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- EMDB-1756: Electron crystallography reveals the asymmetry of the trimeric gl... -

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Entry
Database: EMDB / ID: EMD-1756
TitleElectron crystallography reveals the asymmetry of the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum
Map dataThis is a 3D volume reconstructed from 2D crystals of BetP.
Sample
  • Sample: Sodium/betaine symporter BetP with 45 amino acids truncated at the C-terminus
  • Protein or peptide: Sodium betaine symporter
KeywordsBCCT family / secondary transporter / osmotic stress
Biological speciesCorynebacterium glutamicum (bacteria)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 8.0 Å
AuthorsTsai C-J / Khafizov KF / Hakulinen J / Forrest LR / Kraemer R / Kuehlbrandt W / Ziegler C
CitationJournal: J Mol Biol / Year: 2011
Title: Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum.
Authors: Ching-Ju Tsai / Kamil Khafizov / Jonna Hakulinen / Lucy R Forrest / Lucy R Forrest / Reinhard Krämer / Werner Kühlbrandt / Christine Ziegler /
Abstract: The Na(+)-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own ...The Na(+)-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.
History
DepositionJul 7, 2010-
Header (metadata) releaseJul 23, 2010-
Map releaseJul 23, 2010-
UpdateFeb 24, 2012-
Current statusFeb 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.077
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.077
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-1765.jpg

Downloads & links

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Map

FileDownload / File: emd_1756.map.gz / Format: CCP4 / Size: 326.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D volume reconstructed from 2D crystals of BetP.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
2.26 Å/pix.
x 50 pix.
= 154. Å		2.29 Å/pix.
x 50 pix.
= 91.6 Å		2.34 Å/pix.
x 34 pix.
= 150. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 2.29 Å / Y: 2.26471 Å / Z: 2.34375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.077 / Movie #1: 0.077
Minimum - Maximum-0.31079239 - 0.31979445
Average (Standard dev.)-0.00266912 (±0.07689252)
SymmetrySpace group: 4
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin000
Dimensions503450
Spacing684064
CellA: 91.6 Å / B: 154.0 Å / C: 150.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.292.26470588235292.34375
M x/y/z406864
origin x/y/z0.0000.0000.000
length x/y/z91.600154.000150.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ505034
MAP C/R/S312
start NC/NR/NS000
NC/NR/NS345050
D min/max/mean-0.3110.320-0.003

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Supplemental data

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Sample components

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Entire : Sodium/betaine symporter BetP with 45 amino acids truncated at th...

EntireName: Sodium/betaine symporter BetP with 45 amino acids truncated at the C-terminus
Components
  • Sample: Sodium/betaine symporter BetP with 45 amino acids truncated at the C-terminus
  • Protein or peptide: Sodium betaine symporter

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Supramolecule #1000: Sodium/betaine symporter BetP with 45 amino acids truncated at th...

SupramoleculeName: Sodium/betaine symporter BetP with 45 amino acids truncated at the C-terminus
type: sample / ID: 1000
Details: 2D crystals were grown with lipids extracted from Corynebacterium glutamicum
Oligomeric state: One assymetric homotrimer of BetP / Number unique components: 1
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Sodium betaine symporter

MacromoleculeName: Sodium betaine symporter / type: protein_or_peptide / ID: 1 / Name.synonym: BetP / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Corynebacterium glutamicum (bacteria) / Location in cell: Cell membrane
Molecular weightTheoretical: 180 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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 mg/mL
BufferpH: 7.5
Details: 50mM pH 7.5 Tris-HCl, 200mM NaCl, 4mM CaCl2, 5% glycerol, 5% MPD, 3mM NaN3
StainingType: NEGATIVE / Details: Samples embedded in 5% glucose
GridDetails: 400 mesh copper or molybdenum grid
VitrificationCryogen name: NITROGEN / Chamber humidity: 20 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Manual freezing

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Electron microscopy

MicroscopeJEOL 3000SFF
TemperatureMin: 4.2 K / Max: 20 K / Average: 4.2 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 72 / Average electron dose: 10 e/Å2 / Details: Image size 6000 pixel. / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 1.6 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 45000
Sample stageSpecimen holder: Cartridges with tilt angles from 0-50 degrees
Specimen holder model: JEOL / Tilt angle min: -50 / Tilt angle max: 50 / Tilt series - Axis1 - Min angle: -50 ° / Tilt series - Axis1 - Max angle: 50 °

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Image processing

DetailsImages were processed using Image2000 package.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Software - Name: Image2000 / Details: B factor was set to -300.
Crystal parametersUnit cell - A: 92.1 Å / Unit cell - B: 155.1 Å / Unit cell - C: 150 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Plane group: P 1 21
CTF correctionDetails: CTF correction of each image

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