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- PDB-6ww5: Structure of VcINDY-Na-Fab84 in nanodisc -

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Basic information

Entry
Database: PDB / ID: 6ww5
TitleStructure of VcINDY-Na-Fab84 in nanodisc
Components
  • DASS family sodium-coupled anion symporter
  • Fab84 Heavy Chain
  • Fab84 Light Chain
KeywordsMEMBRANE PROTEIN / Transporter
Function / homologySolute carrier family 13 / Sodium/sulphate symporter, conserved site / transmembrane transporter activity / integral component of membrane / Transporter, NadC family
Function and homology information
Biological speciesVibrio cholerae (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsSauer, D.B. / Marden, J. / Song, J.M. / Koide, A. / Koide, S. / Wang, D.N.
Funding support United States, 3items
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
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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: DASS family sodium-coupled anion symporter
B: DASS family sodium-coupled anion symporter
C: Fab84 Heavy Chain
E: Fab84 Heavy Chain
F: Fab84 Light Chain
D: Fab84 Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,40212
Polymers204,1806
Non-polymers1,2226
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18750 Å2
ΔGint-77 kcal/mol
Surface area66520 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_1ens_2chain "C"
d_2ens_2chain "E"
d_1ens_3chain "F"
d_2ens_3chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LEUGLNA1 - 445
d_21ens_1LEUGLNB1 - 445
d_11ens_2GLULYSC1 - 228
d_21ens_2GLULYSD1 - 228
d_11ens_3METGLYE1 - 209
d_21ens_3METGLYL1 - 209

NCS ensembles:
ID
ens_1
ens_2
ens_3

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein DASS family sodium-coupled anion symporter / Transporter / NadC family


Mass: 48157.359 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Gene: VC0395_0108, GG844_00510 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0H3AG83

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Antibody , 2 types, 4 molecules CEFD

#2: Antibody Fab84 Heavy Chain


Mass: 28009.484 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab84 Light Chain


Mass: 25923.102 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 6 molecules

#4: Chemical ChemComp-6PE / 1,2-DIHEXANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE


Mass: 410.420 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H33NO8P / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-HEX / HEXANE / Hexane


Mass: 86.175 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14
#6: Chemical ChemComp-OCT / N-OCTANE / Octane


Mass: 114.229 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H18

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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: Complex of VcINDY and Fab84 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Vibrio cholerae (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 100 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 10 sec. / Electron dose: 44 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 826

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3855refinement
PHENIX1.18_3855refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC2.12CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC2.12initial Euler assignment
11cryoSPARC2.12final Euler assignment
12cryoSPARC2.12classification
13cryoSPARC2.123D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92239 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5UL9
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.58 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.012613692
ELECTRON MICROSCOPYf_angle_d0.934318630
ELECTRON MICROSCOPYf_chiral_restr0.06262198
ELECTRON MICROSCOPYf_plane_restr0.00832304
ELECTRON MICROSCOPYf_dihedral_angle_d11.63171896
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefinement-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000712566409578
ens_2d_2CELECTRON MICROSCOPYNCS constraints0.000709774174697
ens_3d_2EELECTRON MICROSCOPYNCS constraints0.0791440733326

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