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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: 8313
TitleStructure of the SLC4 transporter Bor1p in an inward-facing conformation
Map dataBor1p helical tube density map (Type 1)
SampleBor1p dimer in an inward-facing conformation:
Bor1p boron transporter
SourceSaccharomyces mikatae (yeast)
Methodhelical reconstruction / cryo EM / 5.9 Å resolution
AuthorsCoudray N / Seyler S
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.
Validation ReportPDB-ID: 5sv9

SummaryFull reportAbout validation report
DateDeposition: Aug 5, 2016 / Header (metadata) release: Aug 17, 2016 / Map release: Aug 17, 2016 / Last update: Sep 13, 2017

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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
  • Download
  • 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

Downloads & links

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Map

Fileemd_8313.map.gz (map file in CCP4 format, 4122 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
101 pix
1.82 Å/pix.
= 183.82 Å
101 pix
1.82 Å/pix.
= 183.82 Å
101 pix
1.82 Å/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
Contour Level:130 (by author), 130 (movie #1):
Minimum - Maximum-365.12125 - 478.5021
Average (Standard dev.)0.7892577 (24.340841)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions101101101
Origin-50-50-50
Limit505050
Spacing101101101
CellA=B=C: 183.82 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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 / Number of components: 2
MassTheoretical: 60 kDa

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Component #1: protein, Bor1p dimer in an inward-facing conformation

ProteinName: Bor1p dimer in an inward-facing conformation / Recombinant expression: No
MassTheoretical: 60 kDa
SourceSpecies: Saccharomyces mikatae (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: pSGP46

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Component #2: protein, Bor1p boron transporter

ProteinName: Bor1p boron transporter / Recombinant expression: No
MassTheoretical: 53.829766 kDa
Source (engineered)Expression System: Saccharomyces mikatae (yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 4.8 Å / Delta phi: 37.35 deg.
Sample solutionSpecimen conc.: 0.25 mg/ml / Buffer solution: Buffer was changed twice per day. / pH: 7
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 19000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2500 nm
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 252

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: FREALIX, SPARX/EMAN2 / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF
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.
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: cross-correlation coefficient / Refinement space: REAL
Details: A 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.
Input PDB model: 4YZF
Chain ID: A,B
Output model

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