral_angle_d14.5252176
ELECTRON MICROSCOPYf_chiral_restr0.052100
ELECTRON MICROSCOPYf_plane_restr0.0082204

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