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- EMDB-14118: Outward-facing apo-form of auxin transporter PIN8 in detergent -

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Basic information

Entry
Database: EMDB / ID: EMD-14118
TitleOutward-facing apo-form of auxin transporter PIN8 in detergent
Map data
Sample
  • Complex: Apo-form of PIN8
    • Protein or peptide: Auxin efflux carrier component 8 (PIN-FORMED auxin transporter 8)
KeywordsAuxin transport / AEC family / BART superfamily / MEMBRANE PROTEIN
Function / homology
Function and homology information


auxin export across the plasma membrane / pollen development / auxin-activated signaling pathway / endoplasmic reticulum membrane / endoplasmic reticulum / plasma membrane
Similarity search - Function
Membrane transport protein / Auxin efflux carrier, plant type / Membrane transport protein
Similarity search - Domain/homology
Auxin efflux carrier component 8
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsUng KL / Winkler MBL / Dedic E / Stokes DL / Pedersen BP
Funding supportEuropean Union, 1 items
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, 2022-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14118.png

Downloads & links

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Map

FileDownload / File: emd_14118.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 208 pix.
= 271.648 Å		1.31 Å/pix.
x 208 pix.
= 271.648 Å		1.31 Å/pix.
x 208 pix.
= 271.648 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.306 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-1.2060552 - 2.060951
Average (Standard dev.)0.0006299099 (±0.04166383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 271.648 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14118_msk_1.map
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Half map: #1

Fileemd_14118_half_map_1.map
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Half map: #2

Fileemd_14118_half_map_2.map
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Sample components

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Entire : Apo-form of PIN8

EntireName: Apo-form of PIN8
Components
  • Complex: Apo-form of PIN8
    • Protein or peptide: Auxin efflux carrier component 8 (PIN-FORMED auxin transporter 8)

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Supramolecule #1: Apo-form of PIN8

SupramoleculeName: Apo-form of PIN8 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: dimer form of PIN8
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 81 KDa

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Macromolecule #1: Auxin efflux carrier component 8 (PIN-FORMED auxin transporter 8)

MacromoleculeName: Auxin efflux carrier component 8 (PIN-FORMED auxin transporter 8)
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGISWLDIYH VVSATVPLYV SMTLGFLSAR HLKLFSPEQC AGINKFVAKF SIPLLSFQII SENNPFKMSP KLILSDILQK FLVVVVLAMV LRFWHPTGGR GGKLGWVITG LSISVLPNTL ILGMPILSAI YGDEAASILE QIVVLQSLIW YTILLFLFEL NAARALPSSG ...String:
MGISWLDIYH VVSATVPLYV SMTLGFLSAR HLKLFSPEQC AGINKFVAKF SIPLLSFQII SENNPFKMSP KLILSDILQK FLVVVVLAMV LRFWHPTGGR GGKLGWVITG LSISVLPNTL ILGMPILSAI YGDEAASILE QIVVLQSLIW YTILLFLFEL NAARALPSSG ASLEHTGNDQ EEANIEDEPK EEEDEEEVAI VRTRSVGTMK ILLKAWRKLI INPNTYATLI GIIWATLHFR LGWNLPEMID KSIHLLSDGG LGMAMFSLGL FMASQSSIIA CGTKMAIITM LLKFVLGPAL MIASAYCIRL KSTLFKVAIL QAALPQGVVP FVFAKEYNLH PEIISTGVIF GMLIALPTTL AYYFLLDLPG ENLYFQ

UniProtKB: Auxin efflux carrier component 8

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
150.0 mMNaClsodium chloride
0.5 mMC10H16N2O8EDTA
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: The grid was glow-discharge at 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Wait 4 seconds after sample loading, Blotting time 4 seconds with blotting force of -1 before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 7808 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 973540
Startup modelType of model: INSILICO MODEL / In silico model: RoseTTAfold model (Baek et al.,2021) / Details: Sequence used: UniProt Q9LFP6
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Non-uniform refinement job / Number images used: 74743
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: ab initio job / Details: Cryosparc ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: non-uniform refinement job / Details: Cryosparc NU refinement
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Heterogenous Refinement Job
FSC plot (resolution estimation)

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