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

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Basic information

Entry
Database: PDB / ID: 5sv9
TitleStructure of the SLC4 transporter Bor1p in an inward-facing conformation
ComponentsBor1p boron transporter
KeywordsTRANSPORT PROTEIN / boron transporter / anion exchanger family / alternating access mechanism / Structural Genomics / PSI-Biology / Transcontinental EM Initiative for Membrane Protein Structure / TEMIMPS
Function / homologyBicarbonate transporter, eukaryotic / Bicarbonate transporter-like, transmembrane domain / HCO3- transporter family / solute:inorganic anion antiporter activity / membrane => GO:0016020 / Bor1p boron transporter
Function and homology information
Biological speciesSaccharomyces mikatae (yeast)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsCoudray, N. / Seyler, S. / Lasala, R. / Zhang, Z. / Clark, K.M. / Dumont, M.E. / Rohou, A. / Beckstein, O. / Stokes, D.L. / Transcontinental EM Initiative for Membrane Protein Structure (TEMIMPS)
Funding support United States, 4items
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 17, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2016Group: Database references
Revision 1.2Jan 11, 2017Group: Database references
Revision 1.3Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Jan 23, 2019Group: Data collection / Refinement description / Category: em_3d_fitting_list
Revision 1.5Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Bor1p boron transporter
B: Bor1p boron transporter


Theoretical massNumber of molelcules
Total (without water)107,6602
Polymers107,6602
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2330 Å2
ΔGint-15 kcal/mol
Surface area46170 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: yes / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 4.8 Å / Rotation per n subunits: 37.35 °)

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Components

#1: Protein Bor1p boron transporter


Mass: 53829.766 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces mikatae (yeast) / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A1C7D1B8*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Bor1p dimer in an inward-facing conformation / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.06 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces mikatae (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pSGP46
Buffer solutionpH: 7 / Details: Buffer was changed twice per day.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidC8H18N2O41
2100 mMsodium chlorideNaClSodium chloride1
32 mMboric acidH3BO31
45 %sodium azideNaN31
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The 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.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 19000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 850 nm / Calibrated defocus max: 2700 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 6.75 sec. / Electron dose: 45 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 252
Image scansWidth: 3710 / Height: 3710 / Movie frames/image: 27 / Used frames/image: 2-27

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Processing

EM software
IDNameVersionCategoryDetails
1SPARX/EMAN2EMAN2.1particle selectionsxhelixboxer.py
4CTFFIND3CTF correction
5FREALIX1.2.0CTF correctionflx_wrap.rb
8Situs2.8model fitting
9NAMD2.11model fittingMDFF
11FREALIX1.2.0initial Euler assignmentflx_wrap.rb
12FREALIX1.2.0final Euler assignmentflx_wrap.rb
14FREALIX1.2.03D reconstructionflx_wrap.rb
15SPARX/EMAN2EMAN2.13D reconstructionfilt_table and filt_tanl
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 37.35 ° / Axial rise/subunit: 4.8 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 87
3D reconstructionResolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75
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.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
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 ...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.
Atomic model building

3D fitting-ID: 1 / Accession code: 4YZF / Initial refinement model-ID: 1 / Pdb chain residue range: 418-911 / PDB-ID: 4YZF

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B

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