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- PDB-7rtm: Cryo-EM Structure of the Sodium-driven Chloride/Bicarbonate Excha... -

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Basic information

Entry
Database: PDB / ID: 7rtm
TitleCryo-EM Structure of the Sodium-driven Chloride/Bicarbonate Exchanger NDCBE (SLC4A8)
ComponentsElectroneutral sodium bicarbonate exchanger 1
KeywordsMEMBRANE PROTEIN / NDCBE / sodium-driven chloride/bicarbonate exchanger / cryo-EM
Function / homologyCARBONATE ION
Function and homology information
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWang, W.G. / Tsirulnikov, K. / Zhekova, H. / Kayik, G. / Muhammad-Khan, H. / Azimov, R. / Abuladze, N. / Kao, L. / Newman, D. / Noskov, S.Y. ...Wang, W.G. / Tsirulnikov, K. / Zhekova, H. / Kayik, G. / Muhammad-Khan, H. / Azimov, R. / Abuladze, N. / Kao, L. / Newman, D. / Noskov, S.Y. / Zhou, Z.H. / Pushkin, A. / Kurtz, I.
Funding support United States, Canada, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01 DK077162 United States
Other governmentNo. RGPIN-2021-02439 Canada
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE.
Authors: Weiguang Wang / Kirill Tsirulnikov / Hristina R Zhekova / Gülru Kayık / Hanif Muhammad Khan / Rustam Azimov / Natalia Abuladze / Liyo Kao / Debbie Newman / Sergei Yu Noskov / Z Hong Zhou / ...Authors: Weiguang Wang / Kirill Tsirulnikov / Hristina R Zhekova / Gülru Kayık / Hanif Muhammad Khan / Rustam Azimov / Natalia Abuladze / Liyo Kao / Debbie Newman / Sergei Yu Noskov / Z Hong Zhou / Alexander Pushkin / Ira Kurtz /
Abstract: SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) ...SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.
History
DepositionAug 13, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 29, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Electroneutral sodium bicarbonate exchanger 1
B: Electroneutral sodium bicarbonate exchanger 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,47210
Polymers128,6082
Non-polymers1,8648
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5870 Å2
ΔGint-41 kcal/mol
Surface area56270 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LEULYSA1 - 571
d_21ens_1LEULYSB1 - 571

NCS oper: (Code: givenMatrix: (-0.999999762042, 0.000477641228076, -0.000497768858412), (-0.000477422565083, -0.999999789536, -0.000439312482978), (-0.000497978587404, -0.000439074732355, 0. ...NCS oper: (Code: given
Matrix: (-0.999999762042, 0.000477641228076, -0.000497768858412), (-0.000477422565083, -0.999999789536, -0.000439312482978), (-0.000497978587404, -0.000439074732355, 0.999999779615)
Vector: 132.743552839, 85.5869142575, 0.172902338478)

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Components

#1: Protein Electroneutral sodium bicarbonate exchanger 1


Mass: 64304.094 Da / Num. of mol.: 2 / Mutation: None
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Production host: Homo sapiens (human)
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 627.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[a2122h-1b_1-5_2*NCC/3=O]/1-1-1/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CO3 / CARBONATE ION


Mass: 60.009 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CO3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: NDCBE(SLC4A8) dimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTris-HClTris-HCl1
30.01 %LMNGC47H88O221
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: sample in 0.01% LMNG.
Specimen supportDetails: Glow discharged with Easyglow. / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot time 2 seconds. blot force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 130000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52 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: 7545
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2SerialEM3.8image acquisitionSerialEM was used for automatic data collection
4CTFFIND4.1CTF correction
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Coot0.8.9model refinementwindows version
14PHENIX1.18.2model refinementreal space refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2739162 / Details: particles were autopicked in Relion
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 380776 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 67.91 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0079412
ELECTRON MICROSCOPYf_angle_d0.816212796
ELECTRON MICROSCOPYf_chiral_restr0.04841507
ELECTRON MICROSCOPYf_plane_restr0.00471548
ELECTRON MICROSCOPYf_dihedral_angle_d24.0431262
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.000708577549399 Å

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