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- PDB-7n5e: Structure of Mechanosensitive Ion Channel Flycatcher1 in GDN -

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Basic information

Entry
Database: PDB / ID: 7n5e
TitleStructure of Mechanosensitive Ion Channel Flycatcher1 in GDN
ComponentsMechanosensitive ion channel Flycatcher1
KeywordsMEMBRANE PROTEIN / mechanically activated ion channel
Function / homologyPALMITIC ACID
Function and homology information
Biological speciesDionaea muscipula (Venus flytrap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJojoa-Cruz, S. / Saotome, K. / Lee, W.H. / Patapoutian, A. / Ward, A.B.
Funding support United States, United Kingdom, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL143297 United States
Wellcome Trust208361/Z/17/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N000145/1 United Kingdom
Engineering and Physical Sciences Research CouncilEP/R004722/1 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Structural insights into the Venus flytrap mechanosensitive ion channel Flycatcher1.
Authors: Sebastian Jojoa-Cruz / Kei Saotome / Che Chun Alex Tsui / Wen-Hsin Lee / Mark S P Sansom / Swetha E Murthy / Ardem Patapoutian / Andrew B Ward /
Abstract: Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is a larger protein and its ...Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is a larger protein and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterize FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion preference and conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt 'up' or 'down' conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1.
History
DepositionJun 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Mechanosensitive ion channel Flycatcher1
B: Mechanosensitive ion channel Flycatcher1
C: Mechanosensitive ion channel Flycatcher1
D: Mechanosensitive ion channel Flycatcher1
E: Mechanosensitive ion channel Flycatcher1
F: Mechanosensitive ion channel Flycatcher1
G: Mechanosensitive ion channel Flycatcher1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)609,49714
Polymers607,7027
Non-polymers1,7957
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37060 Å2
ΔGint-231 kcal/mol
Surface area110750 Å2

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Components

#1: Protein
Mechanosensitive ion channel Flycatcher1


Mass: 86814.523 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dionaea muscipula (Venus flytrap) / Tissue: Trigger hair / Organ: Trap / Plasmid: pEG / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Chemical
ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C16H32O2
Has ligand of interestN
Sequence detailsThe last 10 residues (GSGSLEVLFQ) are leftover of a linker and protease site.

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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: FLYC1 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.607 MDa / Experimental value: NO
Source (natural)Organism: Dionaea muscipula (Venus flytrap) / Organ: Trap / Tissue: Trigger hair
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pEG
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
2150 mMNaClSodium chloride1
30.0004 mg/mLleupeptin1
40.0004 mg/mLaprotinin1
50.0004 mMpepstatin1
60.2 mMphenylmethylsulfonyl fluoridePMSF1
70.4 mMdiothiothreitol1
80.04 %GDN1
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 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 FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 43

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2particle selection
2Leginonimage acquisition
4Gctf1.06CTF correctionFor CryoSPARC
5CTFFIND4CTF correctionFor RELION
8Coot0.9model fitting
10cryoSPARC2initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14Coot0.9model refinement
15PHENIX1.18.2-3874model refinement
16Rosetta3.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1100000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 255359 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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