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- PDB-8s5z: Cryo-EM structure of hydroxychloroquine-bound human SLC19A3 in in... -

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Basic information

Entry
Database: PDB / ID: 8s5z
TitleCryo-EM structure of hydroxychloroquine-bound human SLC19A3 in inward-open state
Components
  • Nb3.3
  • 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 Å
AuthorsGabriel, F. / Loew, C.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)10251European Union
CitationJournal: To Be Published
Title: Structural basis of substrate transport and drug recognition by the human thiamine transporter SLC19A3
Authors: Gabriel, F. / Loew, C.
History
DepositionFeb 26, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Nb3.3
A: Thiamine transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,4504
Polymers74,8932
Non-polymers5572
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1250 Å2
ΔGint-3 kcal/mol
Surface area22370 Å2
MethodPISA

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Components

#1: Antibody Nb3.3


Mass: 15205.778 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 / Mutation: N45Q, N166Q
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
#4: Chemical ChemComp-A1H5D / Hydroxychloroquine / 2-[[(4~{R})-4-[(7-chloranylquinolin-4-yl)amino]pentyl]-ethyl-amino]ethanol


Mass: 335.872 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H26ClN3O / 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

Component
IDNameTypeEntity IDParent-IDSource
1SLC19A3:Nb3.7:hydroxychloroquineCOMPLEX#10RECOMBINANT
2SLC19A3COMPLEX#21RECOMBINANT
3Nb3.3COMPLEX#11RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
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
50.25 mMhydroxychloroqine1
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: 215000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.1 sec. / Electron dose: 58 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9492
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage 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 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 249857 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementResolution: 3→3 Å / Cor.coef. Fo:Fc: 0.486 / WRfactor Rwork: 0.485 / Average fsc free: 0 / Average fsc overall: 0.5433 / Average fsc work: 0.5433 / ESU R: 0.067
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.4848 1948025 -
all0.485 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 39.364 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.0124181
ELECTRON MICROSCOPYr_bond_other_d0.0020.0163932
ELECTRON MICROSCOPYr_angle_refined_deg1.3611.795703
ELECTRON MICROSCOPYr_angle_other_deg0.9041.7018970
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.0365504
ELECTRON MICROSCOPYr_dihedral_angle_2_deg0.559518
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.90710632
ELECTRON MICROSCOPYr_dihedral_angle_6_deg16.910171
ELECTRON MICROSCOPYr_chiral_restr0.0540.2646
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.024877
ELECTRON MICROSCOPYr_gen_planes_other0.0110.021070
ELECTRON MICROSCOPYr_nbd_refined0.2330.21073
ELECTRON MICROSCOPYr_symmetry_nbd_other0.2210.23897
ELECTRON MICROSCOPYr_nbtor_refined0.2110.22201
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0840.22315
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1570.262
ELECTRON MICROSCOPYr_mcbond_it042025
ELECTRON MICROSCOPYr_mcbond_other042025
ELECTRON MICROSCOPYr_mcangle_it07.1742526
ELECTRON MICROSCOPYr_mcangle_other07.1742527
ELECTRON MICROSCOPYr_scbond_it042156
ELECTRON MICROSCOPYr_scbond_other042157
ELECTRON MICROSCOPYr_scangle_it07.3713177
ELECTRON MICROSCOPYr_scangle_other07.3713178
ELECTRON MICROSCOPYr_lrange_it047.75117501
ELECTRON MICROSCOPYr_lrange_other047.75217502
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
3-3.0780.6261443380.6261443380.1950.626
3.078-3.1620.5861407250.5861407250.2350.586
3.162-3.2540.5511364090.5511364090.2930.551
3.254-3.3540.5311324060.5311324060.3470.531
3.354-3.4640.5111283210.5111283210.4160.511
3.464-3.5860.491245460.491245460.4890.49
3.586-3.7210.4761203770.4761203770.5640.476
3.721-3.8730.4631155650.4631155650.6340.463
3.873-4.0450.4541107170.4541107170.6870.454
4.045-4.2430.4441060220.4441060220.7350.444
4.243-4.4720.4281008200.4281008200.7740.428
4.472-4.7430.409949700.409949700.7950.409
4.743-5.0710.395895940.395895940.7790.395
5.071-5.4770.398831060.398831060.7310.398
5.477-5.9990.447766730.447766730.6480.447
5.999-6.7070.464693510.464693510.6220.464
6.707-7.7440.462607660.462607660.6660.462
7.744-9.4830.487517870.487517870.7130.487
9.483-13.4040.513398630.513398630.8290.513
13.404-292.8640.952218610.952218610.6550.952

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