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- PDB-8t6u: Cryo-EM structure of human Anion Exchanger 1 bound to Dipyridamole -

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Basic information

Entry
Database: PDB / ID: 8t6u
TitleCryo-EM structure of human Anion Exchanger 1 bound to Dipyridamole
ComponentsBand 3 anion transport protein
KeywordsTRANSPORT PROTEIN / Transmembrane
Function / homology
Function and homology information


pH elevation / Defective SLC4A1 causes hereditary spherocytosis type 4 (HSP4), distal renal tubular acidosis (dRTA) and dRTA with hemolytic anemia (dRTA-HA) / negative regulation of urine volume / Bicarbonate transporters / intracellular monoatomic ion homeostasis / ankyrin-1 complex / plasma membrane phospholipid scrambling / monoatomic anion transmembrane transporter activity / chloride:bicarbonate antiporter activity / solute:inorganic anion antiporter activity ...pH elevation / Defective SLC4A1 causes hereditary spherocytosis type 4 (HSP4), distal renal tubular acidosis (dRTA) and dRTA with hemolytic anemia (dRTA-HA) / negative regulation of urine volume / Bicarbonate transporters / intracellular monoatomic ion homeostasis / ankyrin-1 complex / plasma membrane phospholipid scrambling / monoatomic anion transmembrane transporter activity / chloride:bicarbonate antiporter activity / solute:inorganic anion antiporter activity / bicarbonate transmembrane transporter activity / bicarbonate transport / monoatomic anion transport / chloride transport / chloride transmembrane transporter activity / erythrocyte development / negative regulation of glycolytic process through fructose-6-phosphate / ankyrin binding / hemoglobin binding / cortical cytoskeleton / protein-membrane adaptor activity / chloride transmembrane transport / protein localization to plasma membrane / regulation of intracellular pH / transmembrane transport / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / cytoplasmic side of plasma membrane / Z disc / blood coagulation / basolateral plasma membrane / blood microparticle / protein homodimerization activity / extracellular exosome / membrane / plasma membrane
Similarity search - Function
Anion exchange protein 1 / Anion exchange protein / Anion exchange, conserved site / Anion exchangers family signature 1. / Anion exchangers family signature 2. / Band 3 cytoplasmic domain / Band 3 cytoplasmic domain / Bicarbonate transporter, eukaryotic / Bicarbonate transporter-like, transmembrane domain / HCO3- transporter family / Phosphotransferase/anion transporter
Similarity search - Domain/homology
CHOLESTEROL / Chem-H9F / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Band 3 anion transport protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsCapper, M.J. / Zilberg, G. / Mathiharan, Y.K. / Yang, S. / Stone, A.C. / Wacker, D.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM133504 United States
Other privateEdward Mallinckrodt, Jr Foundation Grant
Other privateMcKnight Endowment Fund for Neuroscience Scholarship
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Substrate binding and inhibition of the anion exchanger 1 transporter.
Authors: Michael J Capper / Shifan Yang / Alexander C Stone / Sezen Vatansever / Gregory Zilberg / Yamuna Kalyani Mathiharan / Raul Habib / Keino Hutchinson / Yihan Zhao / Avner Schlessinger / Mihaly ...Authors: Michael J Capper / Shifan Yang / Alexander C Stone / Sezen Vatansever / Gregory Zilberg / Yamuna Kalyani Mathiharan / Raul Habib / Keino Hutchinson / Yihan Zhao / Avner Schlessinger / Mihaly Mezei / Roman Osman / Bin Zhang / Daniel Wacker /
Abstract: Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO transport between lungs and tissues. Previous ...Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.
History
DepositionJun 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.2Nov 1, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Band 3 anion transport protein
B: Band 3 anion transport protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,04016
Polymers116,5082
Non-polymers6,53214
Water64936
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Band 3 anion transport protein / / Anion exchange protein 1 / AE 1 / Anion exchanger 1 / Solute carrier family 4 member 1


Mass: 58254.105 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC4A1, AE1, DI, EPB3 / Plasmid: pFB / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P02730
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][a-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 48 molecules

#3: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE / Phosphatidylcholine


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#5: Chemical ChemComp-H9F / 2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol / Dipyridamole


Mass: 504.626 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H40N8O4 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, inhibitor*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Dimeric anion exchanger 1 (SLC4A1) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.203 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pFB
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2100 mMsodium chloride1
30.001 % (w/v)LMNG1
40.0001 % (w/v)CHS1
50.0001 % (w/v)GDN1
SpecimenConc.: 4.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was monodisperse following gel filtration and immediately concentrated for grid preparation
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 285 K
Details: Blot force 3 for 3-5 seconds was used and subsequent grids were screened for ice thickness prior to data collection

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51.69 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4570

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9REFMACservalcatmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2499027
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270791 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model buildingPDB-ID: 4YZF

4yzf


Accession code: 4YZF / Source name: PDB / Type: experimental model
RefinementResolution: 3.13→139.88 Å / Cor.coef. Fo:Fc: 0.883 / SU B: 15.851 / SU ML: 0.264 / ESU R: 0.529
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34403 --
obs0.34403 70400 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 103.19 Å2
Refinement stepCycle: 1 / Total: 8647
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0128848
ELECTRON MICROSCOPYr_bond_other_d00.0168868
ELECTRON MICROSCOPYr_angle_refined_deg1.3311.6312058
ELECTRON MICROSCOPYr_angle_other_deg0.4571.54920558
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.9151028
ELECTRON MICROSCOPYr_dihedral_angle_2_deg8.7778.96658
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.497101426
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0580.21446
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.029342
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021760
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it13.2958.8584124
ELECTRON MICROSCOPYr_mcbond_other13.2948.8574124
ELECTRON MICROSCOPYr_mcangle_it19.36113.3545148
ELECTRON MICROSCOPYr_mcangle_other19.3613.3585149
ELECTRON MICROSCOPYr_scbond_it15.32111.5514724
ELECTRON MICROSCOPYr_scbond_other15.31911.5534725
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other24.37316.6016911
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.13→3.212 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.875 5291 -
obs--100 %

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