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- PDB-2vdc: THE 9.5 A RESOLUTION STRUCTURE OF GLUTAMATE SYNTHASE FROM CRYO-EL... -

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Entry
Database: PDB / ID: 2vdc
TitleTHE 9.5 A RESOLUTION STRUCTURE OF GLUTAMATE SYNTHASE FROM CRYO-ELECTRON MICROSCOPY AND ITS OLIGOMERIZATION BEHAVIOR IN SOLUTION: FUNCTIONAL IMPLICATIONS.
Components(GLUTAMATE SYNTHASE [NADPH] ...) x 2
KeywordsOXIDOREDUCTASE / AMIDOTRANSFERASE / AMMONIA ASSIMILATION / NADP / IRON / ZYMOGEN / NADPH-DEPENDENT GLUTAMATE SYNTHASE / IRON SULPHUR FLAVOPROTEIN / GLUTAMINE AMIDOTRANSFERASE / GLUTAMATE BIOSYNTHESIS / AMINO-ACID BIOSYNTHESIS
Function / homology
Function and homology information


glutamate synthase (NADPH) activity / glutamate synthase (NADPH) / L-glutamate biosynthetic process / glutamine metabolic process / 3 iron, 4 sulfur cluster binding / 4 iron, 4 sulfur cluster binding / metal ion binding
Pyridine nucleotide-disulphide oxidoreductase / Aldolase-type TIM barrel / Glutamate synthase central domain / Conserved region in glutamate synthase / GXGXG motif / Glutamine amidotransferases class-II / Glutamate synthase, alpha subunit, C-terminal domain superfamily / FAD/NAD(P)-binding domain superfamily / Nucleophile aminohydrolases, N-terminal / Dihydroprymidine dehydrogenase domain II ...Pyridine nucleotide-disulphide oxidoreductase / Aldolase-type TIM barrel / Glutamate synthase central domain / Conserved region in glutamate synthase / GXGXG motif / Glutamine amidotransferases class-II / Glutamate synthase, alpha subunit, C-terminal domain superfamily / FAD/NAD(P)-binding domain superfamily / Nucleophile aminohydrolases, N-terminal / Dihydroprymidine dehydrogenase domain II / FAD/NAD(P)-binding domain / Glutamine amidotransferase type 2 domain / Alpha-helical ferredoxin / Glutamate synthase, central-N / Glutamate synthase NADPH small chain-like / Glutamate synthase domain / Glutamate synthase, alpha subunit, C-terminal
Glutamate synthase [NADPH] large chain / Glutamate synthase [NADPH] small chain
Biological speciesAZOSPIRILLUM BRASILENSE (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.5 Å
AuthorsCottevieille, M. / Larquet, E. / Jonic, S. / Petoukhov, M.V. / Caprini, G. / Paravisi, S. / Svergun, D.I. / Vanoni, M.A. / 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 4, 2007Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2008Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Aug 23, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 1.4Oct 23, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB
Revision 1.5Nov 20, 2019Group: Advisory / Derived calculations
Category: database_PDB_caveat / pdbx_struct_conn_angle ...database_PDB_caveat / pdbx_struct_conn_angle / pdbx_validate_close_contact / struct_conn
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AJ" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AJ" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "BH" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "CJ" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "DH" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "EJ" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "FH" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN THE SHEET RECORDS BELOW, TWO SHEETS ARE DEFINED.

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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)1,281,31054
Polymers1,265,90012
Non-polymers15,41042
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31600 Å2
ΔGint-70.1 kcal/mol
Surface area461030 Å2
MethodPQS

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Components

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GLUTAMATE SYNTHASE [NADPH] ... , 2 types, 12 molecules ABCDEFGHIJKL

#1: Protein
GLUTAMATE SYNTHASE [NADPH] LARGE CHAIN / GLUTAMATE SYNTHASE ALPHA SUBUNIT / NADPH-GOGAT / GLTS ALPHA CHAIN


Mass: 161615.875 Da / Num. of mol.: 6 / Fragment: RESIDUES 37-1508, ALPHA SUBUNIT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) AZOSPIRILLUM BRASILENSE (bacteria) / Strain: SP7 / Plasmid: PET11A / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: Q05755, glutamate synthase (NADPH)
#2: Protein
GLUTAMATE SYNTHASE [NADPH] SMALL CHAIN / GLUTAMATE SYNTHASE BETA SUBUNIT / NADPH-GOGAT / GLTS BETA CHAIN


