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- EMDB-2636: 3D EM map of the sodium proton antiporter MjNhaP1 from Methanocal... -

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Basic information

Entry
Database: EMDB / ID: EMD-2636
Title3D EM map of the sodium proton antiporter MjNhaP1 from Methanocaldococcus jannaschii
Map dataA B-factor of -200 was applied.
Sample
  • Sample: 3D EM map of the sodium/proton antiporter MjNhaP1
  • Protein or peptide: MjNhaP1
Keywordsmembrane protein / antiporter / transporter / exchanger / CPA
Function / homology
Function and homology information


: / transport / antiporter activity / sodium ion transport / monoatomic cation transport / monoatomic ion transport / proton transmembrane transport / transmembrane transport / membrane => GO:0016020 / membrane ...: / transport / antiporter activity / sodium ion transport / monoatomic cation transport / monoatomic ion transport / proton transmembrane transport / transmembrane transport / membrane => GO:0016020 / membrane / identical protein binding / plasma membrane
Similarity search - Function
Sodium/solute symporter superfamily / Cation/H+ exchanger / Cation/H+ exchanger / Sodium/hydrogen exchanger family
Similarity search - Domain/homology
Na(+)/H(+) antiporter 1
Similarity search - Component
Biological speciesMethanocaldococcus jannaschii (archaea)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 6.0 Å
AuthorsPaulino C / Woehlert D / Yildiz O / Kuhlbrandt W
CitationJournal: Elife / Year: 2014
Title: Structure and transport mechanism of the sodium/proton antiporter MjNhaP1.
Authors: Cristina Paulino / David Wöhlert / Ekaterina Kapotova / Özkan Yildiz / Werner Kühlbrandt /
Abstract: Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 ...Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 in two complementary states. The inward-open state was obtained by x-ray crystallography in the presence of sodium at pH 8, where the transporter is highly active. The outward-open state was obtained by electron crystallography without sodium at pH 4, where MjNhaP1 is inactive. Comparison of both structures reveals a 7° tilt of the 6 helix bundle. (22)Na(+) uptake measurements indicate non-cooperative transport with an activity maximum at pH 7.5. We conclude that binding of a Na(+) ion from the outside induces helix movements that close the extracellular cavity, open the cytoplasmic funnel, and result in a ∼5 Å vertical relocation of the ion binding site to release the substrate ion into the cytoplasm.
History
DepositionApr 25, 2014-
Header (metadata) releaseMay 28, 2014-
Map releaseDec 17, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 100
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 100
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4d0a
  • Surface level: 100
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2636.png

Downloads & links

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Map

FileDownload / File: emd_2636.map.gz / Format: CCP4 / Size: 6.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA B-factor of -200 was applied.
Voxel sizeX: 1.01875 Å / Y: 0.99327 Å / Z: 1 Å
Density
Contour LevelBy AUTHOR: 1.8 / Movie #1: 100
Minimum - Maximum-275.961944580000022 - 361.103149410000015
Average (Standard dev.)0.1016627 (±50.196964260000001)
SymmetrySpace group: 18
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin0-100-51
Dimensions82201103
Spacing10480200
CellA: 81.5 Å / B: 103.30008 Å / C: 200.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.018750.993269230769231
M x/y/z80104200
origin x/y/z0.0000.0000.000
length x/y/z81.500103.300200.000
α/β/γ90.00090.00090.000
start NX/NY/NZ0-51-100
NX/NY/NZ82103201
MAP C/R/S312
start NC/NR/NS-1000-51
NC/NR/NS20182103
D min/max/mean-275.962361.1030.102

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Supplemental data

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Sample components

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Entire : 3D EM map of the sodium/proton antiporter MjNhaP1

EntireName: 3D EM map of the sodium/proton antiporter MjNhaP1
Components
  • Sample: 3D EM map of the sodium/proton antiporter MjNhaP1
  • Protein or peptide: MjNhaP1

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Supramolecule #1000: 3D EM map of the sodium/proton antiporter MjNhaP1

SupramoleculeName: 3D EM map of the sodium/proton antiporter MjNhaP1 / type: sample / ID: 1000 / Details: protein was purified in absence of sodium. / Oligomeric state: Dimer / Number unique components: 1
Molecular weightExperimental: 46 KDa / Theoretical: 46 KDa / Method: SDS-PAGE, MS

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Macromolecule #1: MjNhaP1

MacromoleculeName: MjNhaP1 / type: protein_or_peptide / ID: 1 / Name.synonym: NhaP1 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Methanocaldococcus jannaschii (archaea) / synonym: Methanocaldococcus jannaschii / Location in cell: Plasma membrane
Molecular weightExperimental: 46 KDa / Theoretical: 46 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET26b
SequenceUniProtKB: Na(+)/H(+) antiporter 1
GO: transport, monoatomic ion transport, monoatomic cation transport, sodium ion transport, transmembrane transport, proton transmembrane transport, antiporter activity, GO: 0015299, identical ...GO: transport, monoatomic ion transport, monoatomic cation transport, sodium ion transport, transmembrane transport, proton transmembrane transport, antiporter activity, GO: 0015299, identical protein binding, plasma membrane, membrane, membrane => GO:0016020
InterPro: Cation/H+ exchanger

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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: 4 / Details: 25mM KAc pH4, 200mM KCl, 5mM glycerol, 5mM MPD
StainingType: NEGATIVE / Details: back injection method with 4% trehalose
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NITROGEN / Chamber humidity: 20 % / Chamber temperature: 77 K / Instrument: OTHER / Details: all buffers used were sodium-free
Timed resolved state: sample was plunge-frozen in liquid nitrogen
Method: back injection method (Wang & Kuhlbrandt, 1991) with 4% trehalose
DetailsE.coli polar lipids with a lipid-to-protein ration (LPR) of 0.4-0.5 were used. 2D crystals were grown by slow removal of detergent (0.15% DM) by dialysis.
Crystal formationDetails: E.coli polar lipids with a lipid-to-protein ration (LPR) of 0.4-0.5 were used. 2D crystals were grown by slow removal of detergent (0.15% DM) by dialysis.

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Electron microscopy

MicroscopeJEOL 3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 53000 / Illumination mode: SPOT SCAN / Imaging mode: OTHER / Cs: 1.6 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.12 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: -
Sample stageSpecimen holder: helium-cooled top entry stage with fixed specimen holder.
Specimen holder model: JEOL / Tilt angle max: 45 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 45 °
TemperatureMin: 4 K / Max: 10 K / Average: 4 K
Alignment procedureLegacy - Astigmatism: objective lens was corrected at 60kx and/or 300kx magnification
Legacy - Electron beam tilt params: -
DateDec 1, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 128 / Average electron dose: 25 e/Å2
Tilt angle min0

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Image processing

Crystal parametersUnit cell - A: 81.5 Å / Unit cell - B: 103.3 Å / Unit cell - C: 200 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 2 21 21
CTF correctionDetails: 2dx
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: OTHER / Software - Name: 2dx
Details: 6A in plane resolution and 14A resolution in the z direction.
DetailsImages were processed with the 2dx software.

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Atomic model buiding 1

Initial modelPDB ID:

4czb


Chain - Chain ID: B
SoftwareName: Coot
DetailsThe X-ray structure of the same protein (4czb) obtained at different conditions was manually fitted to the 3D EM density map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4d0a:
3D EM map of the sodium proton antiporter MjNhaP1 from Methanocaldococcus jannaschii

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