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- EMDB-2387: Structure and substrate induced conformational changes of the sec... -

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Basic information

Entry
Database: EMDB / ID: EMD-2387
TitleStructure and substrate induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography
Map data3D reconstruction of CitS in Sodium Acetate buffer, pH 4.5
Sample
  • Sample: CitS dimer
  • Protein or peptide: CitS
KeywordsCitS / Secondary Transporter / Membrane Protein
Function / homology2-hydroxycarboxylate transporter / 2-hydroxycarboxylate transporter, Proteobacteria/Firmicutes / 2-hydroxycarboxylate transporter family / citrate metabolic process / organic anion transmembrane transporter activity / symporter activity / membrane => GO:0016020 / plasma membrane / Citrate-sodium symporter
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 6.0 Å
AuthorsKebbel F / Kurz M / Arheit M / Gruetter MG / Stahlberg H
CitationJournal: Structure / Year: 2013
Title: Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography.
Authors: Fabian Kebbel / Mareike Kurz / Marcel Arheit / Markus G Grütter / Henning Stahlberg /
Abstract: The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into ...The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 a-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of a helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of ‘‘alternating access’’.
History
DepositionMay 27, 2013-
Header (metadata) releaseJun 19, 2013-
Map releaseJun 19, 2013-
UpdateSep 4, 2013-
Current statusSep 4, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 46.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 46.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-4bpq
  • Surface level: 46.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_2387.tif

Downloads & links

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Map

FileDownload / File: emd_2387.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of CitS in Sodium Acetate buffer, pH 4.5
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 100 pix.
= 100. Å		1 Å/pix.
x 100 pix.
= 100. Å		1 Å/pix.
x 100 pix.
= 100. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 46.200000000000003 / Movie #1: 46.2
Minimum - Maximum-151.810104370000005 - 244.377960209999998
Average (Standard dev.)0.38074851 (±19.640151979999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-49-49-49
Dimensions100100100
Spacing100100100
CellA=B=C: 100.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z100.000100.000100.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-49-49-49
NC/NR/NS100100100
D min/max/mean-151.810244.3780.381

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Supplemental data

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Sample components

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Entire : CitS dimer

EntireName: CitS dimer
Components
  • Sample: CitS dimer
  • Protein or peptide: CitS

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Supramolecule #1000: CitS dimer

SupramoleculeName: CitS dimer / type: sample / ID: 1000 / Oligomeric state: 2 / Number unique components: 1
Molecular weightTheoretical: 95 KDa

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Macromolecule #1: CitS

MacromoleculeName: CitS / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: 2 / Recombinant expression: Yes
Source (natural)Organism: Klebsiella pneumoniae (bacteria) / Location in cell: inner membrane
Molecular weightTheoretical: 95 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C43(DE3)
SequenceUniProtKB: Citrate-sodium symporter / GO: membrane => GO:0016020

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration1.35 mg/mL
BufferpH: 4.5
Details: 20 mM sodium acetate, pH 4.5, 500 mM NaCl, 15 mM MgCl2, 2 mM DTT, 2 mM NaN3
StainingType: NEGATIVE
Details: 20 mM sodium acetate, pH 4.5, 200 mM NaCl, 15 mM MgCl2, 2 mM DTT, 2 mM NaN3
GridDetails: 400 mesh Co grid with 2 micrometer holes from Quantifoil, covered with a very thin additional continuous carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 80 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 4.5 seconds
DetailsDialysis
Crystal formationDetails: Dialysis

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureMin: 85 K / Max: 90 K / Average: 87 K
DetailsPre-exposure recorded on TVIPS-F416 of 0.1 ms, to reduce beam-induced resolution loss in subsequent image on film
DateDec 31, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 5 µm / Number real images: 79 / Average electron dose: 6 e/Å2 / Od range: 1.2 / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 45 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 45 °

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Image processing

DetailsImages were unbent using 2dx.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: OTHER / Software - Name: 2dx
Crystal parametersUnit cell - A: 96 Å / Unit cell - B: 106 Å / Unit cell - C: 200 Å / Unit cell - γ: 90 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 2 21 21

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