Mass: 49367.531 Da / Num. of mol.: 6 / Fragment: RESIDUES 27-482, BETA SUBUNIT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) AZOSPIRILLUM BRASILENSE (bacteria) / Strain: SP7 / Plasmid: PET11A / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: Q05756, glutamate synthase (NADPH)

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Non-polymers , 6 types, 42 molecules

#3: Chemical
ChemComp-OMT / S-DIOXYMETHIONINE


Type: L-peptide linking / Mass: 181.210 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C5H11NO4S
#4: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C17H21N4O9P
#5: Chemical
ChemComp-AKG / 2-OXOGLUTARIC ACID / Alpha-Ketoglutaric acid


Mass: 146.098 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C5H6O5
#6: Chemical
ChemComp-F3S / FE3-S4 CLUSTER / Iron–sulfur cluster


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


Mass: 351.640 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Fe4S4
#8: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


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

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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: AZOSPIRILLUM BRASILENSE GLUTAMATE SYNTHASE / Type: COMPLEX
Buffer solutionName: 25 MM HEPES/KOH, 1 MM EDTA, 1 MM DTT / pH: 7.5 / Details: 25 MM HEPES/KOH, 1 MM EDTA, 1 MM DTT
SpecimenConc.: 9.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Details: NLIQUID ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL 2010UHR / Date: May 10, 2005
Details: CC =1.1 MM, FOCAL -LEN-1.9 M. MICROGRAPH WITH ANISOTROPIC OR NO DIFFRACTION RINGS WERE REMOVED USING THE ENHANCED POWER SPECTRA SORTING METHOD (JONIC S,SORZANO CO,COTTEVIEILLE M, LARQUET E, ...Details: CC =1.1 MM, FOCAL -LEN-1.9 M. MICROGRAPH WITH ANISOTROPIC OR NO DIFFRACTION RINGS WERE REMOVED USING THE ENHANCED POWER SPECTRA SORTING METHOD (JONIC S,SORZANO CO,COTTEVIEILLE M, LARQUET E, BOISSET N.,A NOVEL METHOD FOR IMPROVEMENT OF VISUALIZATION OF POWER SPECTRA FOR SORTING CRYO-ELECTRON MICROGRAPHS AND THEIR LOCAL AREAS. J STRUCT BIOL. 2007, 157(1),156-67.)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1700 nm / Cs: 0.5 mm
Specimen holderTemperature: 93.15 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 10 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 151
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: WIENER FILTERING OF VOLUMES FROM FOCAL SERIES
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: RANDOM CONICAL TILT SERIES (CRYOEM) AND REFINEMENT BY PROJECTION MATCHING AND CTF CORRECTION USING WIENER FILTERING OF VOLUMES FROM FOCAL SERIES
Resolution: 9.5 Å / Num. of particles: 12800 / Nominal pixel size: 1.59 Å
Details: THE CHAINS A,B,C,D,E,F OF THE COMPLEX CORRESPOND TO THE ALPHA SUBUNITS, SOLVED BY X-RAY (PDB CODE 1EA0). THREE DISORDERED REGIONS OF THIS X-RAY STRUCTURE (CHAINS A,B,C,D,E, F), IE RESIDUES ...Details: THE CHAINS A,B,C,D,E,F OF THE COMPLEX CORRESPOND TO THE ALPHA SUBUNITS, SOLVED BY X-RAY (PDB CODE 1EA0). THREE DISORDERED REGIONS OF THIS X-RAY STRUCTURE (CHAINS A,B,C,D,E, F), IE RESIDUES 305-307, 1172-1179 AND 1194-1202 WERE MODELLED WITH MODELLER. THE CHAINS G,H,I,J,K,L CORRESPOND TO THE HOMOLOGY MODEL OF THE BETA SUBUNIT (GENERATED BY MODELLER).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: METHOD--REAL-SPACE LOCAL OPTIMIZATION REFINEMENT PROTOCOL--X-RAY AND HOMOLOGY MODELLING
Atomic model buildingPDB-ID: 2VDC
RefinementHighest resolution: 9.5 Å
Refinement stepCycle: LAST / Highest resolution: 9.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms88830 0 768 0 89598

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