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- PDB-7tea: Crystal structure of S. aureus GlnR-DNA complex -

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Basic information

Entry
Database: PDB / ID: 7tea
TitleCrystal structure of S. aureus GlnR-DNA complex
Components
  • DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*AP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
  • DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*TP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
  • Glutamine synthetase repressor
KeywordsDNA BINDING PROTEIN/DNA / GlnR / winged-HTH / S. aureus / femC / glutamine synthetase / transcription repressor / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA-binding transcription factor activity / DNA binding
Similarity search - Function
: / MerR HTH family regulatory protein / MerR-type HTH domain profile. / helix_turn_helix, mercury resistance / MerR-type HTH domain / Putative DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Glutamine synthetase repressor
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.35 Å
AuthorsSchumacher, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM130290 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 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _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
B: Glutamine synthetase repressor
E: Glutamine synthetase repressor
G: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*AP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
F: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*TP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
A: Glutamine synthetase repressor
C: Glutamine synthetase repressor
D: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*AP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
H: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*TP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,41810
Polymers66,3388
Non-polymers802
Water1,63991
1
B: Glutamine synthetase repressor
E: Glutamine synthetase repressor
G: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*AP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
F: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*TP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,2095
Polymers33,1694
Non-polymers401
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
A: Glutamine synthetase repressor
C: Glutamine synthetase repressor
D: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*AP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
H: DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*TP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,2095
Polymers33,1694
Non-polymers401
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)66.502, 99.265, 238.922
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number24
Space group name H-MI212121
Components on special symmetry positions
IDModelComponents
11E-201-

HOH

21C-201-

HOH

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Components

#1: Protein
Glutamine synthetase repressor / Glutamine synthetase / Glutamine synthetase repressor / HTH-type transcriptional regulator GlnR / ...Glutamine synthetase / Glutamine synthetase repressor / HTH-type transcriptional regulator GlnR / MerR family transcriptional regulator / glutamine synthetase repressor / Transcriptional regulator (Nitrogen metabolism) / Transcriptional regulator / MerR family / MerR family protein / FemC / factor involved in methicillin resistance


Mass: 10141.709 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: glnR, femC, BN1321_240156, BSG37_06895, BTN44_06150, C7P97_11270, CFN13_06110, CSC87_13065, CV021_11625, DD547_01329, DQU54_07135, E3A28_02075, E3K14_06475, E4U00_04150, E5491_06810, ELP52_ ...Gene: glnR, femC, BN1321_240156, BSG37_06895, BTN44_06150, C7P97_11270, CFN13_06110, CSC87_13065, CV021_11625, DD547_01329, DQU54_07135, E3A28_02075, E3K14_06475, E4U00_04150, E5491_06810, ELP52_06590, FA040_05040, FAF32_003415, FPP26_06465, FVP29_03765, G6X35_05925, G6X37_15500, G6Y24_07775, GO782_03725, GO788_03065, GO793_18140, GO810_03920, GO814_05910, GO941_02620, GO942_07700, GQX37_02050, GZ128_03000, GZ145_02540, HMPREF3211_01353, NCTC10654_01351, NCTC10702_02054, NCTC5664_02143, NCTC6133_01601, RK64_06965, SA0759_02803, SA950122_02714, SAGV51_01473, SAGV69_01244, SAHC1335_02241, SAJPND4_01239, SAMEA1029536_01443, SAMEA103891420_00579, SAMEA103891454_00903, SAMEA2076468_01609, SAMEA2076503_01121, SAMEA2077334_01678, SAMEA2078260_01440, SAMEA2078588_01695, SAMEA2079501_01495, SAMEA2080344_01609, SAMEA2081063_01288, SAMEA4008569_01468, SAMEA70146418_02626, SAMEA70245418_01181
Production host: Escherichia coli (E. coli) / References: UniProt: Q53687
#2: DNA chain DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*AP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')


Mass: 6447.185 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: synthetic GlnR operator DNA / Source: (synth.) Staphylococcus aureus (bacteria)
#3: DNA chain DNA (5'-D(*CP*GP*TP*GP*TP*CP*AP*GP*AP*TP*TP*AP*TP*CP*TP*GP*AP*CP*AP*CP*G)-3')


Mass: 6438.171 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria)
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 91 / 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: 2.97 Å3/Da / Density % sol: 58.61 %
Crystal growTemperature: 275 K / Method: vapor diffusion, hanging drop
Details: 50 mM sodium cacodylate pH 6.5, 13% PEG 8000, 0.15 M calcium acetate, 20% glycerol

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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: Mar 1, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.09 Å / Relative weight: 1
ReflectionResolution: 2.35→49.63 Å / Num. obs: 31531 / % possible obs: 98.6 % / Redundancy: 6.2 % / CC1/2: 1 / Rpim(I) all: 0.027 / Rsym value: 0.042 / Net I/σ(I): 21.4
Reflection shell

Diffraction-ID: 1

Resolution (Å)Mean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRsym valueNet I/σ(I) obs
2.47-2.561.926060.8180.4870.536
2.35-2.471.450.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX1.17.1_3660refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4R4E

4r4e


Resolution: 2.35→49.63 Å / SU ML: 0.47 / Cross valid method: THROUGHOUT / σ(F): 0 / Phase error: 38.36 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2635 1995 6.33 %
Rwork0.2126 29536 -
obs0.2159 31531 94.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 165.69 Å2 / Biso mean: 73.7533 Å2 / Biso min: 30 Å2
Refinement stepCycle: final / Resolution: 2.35→49.63 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2565 1710 2 91 4368
Biso mean--63.52 68.76 -
Num. residues----392
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0094537
X-RAY DIFFRACTIONf_angle_d1.1166458
X-RAY DIFFRACTIONf_dihedral_angle_d28.4071170
X-RAY DIFFRACTIONf_chiral_restr0.053734
X-RAY DIFFRACTIONf_plane_restr0.006525
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 (%)
2.35-2.410.4415830.51261244132757
2.41-2.480.52231250.45641836196183
2.48-2.550.41191350.40161997213292
2.55-2.630.44531450.35522154229998
2.63-2.730.37371490.330922012350100
2.73-2.840.32751480.279121742322100
2.84-2.970.31881470.275422012348100
2.97-3.120.33961470.27222082355100
3.12-3.320.26331510.23182216236799
3.32-3.570.25071480.20982179232799
3.57-3.930.26361520.204222442396100
3.93-4.50.23871520.160822412393100
4.5-5.670.22471540.158822722426100
5.67-49.630.21161590.17692369252899
Refinement TLS params.Method: refined / Origin x: 41.7984 Å / Origin y: 61.1108 Å / Origin z: 91.5599 Å
111213212223313233
T0.2738 Å20.1125 Å20.0497 Å2-0.3934 Å2-0.0114 Å2--0.368 Å2
L0.8718 °20.9619 °20.0279 °2-1.7811 °20.2246 °2--0.5197 °2
S-0.0477 Å °0.0901 Å °0.06 Å °-0.0448 Å °0.1156 Å °0.1203 Å °0.0162 Å °0.0346 Å °-0.0574 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allB6 - 83
2X-RAY DIFFRACTION1allE6 - 83
3X-RAY DIFFRACTION1allG0 - 20
4X-RAY DIFFRACTION1allF0 - 20
5X-RAY DIFFRACTION1allA6 - 82
6X-RAY DIFFRACTION1allC6 - 81
7X-RAY DIFFRACTION1allD0 - 20
8X-RAY DIFFRACTION1allH0 - 20
9X-RAY DIFFRACTION1allY1 - 2
10X-RAY DIFFRACTION1allS1 - 109

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