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- EMDB-8313: Structure of the SLC4 transporter Bor1p in an inward-facing confo... -

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Basic information

Entry
Database: EMDB / ID: EMD-8313
TitleStructure of the SLC4 transporter Bor1p in an inward-facing conformation
Map dataBor1p helical tube density map (Type 1)
Sample
  • Complex: Bor1p dimer in an inward-facing conformation
    • Protein or peptide: Bor1p boron transporter
Keywordsboron transporter / anion exchanger family / alternating access mechanism / Structural Genomics / PSI-Biology / Transcontinental EM Initiative for Membrane Protein Structure / TEMIMPS / TRANSPORT PROTEIN
Function / homologyBicarbonate transporter, eukaryotic / Bicarbonate transporter-like, transmembrane domain / HCO3- transporter integral membrane domain / solute:inorganic anion antiporter activity / monoatomic anion transport / membrane / Bor1p boron transporter
Function and homology information
Biological speciesSaccharomyces mikatae (yeast)
Methodhelical reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsCoudray N / Seyler S
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U54 GM094598 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM095747 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U54 GM094611 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM118772 United States
CitationJournal: Protein Sci / Year: 2017
Title: Structure of the SLC4 transporter Bor1p in an inward-facing conformation.
Authors: Nicolas Coudray / Sean L Seyler / Ralph Lasala / Zhening Zhang / Kathy M Clark / Mark E Dumont / Alexis Rohou / Oliver Beckstein / David L Stokes /
Abstract: Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate ...Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.
History
DepositionAug 5, 2016-
Header (metadata) releaseAug 17, 2016-
Map releaseAug 17, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 130
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 130
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5sv9
  • Surface level: 130
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8313.png

Downloads & links

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Map

FileDownload / File: emd_8313.map.gz / Format: CCP4 / Size: 3.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBor1p helical tube density map (Type 1)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.82 Å/pix.
x 101 pix.
= 183.82 Å		1.82 Å/pix.
x 101 pix.
= 183.82 Å		1.82 Å/pix.
x 101 pix.
= 183.82 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 130.0 / Movie #1: 130
Minimum - Maximum-365.121249999999975 - 478.502099999999984
Average (Standard dev.)0.7892577 (±24.340841000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions101101101
Spacing101101101
CellA=B=C: 183.82 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.821.821.82
M x/y/z101101101
origin x/y/z0.0000.0000.000
length x/y/z183.820183.820183.820
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS101101101
D min/max/mean-365.121478.5020.789

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Supplemental data

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Sample components

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Entire : Bor1p dimer in an inward-facing conformation

EntireName: Bor1p dimer in an inward-facing conformation
Components
  • Complex: Bor1p dimer in an inward-facing conformation
    • Protein or peptide: Bor1p boron transporter

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Supramolecule #1: Bor1p dimer in an inward-facing conformation

SupramoleculeName: Bor1p dimer in an inward-facing conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces mikatae (yeast)
Molecular weightTheoretical: 60 KDa

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Macromolecule #1: Bor1p boron transporter

MacromoleculeName: Bor1p boron transporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces mikatae (yeast)
Molecular weightTheoretical: 53.829766 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: IWLDLKDRIP YYKSDWVDAF NYRVIPSTVD TYFNNLLPAI AFAQDMFDRT DNSYGVNEVL LSSAMAGIVF GVLAGQPLCI VGVTGPISI FNYTVYEIIK PLNTSYFGFM FWICLWSMIF HLLLAFTNVV CLLQYVTTFP CDIFGLFINV VYIQKGIQIL T RQFHNTSG ...String:
IWLDLKDRIP YYKSDWVDAF NYRVIPSTVD TYFNNLLPAI AFAQDMFDRT DNSYGVNEVL LSSAMAGIVF GVLAGQPLCI VGVTGPISI FNYTVYEIIK PLNTSYFGFM FWICLWSMIF HLLLAFTNVV CLLQYVTTFP CDIFGLFINV VYIQKGIQIL T RQFHNTSG EKSVQDGFAS VVVALVMTAF GLFFKSFHHY PLFTHKIRTF ISDYSTALSV LFWSSFTHFG GYLNDVKFKK LP ITKSFFP TSKFNRPQNT WLAYEPIPVK DVFIALPFGI ILTILFYFDH NVSSLMAQRH QYKLRKPSSF HYDFALLGLT TCI SGVLGI PAPNGLIPQA PLHTETLLVR DSNQNVVRCV EQRLTNTFQG LMILGTMTRP LLVCLGEIPQ AVLSGLFFIM GING LMTNV IIHRIVFLFS DPKRRDNNSP LAKISKRSMV IFLCFSLAGF TGEFAITNTI AAIGFPLVLL LSVIVSFSFT Y

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
100.0 mMC8H18N2O44-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMNaClsodium chloride
2.0 mMH3BO3boric acid
5.0 %NaN3sodium azide

Details: Buffer was changed twice per day.
GridModel: EMS / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: HOMEMADE PLUNGER
DetailsThe protein was solubilized in 1% n-Dodecyl beta-D-maltoside and exchanged into heptaethyleneglycol-n-dodecylether (C12E7) while bound to the IgG Sepharose affinity column.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 2-27 / Number grids imaged: 2 / Number real images: 252 / Average exposure time: 6.75 sec. / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.7 µm / Calibrated defocus min: 0.85 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 19000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 4.8 Å
Applied symmetry - Helical parameters - Δ&Phi: 37.35 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
FREALIX (ver. 1.2.0)flx_wrap.rb
SPARX/EMAN2 (ver. EMAN2.1)filt_table and filt_tanl

Details: Reconstruction was done using D1 symmetry because of an existing two-fold symmetry in the unit cells composing the helical lattice. This two-fold axis runs perpendicular to the helical axis. ...Details: Reconstruction was done using D1 symmetry because of an existing two-fold symmetry in the unit cells composing the helical lattice. This two-fold axis runs perpendicular to the helical axis. For the final structure, a filter was applied to the map in order to compensate for resolution-dependent amplitude falloff. To do so, we built a model by arranging UraA in a helical assembly in order to mimic the mass distribution in Bor1p tubes. Fourier transforms from this model and from the experimental maps were then rotationally averaged to produce 1D scattering profiles. The resolution-dependent amplitude ratio from these profiles was used as a filter that was applied to the experimental amplitudes using SPARX routines. Finally, a low-pass filter was applied with a 5 Angstrom stop-band frequency.
Number images used: 75
Segment selectionNumber selected: 87 / Software - Name: SPARX/EMAN2 (ver. EMAN2.1) / Software - details: sxhelixboxer.py
Startup modelType of model: OTHER
Details: Initial model was created using the Fourier-Bessel method using 41 tube sections.
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIX (ver. 1.2.0) / Software - details: flx_wrap.rb
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4yzf

chain_id: A, residue_range: 418-911, source_name: PDB, initial_model_type: experimental model

4yzf

chain_id: B, residue_range: 418-911, source_name: PDB, initial_model_type: experimental model
DetailsA homology model was first created using human AE1 (PDB entry 4YZF) as a template using MODELLER, placed into the density map using SITUS, and then fitted into the map using the Molecular Dynamics Flexible Fitting (MDFF) method.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation coefficient
Output model

PDB-5sv9:
Structure of the SLC4 transporter Bor1p in an inward-facing conformation

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