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- PDB-7tdp: Structure of Paenibacillus polymyxa GS bound to Met-Sox-P-ADP (Tr... -

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Basic information

Entry
Database: PDB / ID: 7tdp
TitleStructure of Paenibacillus polymyxa GS bound to Met-Sox-P-ADP (Transition state complex) to 1.98 Angstom
ComponentsGlutamine synthetase
KeywordsLIGASE / glutamate-ammonium ligase / GlnR / GS / feedback inhibition / transcription coregulator / glnRA
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 (GS) beta-grasp domain profile. / Glutamine synthetase (GS) catalytic domain profile. / 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, N-terminal domain / Glutamine synthetase, N-terminal domain superfamily ...Glutamine synthetase type I / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase (GS) catalytic domain profile. / 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, N-terminal domain / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / 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 speciesPaenibacillus polymyxa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.98 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 2, 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

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Assembly

Deposited unit
A: Glutamine synthetase
C: Glutamine synthetase
B: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,29818
Polymers151,0173
Non-polymers2,28115
Water14,394799
1
A: Glutamine synthetase
C: Glutamine synthetase
B: Glutamine synthetase
hetero molecules

A: Glutamine synthetase
C: Glutamine synthetase
B: Glutamine synthetase
hetero molecules

A: Glutamine synthetase
C: Glutamine synthetase
B: Glutamine synthetase
hetero molecules

A: Glutamine synthetase
C: Glutamine synthetase
B: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)613,19272
Polymers604,06812
Non-polymers9,12460
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation7_556y,x,-z+11
crystal symmetry operation8_556-y,-x,-z+11
Buried area78420 Å2
ΔGint-764 kcal/mol
Surface area173310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)164.009, 164.009, 141.528
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number94
Space group name H-MP42212

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Components

#1: Protein Glutamine synthetase /


Mass: 50338.988 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paenibacillus polymyxa (bacteria) / Gene: glnA2, LK13_01575, NCTC10343_02989 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0F0G8G2, glutamine synthetase
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / 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.: 3 / Source method: obtained synthetically / Formula: C5H13N2O6PS / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 799 / Source method: isolated from a natural source / Formula: H2O
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: 3.15 Å3/Da / Density % sol: 60.97 %
Crystal growTemperature: 278 K / Method: vapor diffusion, hanging drop / Details: 30% PEG mono methyl ether 2000, 0.1 M Tris pH 8.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.08 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Sep 12, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.08 Å / Relative weight: 1
ReflectionResolution: 1.98→48.7 Å / Num. obs: 132443 / % possible obs: 98.5 % / Redundancy: 13.1 % / CC1/2: 0.999 / Rpim(I) all: 0.03 / Rsym value: 0.108 / Net I/σ(I): 15.4
Reflection shellResolution: 1.98→2.05 Å / Redundancy: 4.6 % / Mean I/σ(I) obs: 1.4 / Num. unique obs: 12010 / CC1/2: 0.486 / Rpim(I) all: 0.544 / Rsym value: 1.154

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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: 1.98→48.7 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 21.31 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2006 1992 1.51 %
Rwork0.1708 130336 -
obs0.1713 132328 98.24 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 111.37 Å2 / Biso mean: 42.1516 Å2 / Biso min: 20.44 Å2
Refinement stepCycle: final / Resolution: 1.98→48.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10524 0 135 799 11458
Biso mean--32.37 45.85 -
Num. residues----1317
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.98-2.020.38651100.38997295740578
2.02-2.080.35841400.27719219935999
2.08-2.140.26341440.244693529496100
2.14-2.210.241440.220194119555100
2.21-2.290.23841410.226393119452100
2.29-2.380.23221430.19194139556100
2.38-2.490.20671450.185893849529100
2.49-2.620.22041440.183594119555100
2.62-2.780.22251440.185194289572100
2.78-30.23251450.182994569601100
3-3.30.22031460.170995009646100
3.3-3.780.19221450.151395409685100
3.78-4.760.13911480.124996339781100
4.76-48.70.16451530.144998310136100
Refinement TLS params.Method: refined / Origin x: 17.1311 Å / Origin y: 17.0315 Å / Origin z: 41.6451 Å
111213212223313233
T0.1909 Å2-0.0288 Å20.0256 Å2-0.4373 Å20.0196 Å2--0.1896 Å2
L0.5589 °20.0465 °20.0717 °2-0.463 °20.0408 °2--0.1342 °2
S-0.0381 Å °0.3209 Å °0.01 Å °-0.0572 Å °0.1005 Å °-0.0058 Å °-0.0132 Å °0.0235 Å °-0.0634 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA2 - 601
2X-RAY DIFFRACTION1allC1 - 601
3X-RAY DIFFRACTION1allB2 - 601
4X-RAY DIFFRACTION1allD1 - 9
5X-RAY DIFFRACTION1allS1 - 821

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