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- EMDB-1440: The subnanometer resolution structure of the glutamate synthase 1... -

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Basic information

Entry
Database: EMDB / ID: EMD-1440
TitleThe subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications.
Map dataThis is the 3D map file of Azospirillum brasilense NADPH-GltS (glutamate synthase)
Sample
  • Sample: Azospirillum brasilense NADPH-GltS
  • Protein or peptide: NADPH-glutamate synthase
Function / homology
Function and homology information


glutamate synthase (NADPH) / glutamate synthase (NADPH) activity / L-glutamate biosynthetic process / 3 iron, 4 sulfur cluster binding / glutamine metabolic process / 4 iron, 4 sulfur cluster binding / metal ion binding
Similarity search - Function
Glutamate synthase NADPH small chain-like / : / Glutamate synthase, central-N / Glutamate synthase domain / Glutamate synthase, central-N / Glutamine amidotransferases class-II / Conserved region in glutamate synthase / Glutamate synthase central domain / Glutamate synthase, alpha subunit, C-terminal / : ...Glutamate synthase NADPH small chain-like / : / Glutamate synthase, central-N / Glutamate synthase domain / Glutamate synthase, central-N / Glutamine amidotransferases class-II / Conserved region in glutamate synthase / Glutamate synthase central domain / Glutamate synthase, alpha subunit, C-terminal / : / GXGXG motif / Glutamate synthase, alpha subunit, C-terminal domain superfamily / Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / Glutamine amidotransferase type 2 domain profile. / Glutamine amidotransferase type 2 domain / Alpha-helical ferredoxin / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / Nucleophile aminohydrolases, N-terminal / FAD/NAD(P)-binding domain superfamily / Aldolase-type TIM barrel
Similarity search - Domain/homology
Glutamate synthase [NADPH] large chain / Glutamate synthase [NADPH] small chain
Similarity search - Component
Biological speciesAzospirillum brasilense (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.5 Å
AuthorsCottevieille M / Larquet E / Jonic S / Petoukhov MV / Caprini G / Paravisi S / Svergun DI / Vanoni MA / Boisset N
CitationJournal: J Biol Chem / Year: 2008
Title: The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications.
Authors: Magali Cottevieille / Eric Larquet / Slavica Jonic / Maxim V Petoukhov / Gianluca Caprini / Stefano Paravisi / Dmitri I Svergun / Maria A Vanoni / Nicolas Boisset /
Abstract: The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, ...The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alphabeta protomer only 3-fold by increasing the K(m) values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (alphabeta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.
History
DepositionApr 18, 2007-
Header (metadata) releaseOct 4, 2007-
Map releaseJan 9, 2008-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 125
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 125
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2vdc
  • Surface level: 125
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1440.gif

emd_1440.tif

emd_1440_1.tif

Downloads & links

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Map

FileDownload / File: emd_1440.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the 3D map file of Azospirillum brasilense NADPH-GltS (glutamate synthase)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.59 Å/pix.
x 192 pix.
= 304.8 Å		1.59 Å/pix.
x 192 pix.
= 304.8 Å		1.59 Å/pix.
x 192 pix.
= 304.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5875 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 125.0 / Movie #1: 125
Minimum - Maximum-799.538000000000011 - 1332.420000000000073
Average (Standard dev.)5.0626 (±98.685500000000005)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 304.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.58751.58751.5875
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z304.800304.800304.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-60-60-59
NX/NY/NZ120120120
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-799.5381332.4185.063

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Supplemental data

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Sample components

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Entire : Azospirillum brasilense NADPH-GltS

EntireName: Azospirillum brasilense NADPH-GltS
Components
  • Sample: Azospirillum brasilense NADPH-GltS
  • Protein or peptide: NADPH-glutamate synthase

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Supramolecule #1000: Azospirillum brasilense NADPH-GltS

SupramoleculeName: Azospirillum brasilense NADPH-GltS / type: sample / ID: 1000 / Oligomeric state: Glutamate synthase is a heterohexamer / Number unique components: 1
Molecular weightExperimental: 1.2 MDa / Theoretical: 1.29 MDa / Method: small-angle X-ray scattering

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Macromolecule #1: NADPH-glutamate synthase

MacromoleculeName: NADPH-glutamate synthase / type: protein_or_peptide / ID: 1 / Name.synonym: bacterial glutamate synthase
Details: Additional InterPro entries: IPR006982 Glutamate synthase, central-N IPR012220 Glutamate synthase, eukaryotic IPR012061 Glutamate synthase, large subunit region 3, putative IPR012375 ...Details: Additional InterPro entries: IPR006982 Glutamate synthase, central-N IPR012220 Glutamate synthase, eukaryotic IPR012061 Glutamate synthase, large subunit region 3, putative IPR012375 Glutamate synthase, large subunit region 1 amidotransferase, putative IPR002932 Glutamate synthase, central-C IPR002489 Glutamate synthase, alpha subunit, C-terminal IPR014666 Ferredoxin-dependent glutamate synthase IPR012075 Glutamate synthase, large subunit region 1 and 3, putative IPR006006 Glutamate synthase, NADH/NADPH, small subunit 2 IPR006005 Glutamate synthase, NADH/NADPH, small subunit 1
Number of copies: 6 / Oligomeric state: (alpha)6 (beta)6 / Recombinant expression: Yes
Source (natural)Organism: Azospirillum brasilense (bacteria) / Strain: Sp7 / Location in cell: cytoplasm
Molecular weightExperimental: 1.29 MDa / Theoretical: 1.2 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET11a
SequenceGO: glutamate synthase (NADPH) activity / InterPro: Glutamate synthase, central-N

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration9.25 mg/mL
BufferpH: 7.5 / Details: 25 mM Hepes/KOH, 1 mM EDTA, 1 mM DTT
StainingType: NEGATIVE
Details: CRYOEM : 4 microL were applied on a 200 mesh copper grid, coated with a thin holey carbon film. After blotting the excess of solution with Whatman paper, the grid was rapidly plunged into liquid ethane
GridDetails: 200 mesh copper grid, coated with a thin holey
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: manual plunger / Method: Manual single-sided blotting

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Electron microscopy

MicroscopeJEOL 2010UHR
TemperatureMin: 91.15 K / Max: 93.15 K / Average: 93.15 K
Detailslow-dose illumination
DateMay 10, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 1.59 µm / Number real images: 151 / Average electron dose: 10 e/Å2 / Details: Scanner model : Nikon Coolscan 8000ED / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.5 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsParticles were automatically selected using Roseman algorithm with SPIDER software
CTF correctionDetails: Wiener filtration of volumes from focal series
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 12800
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: Chimera
DetailsProtocol: Rigid Body. One alpha dimer (1EA0) and one beta model (homology modelling) were separately fitted by manual docking using Chimera software. D3 symmetry was used to reconstruct the whole complex.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2vdc:
THE 9.5 A RESOLUTION STRUCTURE OF GLUTAMATE SYNTHASE FROM CRYO-ELECTRON MICROSCOPY AND ITS OLIGOMERIZATION BEHAVIOR IN SOLUTION: FUNCTIONAL IMPLICATIONS.

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