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- PDB-9g0x: auxin transporter PIN8 as asymmetric dimer (inward/outward) with ... -

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Basic information

Entry
Database: PDB / ID: 9g0x
Titleauxin transporter PIN8 as asymmetric dimer (inward/outward) with 4-CPA bound in the inward vestibule prebinding state
ComponentsAuxin efflux carrier component 8
KeywordsMEMBRANE PROTEIN / Auxin transport / AEC family / BART superfamily / TRANSPORT PROTEIN
Function / homology
Function and homology information


auxin export across the plasma membrane / auxin efflux transmembrane transporter activity / pollen development / auxin-activated signaling pathway / cell periphery / endoplasmic reticulum membrane / endoplasmic reticulum / protein homodimerization activity / identical protein binding / plasma membrane
Similarity search - Function
Auxin efflux carrier, plant type / : / Membrane transport protein / Membrane transport protein
Similarity search - Domain/homology
: / 1,2-DILINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Auxin efflux carrier component 8
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsUng, K.L. / Stokes, D.L. / Pedersen, B.P.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)101000936European Union
Citation
Journal: bioRxiv / Year: 2024
Title: Transport of herbicides by PIN-FORMED auxin transporters.
Authors: Lukas Schulz / Kien Lam Ung / Sarah Koutnik-Abele / David L Stokes / Bjørn Panyella Pedersen / Ulrich Z Hammes /
Abstract: Auxins are a group of phytohormones that control plant growth and development . Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as ...Auxins are a group of phytohormones that control plant growth and development . Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides . Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate . Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane and we characterize the molecular determinants of this process using a variety of different substrates as well as protein mutagenesis to control substrate specificity. Finally, we present Cryo-EM structures of PIN8 with 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-chlorophenoxyacetic acid (4-CPA) bound. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with substrate binding and transport across the membrane. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results enable development of novel synthetic auxins and for guiding precision breeding of herbicide resistant crop plants.
#1: Journal: Nat.Plants / Year: 2025
Title: Transport of phenoxyacetic acid herbicides by PIN-FORMED auxin transporters
Authors: Schulz, L. / Ung, K.L. / Zuzic, L. / Koutnik-Abele, S. / Stokes, D.L. / Pedersen, B.P. / Hammes, U.Z.
History
DepositionJul 9, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Auxin efflux carrier component 8
B: Auxin efflux carrier component 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,8335
Polymers83,0822
Non-polymers1,7513
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6560 Å2
ΔGint-62 kcal/mol
Surface area28110 Å2

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Components

#1: Protein Auxin efflux carrier component 8 / AtPIN8


Mass: 41541.039 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: PIN8, PIN5, At5g15100, F2G14_220 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9LFP6
#2: Chemical ChemComp-A1IHP / 2-(4-chloranylphenoxy)ethanoic acid


Mass: 186.592 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H7ClO3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-DLP / 1,2-DILINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DI-LINOLEOYL-3-SN-PHOSPHATIDYLCHOLINE


Mass: 782.082 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H80NO8P / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
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

ComponentName: auxin transporter PIN8 as asymmetric dimer (inward/outward) with 4-CPA bound in the inward vestibule prebinding state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.081 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2100 mMSodium chlorideNaCl1
30.5 mMEDTAC10H16N2O81
40.006 %LMNGC47H88O221
50.0006 %Cholesteryl hemisuccinateC31H50O41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was glow-discharge at 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Wait 4 seconds after sample loading, Blotting time 4 seconds with blotting force of -1 before plunging

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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: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 58.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14665
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.19.2_4158:model refinement
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionDetails: CTF amplitude correction was performed following motion correction using cryosparc-live
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8093919
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115380 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 94.4 / Space: REAL
Atomic model buildingPDB-ID: 7QP9

7qp9


Accession code: 7QP9 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025237
ELECTRON MICROSCOPYf_angle_d0.57094
ELECTRON MICROSCOPYf_dihedral_angle_d9.781743
ELECTRON MICROSCOPYf_chiral_restr0.039854
ELECTRON MICROSCOPYf_plane_restr0.003843

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