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- PDB-6s6x: Structure of Azospirillum brasilense Glutamate Synthase in a6b6 o... -

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Basic information

Entry
Database: PDB / ID: 6s6x
TitleStructure of Azospirillum brasilense Glutamate Synthase in a6b6 oligomeric state.
Components(Glutamate synthase [NADPH] ...) x 2
KeywordsFLAVOPROTEIN / glutamate synthesys / complex / oligomeric assemblies
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsChaves-Sanjuan, A. / Camilloni, C. / Bolognesi, M.
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 Report
SummaryFull reportAbout validation report
History
DepositionJul 3, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Glutamate synthase [NADPH] large chain
B: Glutamate synthase [NADPH] large chain
C: Glutamate synthase [NADPH] large chain
D: Glutamate synthase [NADPH] large chain
E: Glutamate synthase [NADPH] large chain
F: Glutamate synthase [NADPH] large chain
G: Glutamate synthase [NADPH] small chain
H: Glutamate synthase [NADPH] small chain
I: Glutamate synthase [NADPH] small chain
J: Glutamate synthase [NADPH] small chain
K: Glutamate synthase [NADPH] small chain
L: Glutamate synthase [NADPH] small chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,325,34542
Polymers1,311,89912
Non-polymers13,44630
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area54430 Å2
ΔGint-573 kcal/mol
Surface area407100 Å2
MethodPISA

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Components

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Glutamate synthase [NADPH] ... , 2 types, 12 molecules ABCDEFGHIJKL

#1: Protein/peptide
Glutamate synthase [NADPH] large chain / Glutamate synthase subunit alpha / GLTS alpha chain / NADPH-GOGAT


Mass: 166224.734 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azospirillum brasilense (bacteria) / Gene: gltB / Production host: Escherichia coli (E. coli) / References: UniProt: Q05755, glutamate synthase (NADPH)
#2: Protein/peptide
Glutamate synthase [NADPH] small chain / Glutamate synthase subunit beta / GLTS beta chain / NADPH-GOGAT


Mass: 52425.109 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azospirillum brasilense (bacteria) / Gene: gltD / Production host: Escherichia coli (E. coli) / References: UniProt: Q05756, glutamate synthase (NADPH)

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Non-polymers , 4 types, 30 molecules

#3: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C17H21N4O9P / Flavin mononucleotide
#4: Chemical
ChemComp-F3S / FE3-S4 CLUSTER


Mass: 295.795 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Fe3S4
#5: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Fe4S4 / Iron–sulfur cluster
#6: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Flavin adenine dinucleotide / Comment: FAD *YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Glutamate Synthase complex in a6b6 oligomeric state / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Azospirillum brasilense (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software

Classification: refinement / Version: 1.16_3549 / NB:

Name
phenix.real_space_refine
PHENIX
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51384 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 72.78 Å2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.023392100
f_angle_d0.9841124818
f_chiral_restr0.125513890
f_plane_restr0.003916326
f_dihedral_angle_d16.372355116

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