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- EMDB-10104: Structure of Azospirillum brasilense Glutamate Synthase in a4b4 o... -

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Basic information

Entry
Database: EMDB / ID: EMD-10104
TitleStructure of Azospirillum brasilense Glutamate Synthase in a4b4 oligomeric state.
Map data
SampleGlutamate Synthase complex in a4b4 oligomeric state:
(Glutamate synthase [NADPH] ...) x 2 / (ligand) x 4
Function / homology
Function and homology information


glutamate synthase (NADPH) / glutamate synthase (NADPH) activity / L-glutamate biosynthetic process / glutamine metabolic process / 3 iron, 4 sulfur cluster binding / 4 iron, 4 sulfur cluster binding / metal ion binding
FAD/NAD(P)-binding domain / Glutamate synthase, central-N / FAD/NAD(P)-binding domain superfamily / Nucleophile aminohydrolases, N-terminal / Dihydroprymidine dehydrogenase domain II / GXGXG motif / Glutamine amidotransferase type 2 domain / Aldolase-type TIM barrel / Alpha-helical ferredoxin / Glutamate synthase, NADH/NADPH, small subunit 2 ...FAD/NAD(P)-binding domain / Glutamate synthase, central-N / FAD/NAD(P)-binding domain superfamily / Nucleophile aminohydrolases, N-terminal / Dihydroprymidine dehydrogenase domain II / GXGXG motif / Glutamine amidotransferase type 2 domain / Aldolase-type TIM barrel / Alpha-helical ferredoxin / Glutamate synthase, NADH/NADPH, small subunit 2 / Glutamine amidotransferases class-II / Glutamate synthase domain / Glutamate synthase, alpha subunit, C-terminal / Conserved region in glutamate synthase / Glutamate synthase central domain / Pyridine nucleotide-disulphide oxidoreductase / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / Glutamine amidotransferase type 2 domain profile. / Glutamate synthase, alpha subunit, C-terminal domain superfamily
Glutamate synthase [NADPH] large chain / Glutamate synthase [NADPH] small chain
Biological speciesAzospirillum brasilense (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSwuec P
CitationJournal: J. Mol. Biol. / Year: 2019
Title: Cryo-EM Structures of Azospirillum brasilense Glutamate Synthase in Its Oligomeric Assemblies.
Authors: Paolo Swuec / Antonio Chaves-Sanjuan / Carlo Camilloni / Maria Antonietta Vanoni / Martino Bolognesi /
Abstract: Bacterial NADPH-dependent glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive synthesis of two L-Glu molecules from L-Gln and 2-oxo-glutarate. GltS functional ...Bacterial NADPH-dependent glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive synthesis of two L-Glu molecules from L-Gln and 2-oxo-glutarate. GltS functional unit hosts an α-subunit (αGltS) and a β-subunit (βGltS) that assemble in different αβ oligomers in solution. Here, we present the cryo-electron microscopy structures of Azospirillum brasilense GltS in four different oligomeric states (αβ, αβ, αβ and αβ, in the 3.5- to 4.1-Å resolution range). Our study provides a comprehensive GltS model that details the inter-protomeric assemblies and allows unequivocal location of the FAD cofactor and of two electron transfer [4Fe-4S] clusters within βGltS.
Validation ReportPDB-ID: 6s6s

SummaryFull reportAbout validation report
History
DepositionJul 3, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 2019-
UpdateSep 11, 2019-
Current statusSep 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6s6s
  • Surface level: 0.09
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10104.map.gz / Format: CCP4 / Size: 111.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 308 pix.
= 439.208 Å
1.43 Å/pix.
x 308 pix.
= 439.208 Å
1.43 Å/pix.
x 308 pix.
= 439.208 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.426 Å
Density
Contour LevelBy AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum0.0 - 1.0
Average (Standard dev.)0.019289454 (±0.12449379)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions308308308
Spacing308308308
CellA=B=C: 439.208 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.4261.4261.426
M x/y/z308308308
origin x/y/z0.0000.0000.000
length x/y/z439.208439.208439.208
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS308308308
D min/max/mean-0.1890.6610.000

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Supplemental data

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Segmentation: #1

Fileemd_10104_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Glutamate Synthase complex in a4b4 oligomeric state

EntireName: Glutamate Synthase complex in a4b4 oligomeric state / Number of components: 7

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Component #1: protein, Glutamate Synthase complex in a4b4 oligomeric state

ProteinName: Glutamate Synthase complex in a4b4 oligomeric state / Recombinant expression: No
SourceSpecies: Azospirillum brasilense (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Glutamate synthase [NADPH] large chain

ProteinName: Glutamate synthase [NADPH] large chain / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 166.224734 kDa
SourceSpecies: Azospirillum brasilense (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Glutamate synthase [NADPH] small chain

ProteinName: Glutamate synthase [NADPH] small chain / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 52.425109 kDa
SourceSpecies: Azospirillum brasilense (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: ligand, FLAVIN MONONUCLEOTIDE

LigandName: FLAVIN MONONUCLEOTIDE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.456344 kDa

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Component #5: ligand, FE3-S4 CLUSTER

LigandName: FE3-S4 CLUSTER / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.295795 kDa

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Component #6: ligand, IRON/SULFUR CLUSTER

LigandName: IRON/SULFUR CLUSTERIron–sulfur cluster / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 0.35164 kDa

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Component #7: ligand, FLAVIN-ADENINE DINUCLEOTIDE

LigandName: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.78555 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 54434
3D reconstructionSoftware: RELION / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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