|Entry||Database: EMDB / ID: 8313|
|Title||Structure of the SLC4 transporter Bor1p in an inward-facing conformation|
|Map data||Bor1p helical tube density map (Type 1)|
|Sample||Bor1p dimer in an inward-facing conformation:|
Bor1p boron transporter
|Source||Saccharomyces mikatae (yeast)|
|Method||helical reconstruction / cryo EM / 5.9 Å resolution|
|Authors||Coudray N / Seyler S|
|Citation||Journal: 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 Report||PDB-ID: 5sv9|
SummaryFull reportAbout validation report
|Date||Deposition: Aug 5, 2016 / Header (metadata) release: Aug 17, 2016 / Map release: Aug 17, 2016 / Last update: Sep 13, 2017|
|Structure viewer||EM map: |
Downloads & links
|File||emd_8313.map.gz (map file in CCP4 format, 4122 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.82 Å|
CCP4 map header:
-Entire Bor1p dimer in an inward-facing conformation
|Entire||Name: Bor1p dimer in an inward-facing conformation / Number of components: 2|
|Mass||Theoretical: 60 kDa|
-Component #1: protein, Bor1p dimer in an inward-facing conformation
|Protein||Name: Bor1p dimer in an inward-facing conformation / Recombinant expression: No|
|Mass||Theoretical: 60 kDa|
|Source||Species: Saccharomyces mikatae (yeast)|
|Source (engineered)||Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: pSGP46|
-Component #2: protein, Bor1p boron transporter
|Protein||Name: Bor1p boron transporter / Recombinant expression: No|
|Mass||Theoretical: 53.829766 kDa|
|Source (engineered)||Expression System: Saccharomyces mikatae (yeast)|
|Specimen||Specimen state: helical array / Method: cryo EM|
|Helical parameters||Axial symmetry: C1 (asymmetric) / Delta z: 4.8 Å / Delta phi: 37.35 deg.|
|Sample solution||Specimen conc.: 0.25 mg/ml / Buffer solution: Buffer was changed twice per day. / pH: 7|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 %|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 19000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2500 nm|
|Specimen Holder||Model: GATAN LIQUID NITROGEN|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Image acquisition||Number of digital images: 252|
-Atomic model buiding
|Modeling #1||Refinement 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
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