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- PDB-6wtw: Structure of LaINDY crystallized in the presence of alpha-ketoglu... -

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Basic information

Entry
Database: PDB / ID: 6wtw
TitleStructure of LaINDY crystallized in the presence of alpha-ketoglutarate and malate
ComponentsDASS family sodium-coupled anion symporter
KeywordsTRANSPORT PROTEIN / Transporter / Structural Genomics / PSI-Biology / New York Consortium on Membrane Protein Structure / NYCOMPS / MEMBRANE PROTEIN
Function / homologyCitrate carrier CitT-related / Sodium:sulfate symporter transmembrane region / Solute carrier family 13 / transmembrane transporter activity / membrane => GO:0016020 / membrane / DASS family sodium-coupled anion symporter / 2-oxoglutarate-malate translocator
Function and homology information
Biological speciesLactobacillus acidophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.86 Å
AuthorsSauer, D.B. / Cocco, N. / Marden, J.J. / Song, J.M. / Wang, D.N. / New York Consortium on Membrane Protein Structure (NYCOMPS)
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS108151 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121994 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK099023 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Elife / Year: 2020
Title: Structural basis for the reaction cycle of DASS dicarboxylate transporters.
Authors: David B Sauer / Noah Trebesch / Jennifer J Marden / Nicolette Cocco / Jinmei Song / Akiko Koide / Shohei Koide / Emad Tajkhorshid / Da-Neng Wang /
Abstract: Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the ...Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.
History
DepositionMay 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DASS family sodium-coupled anion symporter
B: DASS family sodium-coupled anion symporter


Theoretical massNumber of molelcules
Total (without water)107,0792
Polymers107,0792
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5400 Å2
ΔGint-59 kcal/mol
Surface area36960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.328, 76.609, 96.946
Angle α, β, γ (deg.)90.000, 90.485, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: ALA / Beg label comp-ID: ALA / End auth comp-ID: ILE / End label comp-ID: ILE / Auth seq-ID: 0 - 487 / Label seq-ID: 3 - 490

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1(chain 'A' and (resid 0 through 265 or (resid 266...AA
2chain 'B'BB

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Components

#1: Protein DASS family sodium-coupled anion symporter


Mass: 53539.527 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus acidophilus (bacteria) / Gene: D7H66_01865 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3A9Y7Q2, UniProt: Q5FKK5*PLUS

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3 Å3/Da / Density % sol: 58.97 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 6.8 / Details: 30% Jeffamine ED-2001, 100 mM HEPES pH 6.8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 13, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.86→50.09 Å / Num. obs: 30640 / % possible obs: 99.4 % / Redundancy: 6.3 % / Biso Wilson estimate: 71.52 Å2 / CC1/2: 0.966 / Net I/σ(I): 16
Reflection shellResolution: 2.86→2.9 Å / Num. unique obs: 1410 / CC1/2: 0.8

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Processing

Software
NameVersionClassification
PHENIXdev_3707refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6WU1

6wu1


Resolution: 2.86→50.09 Å / SU ML: 0.4 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 31.6932
RfactorNum. reflection% reflection
Rfree0.2748 2011 6.57 %
Rwork0.2204 --
obs0.2239 30589 97.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 73.46 Å2
Refinement stepCycle: LAST / Resolution: 2.86→50.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7504 0 0 0 7504
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00587718
X-RAY DIFFRACTIONf_angle_d0.939510534
X-RAY DIFFRACTIONf_chiral_restr0.05091242
X-RAY DIFFRACTIONf_plane_restr0.00731254
X-RAY DIFFRACTIONf_dihedral_angle_d6.13541016
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.86-2.930.36611210.30181524X-RAY DIFFRACTION74.7
2.93-3.010.34931450.28082021X-RAY DIFFRACTION97.96
3.01-3.10.34681490.26312038X-RAY DIFFRACTION99.41
3.1-3.20.2841440.24092088X-RAY DIFFRACTION99.82
3.2-3.310.33821280.2412080X-RAY DIFFRACTION99.86
3.31-3.440.28811480.22942061X-RAY DIFFRACTION99.91
3.45-3.60.2871490.22162083X-RAY DIFFRACTION100
3.6-3.790.30171470.21812079X-RAY DIFFRACTION99.96
3.79-4.030.25331500.21482085X-RAY DIFFRACTION99.91
4.03-4.340.25831440.20362064X-RAY DIFFRACTION100
4.34-4.780.26651300.18442129X-RAY DIFFRACTION100
4.78-5.470.2491500.19922079X-RAY DIFFRACTION99.91
5.47-6.880.25411490.24692106X-RAY DIFFRACTION99.96
6.89-50.090.25981570.21772141X-RAY DIFFRACTION99.39
Refinement TLS params.Method: refined / Origin x: -25.6886249193 Å / Origin y: 6.20677519193 Å / Origin z: -24.3539988797 Å
111213212223313233
T0.557159847268 Å20.04080727828 Å20.0424983623239 Å2-0.327313844475 Å2-0.0103285257806 Å2--0.580393361488 Å2
L2.04911468759 °20.685779821006 °2-0.109605286114 °2-0.871780926024 °2-0.096518779062 °2--0.346793023514 °2
S0.102191510102 Å °-0.107628846572 Å °-0.0476691818167 Å °0.22166509773 Å °-0.0764011824466 Å °-0.0794249220596 Å °-0.0325068159649 Å °0.0288044293203 Å °-0.0319878785031 Å °
Refinement TLS groupSelection details: all

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