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- PDB-8s4u: Cryo-EM structure of apo human SLC19A3 in outward-open state -

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Basic information

Entry
Database: PDB / ID: 8s4u
TitleCryo-EM structure of apo human SLC19A3 in outward-open state
Components
  • Nanobody 3.4
  • Thiamine transporter 2
KeywordsMEMBRANE PROTEIN / SLC19A3 / vitamin transporter / thiamine transporter / MFS fold / nanobody complex
Function / homology
Function and homology information


pyridoxine transport / thiamine-containing compound metabolic process / Vitamin B1 (thiamin) metabolism / thiamine transmembrane transport / thiamine transmembrane transporter activity / thiamine transport / thiamine diphosphate biosynthetic process / transmembrane transport / membrane / plasma membrane
Similarity search - Function
Thiamine transporter 2 / Reduced folate carrier / Reduced folate carrier / MFS transporter superfamily
Similarity search - Domain/homology
Thiamine transporter 2
Similarity search - Component
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsGabriel, F. / Loew, C.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)10251European Union
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of thiamine transport and drug recognition by SLC19A3.
Authors: Florian Gabriel / Lea Spriestersbach / Antonia Fuhrmann / Katharina E J Jungnickel / Siavash Mostafavi / Els Pardon / Jan Steyaert / Christian Löw /
Abstract: Thiamine (vitamin B) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell ...Thiamine (vitamin B) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane, which is mainly mediated by the two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters lead to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report seven cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enable us to capture different states of SLC19A3's transport cycle, revealing the molecular details of thiamine recognition and transport. We identify seven previously unknown drug interactions of SLC19A3 and present structures of the transporter in complex with the inhibitors fedratinib, amprolium and hydroxychloroquine. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.
History
DepositionFeb 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details / pdbx_modification_feature
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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Oct 23, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Dec 4, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Nanobody 3.4
A: Thiamine transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,1273
Polymers74,9062
Non-polymers2211
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1930 Å2
ΔGint-5 kcal/mol
Surface area22080 Å2

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Components

#1: Antibody Nanobody 3.4


Mass: 15217.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#2: Protein Thiamine transporter 2 / ThTr-2 / ThTr2 / Solute carrier family 19 member 3


Mass: 59687.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC19A3 / Production host: Homo sapiens (human) / References: UniProt: Q9BZV2
#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.: 1 / 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
Has ligand of interestN
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1SLC19A3:Nb3.4-apoCOMPLEX#1-#20MULTIPLE SOURCES
2SLC19A3COMPLEX#21RECOMBINANT
3Nb3.4-apoCOMPLEX#11RECOMBINANT
Molecular weightValue: 0.075 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
2150 mMsodium chlorideNaCl1
30.002 %LMNG1
40.0002 %CHS1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 283.15 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5859
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 486079 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementResolution: 3.09→3.09 Å / Cor.coef. Fo:Fc: 0.685 / WRfactor Rwork: 0.392 / Average fsc free: 0 / Average fsc overall: 0.8037 / Average fsc work: 0.8037 / ESU R: 0.625
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3923 35165 -
all0.392 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 39.382 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.0124092
ELECTRON MICROSCOPYr_bond_other_d0.0020.0163873
ELECTRON MICROSCOPYr_angle_refined_deg1.3441.7815585
ELECTRON MICROSCOPYr_angle_other_deg0.8781.6918847
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.5165499
ELECTRON MICROSCOPYr_dihedral_angle_2_deg0.747515
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.85810626
ELECTRON MICROSCOPYr_dihedral_angle_6_deg16.82410163
ELECTRON MICROSCOPYr_chiral_restr0.0580.2642
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.024752
ELECTRON MICROSCOPYr_gen_planes_other0.0120.021026
ELECTRON MICROSCOPYr_nbd_refined0.2310.21078
ELECTRON MICROSCOPYr_symmetry_nbd_other0.2210.23882
ELECTRON MICROSCOPYr_nbtor_refined0.2080.22192
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0840.22288
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1790.282
ELECTRON MICROSCOPYr_mcbond_it042005
ELECTRON MICROSCOPYr_mcbond_other042005
ELECTRON MICROSCOPYr_mcangle_it07.1742501
ELECTRON MICROSCOPYr_mcangle_other07.1742502
ELECTRON MICROSCOPYr_scbond_it042087
ELECTRON MICROSCOPYr_scbond_other042088
ELECTRON MICROSCOPYr_scangle_it07.3683084
ELECTRON MICROSCOPYr_scangle_other07.3683085
ELECTRON MICROSCOPYr_lrange_it047.76416990
ELECTRON MICROSCOPYr_lrange_other047.76316991
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
3.5-3.5910.47325910.47325910.7240.473
3.591-3.690.44625140.44625140.7540.446
3.69-3.7960.40924900.40924900.7840.409
3.796-3.9130.38723840.38723840.7930.387
3.913-4.0410.37223450.37223450.8110.372
4.041-4.1830.3922290.3922290.8130.39
4.183-4.340.37721370.37721370.8240.377
4.34-4.5170.37621210.37621210.8390.376
4.517-4.7170.35819920.35819920.8510.358
4.717-4.9470.35118910.35118910.8490.351
4.947-5.2140.37118110.37118110.8340.371
5.214-5.5290.38217450.38217450.8130.382
5.529-5.910.4116130.4116130.780.41
5.91-6.3810.42914950.42914950.7510.429
6.381-6.9870.43613940.43613940.7670.436
6.987-7.8070.40912420.40912420.7710.409
7.807-9.0060.38410990.38410990.8030.384
9.006-11.0060.339520.339520.8750.33
11.006-15.4680.2697220.2697220.9160.269
15.468-97.940.5973980.5973980.9530.597

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