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- PDB-7qp9: Outward-facing apo-form of auxin transporter PIN8 -

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Basic information

Entry
Database: PDB / ID: 7qp9
TitleOutward-facing apo-form of auxin transporter PIN8
ComponentsAuxin efflux carrier component 8
KeywordsMEMBRANE PROTEIN / Auxin transport / AEC family / BART superfamily
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: 2.89 Å
AuthorsUng, K.L. / Winkler, M.B.L. / Dedic, E. / Stokes, D.L. / Pedersen, B.P.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)101000936European Union
CitationJournal: Nature / Year: 2022
Title: Structures and mechanism of the plant PIN-FORMED auxin transporter.
Authors: Kien Lam Ung / Mikael Winkler / Lukas Schulz / Martina Kolb / Dorina P Janacek / Emil Dedic / David L Stokes / Ulrich Z Hammes / Bjørn Panyella Pedersen /
Abstract: Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier ...Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline-proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.
History
DepositionJan 3, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Sep 28, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / 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)86,2106
Polymers83,0822
Non-polymers3,1284
Water2,288127
1


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

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Components

#1: Protein Auxin efflux carrier component 8 / AtPIN8


Mass: 41541.039 Da / Num. of mol.: 2
Mutation: N-terminal tag: First two residues MG are cloning tags. Uniprot sequence aligns from Ile2. Note MG is added as residue 0 and 1, to maintain correct numbering compared to uniprot.
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-DLP / 1,2-DILINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DI-LINOLEOYL-3-SN-PHOSPHATIDYLCHOLINE


Mass: 782.082 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C44H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 127 / Source method: isolated from a natural source / Formula: H2O
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: Apo-form of PIN8 / Type: COMPLEX / Details: dimer form of PIN8 / 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
2150 mMsodium chlorideNaCl1
30.5 mMEDTAC10H16N2O81
SpecimenConc.: 10 mg/ml / Embedding 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: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7900
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
IDNameVersionCategoryDetails
2EPUimage acquisition
4cryoSPARC3.2.0CTF correctioncryosparc-live
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.2.0initial Euler assignmentab initio job
11cryoSPARC3.2.0final Euler assignmentnon-uniform refinement job
12cryoSPARC3.2.0classificationHeterogenous Refinement Job
13cryoSPARC3.2.03D reconstructionNon-uniform refinement job
CTF correctionDetails: CTF amplitude correction was performed following motion correction using cryosparc-live
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2082448
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 327193 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT

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