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- PDB-8zl6: Cryo-EM structure of the Drosophila INDY (apo inward-open, pH 8) -

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Basic information

Entry
Database: PDB / ID: 8zl6
TitleCryo-EM structure of the Drosophila INDY (apo inward-open, pH 8)
ComponentsProtein I'm not dead yet
KeywordsMEMBRANE PROTEIN / INDY / SLC13A5 / citrate transporter
Function / homology
Function and homology information


regulation of triglyceride storage / Sodium-coupled sulphate, di- and tri-carboxylate transporters / pyruvate transport / succinate transport / citrate transmembrane transporter activity / citrate transport / pyruvate transmembrane transporter activity / succinate transmembrane transporter activity / determination of adult lifespan / transmembrane transport ...regulation of triglyceride storage / Sodium-coupled sulphate, di- and tri-carboxylate transporters / pyruvate transport / succinate transport / citrate transmembrane transporter activity / citrate transport / pyruvate transmembrane transporter activity / succinate transmembrane transporter activity / determination of adult lifespan / transmembrane transport / basolateral plasma membrane / plasma membrane
Similarity search - Function
Sodium/sulphate symporter, conserved site / Sodium:sulfate symporter family signature. / Sodium:sulfate symporter transmembrane region / Solute carrier family 13
Similarity search - Domain/homology
PHOSPHATIDYLETHANOLAMINE / Protein I'm not dead yet
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKim, S. / Park, J.G. / Choi, S.H. / Kim, J.W. / Jin, M.S.
Funding support Korea, Republic Of, 4items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2019M3E5D6063908 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF-2021M3A9I4022846 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF-2022R1A2C1091278 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF-2021R1A6A3A01086747 Korea, Republic Of
CitationJournal: Life Sci Alliance / Year: 2025
Title: Cryo-EM structures reveal the H/citrate symport mechanism of INDY.
Authors: Subin Kim / Jun Gyou Park / Seung Hun Choi / Ji Won Kim / Mi Sun Jin /
Abstract: I'm Not Dead Yet (INDY) functions as a transporter for citrate, a key metabolite in the citric acid cycle, across the plasma membrane. Partial deficiency of INDY extends lifespan, akin to the ... I'm Not Dead Yet (INDY) functions as a transporter for citrate, a key metabolite in the citric acid cycle, across the plasma membrane. Partial deficiency of INDY extends lifespan, akin to the effects of caloric restriction. In this work, we use cryo-electron microscopy to determine structures of INDY in the presence and absence of citrate and in complex with the well-known inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) at resolutions ranging from 2.7 to 3.6 Å. Together with functional data obtained in vitro, the INDY structures reveal the H/citrate co-transport mechanism, in which aromatic residue F119 serves as a one-gate element. They also provide insight into how protein-lipid interactions at the dimerization interface affect the stability and function of the transporter, and how DIDS disrupts the transport cycle.
History
DepositionMay 17, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein I'm not dead yet
B: Protein I'm not dead yet
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,1856
Polymers131,2742
Non-polymers1,9104
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Protein I'm not dead yet / INDY transporter protein / drIndy


Mass: 65637.023 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Indy, CG3979 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9VVT2
#2: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1citrate transporter INDY in nanodiscsCOMPLEX#10RECOMBINANT
2INDYCOMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
210.13 MDaYES
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Drosophila melanogaster (fruit fly)7227
32Drosophila melanogaster (fruit fly)7227
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
32Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 8
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 281446 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058730
ELECTRON MICROSCOPYf_angle_d0.60811862
ELECTRON MICROSCOPYf_dihedral_angle_d16.9181242
ELECTRON MICROSCOPYf_chiral_restr0.0431386
ELECTRON MICROSCOPYf_plane_restr0.0051442

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