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- PDB-8hml: Co-crystal structure of the C terminal DNA binding domain of Sacc... -

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Basic information

Entry
Database: PDB / ID: 8hml
TitleCo-crystal structure of the C terminal DNA binding domain of Saccharopolyspora erythraea GlnR in complex with its conserved promoter DNA in 2.95 Angstrom resolution
Components
  • DNA (5'-D(*AP*CP*GP*TP*AP*AP*CP*AP*TP*CP*GP*CP*GP*GP*TP*AP*AP*CP*AP*C)-3')
  • DNA (5'-D(*GP*TP*GP*TP*TP*AP*CP*CP*GP*CP*GP*AP*TP*GP*TP*TP*AP*CP*GP*T)-3')
  • DNA-binding response OmpR family regulator
KeywordsTRANSFERASE/DNA / methylenetetrahydrofolate reductase / TRANSFERASE-DNA COMPLEX
Function / homology
Function and homology information


phosphorelay signal transduction system / negative regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / DNA binding
Similarity search - Function
OmpR/PhoB-type DNA-binding domain profile. / OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein WalR-like / Signal transduction response regulator, C-terminal effector / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-binding response OmpR family regulator
Similarity search - Component
Biological speciesSaccharopolyspora erythraea NRRL 2338 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.95 Å
AuthorsLin, W. / Xu, J.C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81903526 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.
Authors: Jing Shi / Zhenzhen Feng / Juncao Xu / Fangfang Li / Yuqiong Zhang / Aijia Wen / Fulin Wang / Qian Song / Lu Wang / Hong Cui / Shujuan Tong / Peiying Chen / Yejin Zhu / Guoping Zhao / Shuang ...Authors: Jing Shi / Zhenzhen Feng / Juncao Xu / Fangfang Li / Yuqiong Zhang / Aijia Wen / Fulin Wang / Qian Song / Lu Wang / Hong Cui / Shujuan Tong / Peiying Chen / Yejin Zhu / Guoping Zhao / Shuang Wang / Yu Feng / Wei Lin /
Abstract: In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate ...In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory -element DNA and a cryo-EM structure of GlnR-TAC which comprises RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved β flap, σR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.
History
DepositionDec 4, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA-binding response OmpR family regulator
B: DNA-binding response OmpR family regulator
D: DNA (5'-D(*AP*CP*GP*TP*AP*AP*CP*AP*TP*CP*GP*CP*GP*GP*TP*AP*AP*CP*AP*C)-3')
C: DNA (5'-D(*GP*TP*GP*TP*TP*AP*CP*CP*GP*CP*GP*AP*TP*GP*TP*TP*AP*CP*GP*T)-3')


Theoretical massNumber of molelcules
Total (without water)44,6174
Polymers44,6174
Non-polymers00
Water27015
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)46.279, 46.279, 366.859
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-z

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Components

#1: Protein DNA-binding response OmpR family regulator


Mass: 16174.348 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharopolyspora erythraea NRRL 2338 (bacteria)
Gene: glnR / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A4FQD5
#2: DNA chain DNA (5'-D(*AP*CP*GP*TP*AP*AP*CP*AP*TP*CP*GP*CP*GP*GP*TP*AP*AP*CP*AP*C)-3')


Mass: 6111.983 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Saccharopolyspora erythraea NRRL 2338 (bacteria)
#3: DNA chain DNA (5'-D(*GP*TP*GP*TP*TP*AP*CP*CP*GP*CP*GP*AP*TP*GP*TP*TP*AP*CP*GP*T)-3')


Mass: 6155.975 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Saccharopolyspora erythraea NRRL 2338 (bacteria)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O

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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: 2.54 Å3/Da / Density % sol: 51.61 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6 / Details: 0.1 M Bis-tris pH6.0, 20% PEG3350 / PH range: 5.7-6.2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U / Wavelength: 0.987 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 28, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 2.34→50 Å / Num. obs: 9134 / % possible obs: 90.7 % / Redundancy: 5.1 % / CC1/2: 0.76 / Χ2: 0.134 / Net I/σ(I): 7.6
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsΧ2Diffraction-ID% possible all
2.34-2.383.61.727940.552178.3
2.38-2.423.71.1528300.566180.7
2.42-2.473.91.7298660.546185.8
2.47-2.524.31.2338910.585187.5
2.52-2.584.61.5488990.585185.8
2.58-2.644.81.2338670.582189.2
2.64-2.74.81.0429260.677187.5
2.7-2.774.91.5339340.571191.8
2.77-2.865.11.6258840.634187.4
2.86-2.954.71.7549670.602192.7
2.95-3.055.91.5559250.702190.4
3.05-3.185.91.0679750.833193.7
3.18-3.325.50.4359730.959190.3
3.32-3.55.20.3129871.247195.8
3.5-3.715.50.3949761.026195.1
3.71-45.10.3610261.196195.5
4-4.44.40.2759741.251191.4
4.4-5.045.20.18710391.058193.9
5.04-6.356.50.15810880.998199.5
6.35-107.20.08112381.161198.3

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHENIX1.2refinement
PHENIXmodel building
PHENIXphasing
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.95→39.842 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.312 923 10.11 %
Rwork0.2559 --
obs0.2617 9134 86.55 %
Refinement stepCycle: LAST / Resolution: 2.95→39.842 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1524 812 0 15 2351
LS refinement shellResolution: 2.9501→3.1056 Å
RfactorNum. reflection% reflection
Rfree0.403 --
Rwork0.3559 1014 -
obs--76 %

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