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- PDB-7mfm: Glutamate synthase, glutamate dehydrogenase counter-enzyme complex -

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Basic information

Entry
Database: PDB / ID: 7mfm
TitleGlutamate synthase, glutamate dehydrogenase counter-enzyme complex
Components
  • (Glutamate synthase (NADPH) ...) x 2
  • Glutamate dehydrogenase
KeywordsCYTOSOLIC PROTEIN / Counter-enzyme complex / glutamate synthase / glutamate dehydrogenae
Function / homology
Function and homology information


glutamate synthase activity / oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor / glutamate biosynthetic process / glutamate dehydrogenase (NAD+) activity / L-glutamate catabolic process / glutamine metabolic process / 3 iron, 4 sulfur cluster binding / iron-sulfur cluster binding / nucleotide binding / metal ion binding
Similarity search - Function
Glutamate synthase, NADH/NADPH, small subunit 1 / 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 ...Glutamate synthase, NADH/NADPH, small subunit 1 / 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 / Glutamate dehydrogenase / NAD(P) binding domain of glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / Glu / Leu / Phe / Val dehydrogenases active site. / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Alpha-helical ferredoxin / Aminoacid dehydrogenase-like, N-terminal domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / Nucleophile aminohydrolases, N-terminal / FAD/NAD(P)-binding domain superfamily / Aldolase-type TIM barrel / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FE3-S4 CLUSTER / FLAVIN-ADENINE DINUCLEOTIDE / FLAVIN MONONUCLEOTIDE / IRON/SULFUR CLUSTER / Glutamate dehydrogenase / Glutamate synthase (NADPH) small chain / Glutamate synthase (NADPH) large chain
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsJayaraman, V. / Lee, D.J. / Elad, N. / Fraser, J.S. / Tawfik, D.S.
Funding support Israel, United States, 2items
OrganizationGrant numberCountry
Israel Science Foundation2575/20 Israel
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123159 United States
CitationJournal: Nat Chem Biol / Year: 2022
Title: A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.
Authors: Vijay Jayaraman / D John Lee / Nadav Elad / Shay Vimer / Michal Sharon / James S Fraser / Dan S Tawfik /
Abstract: Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from ...Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.
History
DepositionApr 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 5, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-23817
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutamate dehydrogenase
G: Glutamate synthase (NADPH) large chain
H: Glutamate synthase (NADPH) large chain
I: Glutamate synthase (NADPH) small chain
J: Glutamate synthase (NADPH) small chain
B: Glutamate dehydrogenase
C: Glutamate dehydrogenase
D: Glutamate dehydrogenase
E: Glutamate dehydrogenase
F: Glutamate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)741,12020
Polymers736,63810
Non-polymers4,48210
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "F"
d_1ens_2chain "H"
d_2ens_2chain "G"
d_1ens_3chain "I"
d_2ens_3chain "J"
d_1ens_4chain "E"
d_2ens_4chain "B"
d_1ens_5chain "C"
d_2ens_5chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LEUILEA1 - 410
d_21ens_1LEUILET1 - 410
d_11ens_2CYSLYSE1 - 1483
d_12ens_2FMNFMNF
d_13ens_2F3SF3SG
d_21ens_2CYSLYSB1 - 1483
d_22ens_2FMNFMNC
d_23ens_2F3SF3SD
d_11ens_3LYSLEUH1 - 490
d_12ens_3FADFADI
d_13ens_3SF4SF4J
d_14ens_3SF4SF4K
d_21ens_3LYSLEUL1 - 490
d_22ens_3FADFADM
d_23ens_3SF4SF4N
d_24ens_3SF4SF4O
d_11ens_4LEUILES1 - 410
d_21ens_4LEUILEP1 - 410
d_11ens_5LEUILEQ1 - 410
d_21ens_5LEUILER1 - 410

NCS ensembles :
ID
ens_1
ens_2
ens_3
ens_4
ens_5

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Components

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Protein , 1 types, 6 molecules ABCDEF

#1: Protein
Glutamate dehydrogenase


Mass: 47100.715 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria)
Gene: gudB, B4122_2172, ETA10_11605, ETK61_12750, GII81_12565, SC09_Contig24orf00413
Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A0C3GZC9

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Glutamate synthase (NADPH) ... , 2 types, 4 molecules GHIJ

#2: Protein Glutamate synthase (NADPH) large chain


Mass: 169006.375 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: B4417_0011 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A164XVV7
#3: Protein Glutamate synthase (NADPH) small chain


Mass: 58010.598 Da / Num. of mol.: 2 / Mutation: D78G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: B4417_0010 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A164XVU4

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Non-polymers , 4 types, 10 molecules

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


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P
#5: Chemical ChemComp-F3S / FE3-S4 CLUSTER


Mass: 295.795 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe3S4
#6: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#7: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN
Has protein modificationN

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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: GudB6-GltA2-GltB2 / Type: COMPLEX / Details: GudB hexamer binding two GltAB heterodimers. / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.74 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 7.9
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 47.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameVersionCategoryDetails
7UCSF Chimera1.15model fittingRigid-body alignment of monomers.
9ISOLDE1.1.0model refinementBackbone positioning
10PHENIX1.19.1-4122model refinementReal-space refine
11Coot0.9.3model refinementModel examination and refinement
4cisTEM1.0.0betaCTF correctionCTFFIND4 as part of cisTEM suite
12cisTEM1.0.0betainitial Euler assignment
13cisTEM1.0.0betafinal Euler assignment
14cisTEM1.0.0betaclassification
15cisTEM1.0.0beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1353359
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 718672 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Real-space refinement involved iterative rounds of ISOLDE, Coot and PHENIX refinement. 1OFD and 6S6T were used as templates for homology modeling in SWISS-MODEL.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
13K8Z

3k8z

C3K8Z117-424
21OFD

1ofd

A1OFD2
36S6T

6s6t

G6S6T3
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 94.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00351240
ELECTRON MICROSCOPYf_angle_d0.523769260
ELECTRON MICROSCOPYf_chiral_restr0.04267586
ELECTRON MICROSCOPYf_plane_restr0.00359004
ELECTRON MICROSCOPYf_dihedral_angle_d3.90296966
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000706356107965
ens_2d_2HELECTRON MICROSCOPYNCS constraints0.0294635984673
ens_3d_2IELECTRON MICROSCOPYNCS constraints0.000712195610245
ens_4d_2EELECTRON MICROSCOPYNCS constraints0.000710024177079
ens_5d_2CELECTRON MICROSCOPYNCS constraints0.000705817911776

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