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- PDB-7tdv: Crystal structure of S. aureus glutamine synthetase in Met-Sox-P/... -

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Basic information

Entry
Database: PDB / ID: 7tdv
TitleCrystal structure of S. aureus glutamine synthetase in Met-Sox-P/ADP transition state complex
ComponentsGlutamine synthetase
KeywordsLIGASE / Glutamine synthetase / glutamate-ammonium ligase / GlnR / S. aureus / femC
Function / homology
Function and homology information


glutamine synthetase / glutamine biosynthetic process / glutamine synthetase activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Glutamine synthetase type I / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain ...Glutamine synthetase type I / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain / Glutamine synthetase, catalytic domain / Glutamine synthetase (GS) catalytic domain profile. / Glutamine synthetase, catalytic domain / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / L-METHIONINE-S-SULFOXIMINE PHOSPHATE / Glutamine synthetase
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.92 Å
AuthorsSchumacher, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130290 United States
CitationJournal: Nat Commun / Year: 2022
Title: Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.
Authors: Brady A Travis / Jared V Peck / Raul Salinas / Brandon Dopkins / Nicholas Lent / Viet D Nguyen / Mario J Borgnia / Richard G Brennan / Maria A Schumacher /
Abstract: How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine ...How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.
History
DepositionJan 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamine synthetase
C: Glutamine synthetase
D: Glutamine synthetase
E: Glutamine synthetase
B: Glutamine synthetase
H: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)312,08040
Polymers307,3496
Non-polymers4,73134
Water5,513306
1
A: Glutamine synthetase
C: Glutamine synthetase
D: Glutamine synthetase
E: Glutamine synthetase
B: Glutamine synthetase
H: Glutamine synthetase
hetero molecules

A: Glutamine synthetase
C: Glutamine synthetase
D: Glutamine synthetase
E: Glutamine synthetase
B: Glutamine synthetase
H: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)624,16180
Polymers614,69912
Non-polymers9,46268
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_465y-1,x+1,-z1
Buried area81800 Å2
ΔGint-738 kcal/mol
Surface area179560 Å2
MethodPISA
Unit cell
Length a, b, c (Å)154.615, 154.615, 299.295
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212

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Components

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Protein , 1 types, 6 molecules ACDEBH

#1: Protein
Glutamine synthetase


Mass: 51224.895 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: glnA, BSG37_06900, FAF32_003410, G6X35_05920, G6X37_15495, G6Y24_07770, SAMEA103891454_00902
Production host: Escherichia coli (E. coli) / References: UniProt: E3VXC2, glutamine synthetase

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Non-polymers , 5 types, 340 molecules

#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-P3S / L-METHIONINE-S-SULFOXIMINE PHOSPHATE


Mass: 260.205 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C5H13N2O6PS / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 306 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.91 Å3/Da / Density % sol: 57.73 %
Crystal growTemperature: 273 K / Method: vapor diffusion, hanging drop
Details: 28% Peg 400, 0.2 M calcium chloride, 0.1 M HEPES 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.09 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 13, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.09 Å / Relative weight: 1
ReflectionResolution: 2.92→48.89 Å / Num. obs: 79309 / % possible obs: 99.9 % / Redundancy: 28 % / CC1/2: 0.999 / Rpim(I) all: 0.049 / Rsym value: 0.165 / Net I/σ(I): 19.1
Reflection shellResolution: 2.92→3.02 Å / Mean I/σ(I) obs: 2.1 / Num. unique obs: 7783 / CC1/2: 0.744 / Rpim(I) all: 0.517 / Rsym value: 1.78

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
SCALAdata scaling
PHASERphasing
PHENIX1.17.1_3660refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4LNI

4lni


Resolution: 2.92→48.89 Å / SU ML: 0.32 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 26.58 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2561 1880 2.37 %
Rwork0.1984 77429 -
obs0.1997 79309 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 190.94 Å2 / Biso mean: 69.5289 Å2 / Biso min: 30 Å2
Refinement stepCycle: final / Resolution: 2.92→48.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21216 0 410 306 21932
Biso mean--69.42 64.44 -
Num. residues----2654
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 13 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
2.92-30.33021420.268758475989
3-3.090.32951430.262958806023
3.09-3.190.29251420.249158495991
3.19-3.30.29581420.231358896031
3.3-3.430.30111430.219658896032
3.43-3.590.28331430.206759036046
3.59-3.780.20571440.195359006044
3.78-4.010.27421430.187159086051
4.01-4.320.20951450.166459536098
4.32-4.760.19321460.154159716117
4.76-5.450.23081450.165860096154
5.45-6.860.2941480.224360666214
6.86-48.890.26681540.205163656519
Refinement TLS params.Method: refined / Origin x: -59.8408 Å / Origin y: 69.0059 Å / Origin z: 16.0161 Å
111213212223313233
T0.3581 Å2-0.0333 Å2-0.0606 Å2-0.3832 Å20.0261 Å2--0.2958 Å2
L0.3436 °2-0.0258 °2-0.1353 °2-0.351 °20.0481 °2--0.3554 °2
S0.0017 Å °-0.0463 Å °0.0527 Å °0.121 Å °-0.0051 Å °-0.1055 Å °-0.0669 Å °0.1313 Å °0.0047 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA5 - 601
2X-RAY DIFFRACTION1allC4 - 601
3X-RAY DIFFRACTION1allD5 - 601
4X-RAY DIFFRACTION1allE5 - 601
5X-RAY DIFFRACTION1allB4 - 601
6X-RAY DIFFRACTION1allH5 - 601
7X-RAY DIFFRACTION1allG1 - 26
8X-RAY DIFFRACTION1allI1 - 307
9X-RAY DIFFRACTION1allF1

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