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- EMDB-21928: Structure of VcINDY-Na-Fab84 in nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-21928
TitleStructure of VcINDY-Na-Fab84 in nanodisc
Map data
Sample
  • Organelle or cellular component: Complex of VcINDY and Fab84
    • Protein or peptide: DASS family sodium-coupled anion symporter
    • Protein or peptide: Fab84 Heavy Chain
    • Protein or peptide: Fab84 Light Chain
  • Ligand: 1,2-DIHEXANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE
  • Ligand: HEXANE
  • Ligand: N-OCTANEOctane
Function / homology
Function and homology information


succinate transmembrane transporter activity / membrane / identical protein binding
Similarity search - Function
Citrate transporter-like domain / Citrate transporter / Sodium/sulphate symporter, conserved site / Sodium:sulfate symporter family signature. / Solute carrier family 13
Similarity search - Domain/homology
Transporter, NadC family / Transporter, NadC family
Similarity search - Component
Biological speciesVibrio cholerae (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsSauer DB / Marden J / Song JM / Koide A / Koide S / Wang DN
Funding support United States, 3 items
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 Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK099023 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121994 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 7, 2020-
Header (metadata) releaseSep 16, 2020-
Map releaseSep 16, 2020-
UpdateSep 16, 2020-
Current statusSep 16, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.71
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.71
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6ww5
  • Surface level: 4.71
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21928.png

Downloads & links

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Map

FileDownload / File: emd_21928.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.142 Å
Density
Contour LevelBy AUTHOR: 4.71 / Movie #1: 4.71
Minimum - Maximum-28.273418 - 40.597233
Average (Standard dev.)-0.000000000 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 292.352 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1421.1421.142
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z292.352292.352292.352
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ310310310
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-28.27340.597-0.000

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Supplemental data

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Half map: #1

Fileemd_21928_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_21928_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of VcINDY and Fab84

EntireName: Complex of VcINDY and Fab84
Components
  • Organelle or cellular component: Complex of VcINDY and Fab84
    • Protein or peptide: DASS family sodium-coupled anion symporter
    • Protein or peptide: Fab84 Heavy Chain
    • Protein or peptide: Fab84 Light Chain
  • Ligand: 1,2-DIHEXANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE
  • Ligand: HEXANE
  • Ligand: N-OCTANEOctane

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Supramolecule #1: Complex of VcINDY and Fab84

SupramoleculeName: Complex of VcINDY and Fab84 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Vibrio cholerae (bacteria)

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Macromolecule #1: DASS family sodium-coupled anion symporter

MacromoleculeName: DASS family sodium-coupled anion symporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 48.157359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: REWFLHRNSL IVLADVALFL ALYHFLPFEH NVVLGISMLA FIAVLWLTEA LHVTVTAILV PVMAVFFGIF ETQAALNNFA NSIIFLFLG GFALAAAMHH QGLDKVIADK VLAMAQGKMS VAVFMLFGVT ALLSMWISNT ATAAMMLPLV LGVLSKVDAD K QRSTYVFV ...String:
REWFLHRNSL IVLADVALFL ALYHFLPFEH NVVLGISMLA FIAVLWLTEA LHVTVTAILV PVMAVFFGIF ETQAALNNFA NSIIFLFLG GFALAAAMHH QGLDKVIADK VLAMAQGKMS VAVFMLFGVT ALLSMWISNT ATAAMMLPLV LGVLSKVDAD K QRSTYVFV LLGVAYSASI GGIATLVGSP PNAIAAAEVG LSFTDWMKFG LPTAMMMLPM AIAILYFLLK PTLNGMFELD RA PVNWDKG KVVTLGIFGL TVFLWIFSSP INAALGGFKS FDTLVALGAI LMLSFARVVH WKEIQKTADW GVLLLFGGGL CLS NVLKQT GTSVFLANAL SDMVSHMGIF VVILVVATFV VFLTEFASNT ASAALLIPVF ATVAEAFGMS PVLLSVLIAV AASC AFMLP VATPPNAIVF ASGHIKQSEM MRVGLYLNIA CIGLLTAIAM LFWQ

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Macromolecule #2: Fab84 Heavy Chain

MacromoleculeName: Fab84 Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.009484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFTIYSSSI HWVRQAPGKG LEWVASIYSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARYNIHY WRWYNPAGQA MDYWGQGTLV TVFNQIKGGP S VFPLAPSS ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFTIYSSSI HWVRQAPGKG LEWVASIYSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARYNIHY WRWYNPAGQA MDYWGQGTLV TVFNQIKGGP S VFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NH KPSNTKV DKKVEPKSCD KTHT

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Macromolecule #3: Fab84 Light Chain

MacromoleculeName: Fab84 Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.923102 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQRYGLL VTFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQRYGLL VTFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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Macromolecule #4: 1,2-DIHEXANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE

MacromoleculeName: 1,2-DIHEXANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: 6PE
Molecular weightTheoretical: 410.42 Da
Chemical component information

ChemComp-6PE:
1,2-DIHEXANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE

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Macromolecule #5: HEXANE

MacromoleculeName: HEXANE / type: ligand / ID: 5 / Number of copies: 2 / Formula: HEX
Molecular weightTheoretical: 86.175 Da
Chemical component information

ChemComp-HEX:
HEXANE / Hexane

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Macromolecule #6: N-OCTANE

MacromoleculeName: N-OCTANE / type: ligand / ID: 6 / Number of copies: 2 / Formula: OCT
Molecular weightTheoretical: 114.229 Da
Chemical component information

ChemComp-OCT:
N-OCTANE / Octane

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 36000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 826 / Average exposure time: 10.0 sec. / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 2.12)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12) / Number images used: 92239

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Atomic model buiding 1

Initial modelPDB ID:

5ul9

Output model

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

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