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- PDB-6weg: Structure of Ft (MglA-SspA)-ppGpp-PigR peptide complex -

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Basic information

Entry
Database: PDB / ID: 6weg
TitleStructure of Ft (MglA-SspA)-ppGpp-PigR peptide complex
Components
  • MglA
  • Peptide
  • Stringent starvation protein A, regulator of transcription
KeywordsTRANSCRIPTION / Francisella tularensis / bioweapon / MglA-SspA / PigR / ppGpp
Function / homology
Function and homology information


glutathione dehydrogenase (ascorbate) activity / L-ascorbic acid metabolic process / glutathione transferase activity / glutathione metabolic process / metal ion binding / cytoplasm
Similarity search - Function
Stringent starvation protein A, C-terminal / Glutathione S-transferase, N-terminal domain / Glutathione S-transferase, N-terminal domain / Glutathione S-transferase, C-terminal domain / Glutathione transferase family / Glutathione S-transferase, C-terminal / Soluble glutathione S-transferase N-terminal domain profile. / Glutathione S-transferase, C-terminal-like / Soluble glutathione S-transferase C-terminal domain profile. / Glutathione S-transferase, N-terminal ...Stringent starvation protein A, C-terminal / Glutathione S-transferase, N-terminal domain / Glutathione S-transferase, N-terminal domain / Glutathione S-transferase, C-terminal domain / Glutathione transferase family / Glutathione S-transferase, C-terminal / Soluble glutathione S-transferase N-terminal domain profile. / Glutathione S-transferase, C-terminal-like / Soluble glutathione S-transferase C-terminal domain profile. / Glutathione S-transferase, N-terminal / Glutathione S-transferase, C-terminal domain superfamily / Thioredoxin-like superfamily
Similarity search - Domain/homology
GUANOSINE-5',3'-TETRAPHOSPHATE / GST N-terminal domain-containing protein / Macrophage growth locus, subunit A / Stringent starvation protein A, regulator of transcription / Uncharacterized protein
Similarity search - Component
Biological speciesFrancisella tularensis subsp. tularensis (bacteria)
Francisella tularensis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.95 Å
AuthorsSchumacher, M.A. / Brennan, R.
CitationJournal: Mol Cell / Year: 2021
Title: Structural Basis for Virulence Activation of Francisella tularensis.
Authors: Brady A Travis / Kathryn M Ramsey / Samantha M Prezioso / Thomas Tallo / Jamie M Wandzilak / Allen Hsu / Mario Borgnia / Alberto Bartesaghi / Simon L Dove / Richard G Brennan / Maria A Schumacher /
Abstract: The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, ...The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ-associated RNAP holoenzyme (RNAPσ), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ-promoter-DNA, FtRNAPσ-(MglA-SspA)-promoter DNA, and FtRNAPσ-(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσhomodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ-(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
History
DepositionApr 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2020Provider: repository / Type: Initial release
Revision 1.1May 26, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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
Revision 1.2Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Stringent starvation protein A, regulator of transcription
A: MglA
D: Stringent starvation protein A, regulator of transcription
B: MglA
P: Peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,3279
Polymers98,0725
Non-polymers1,2554
Water00
1
C: Stringent starvation protein A, regulator of transcription
A: MglA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,3324
Polymers47,7052
Non-polymers6272
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2850 Å2
ΔGint-18 kcal/mol
Surface area19410 Å2
MethodPISA
2
D: Stringent starvation protein A, regulator of transcription
B: MglA
P: Peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,9955
Polymers50,3683
Non-polymers6272
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4570 Å2
ΔGint-31 kcal/mol
Surface area20850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.756, 113.562, 141.073
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Stringent starvation protein A, regulator of transcription


Mass: 24110.094 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4) (bacteria)
Strain: SCHU S4 / Schu 4 / Gene: sspA, FTT_0458 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5NHJ6
#2: Protein MglA


Mass: 23594.529 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis (bacteria) / Gene: DR86_1530 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0E2ZLH6, UniProt: Q5NFG1*PLUS
#3: Protein/peptide Peptide


Mass: 2663.193 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4) (bacteria)
Strain: SCHU S4 / Schu 4 / Gene: FTT_0383 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5NHR4
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-G4P / GUANOSINE-5',3'-TETRAPHOSPHATE / guanosine tetraphosphate;ppGpp


Type: RNA linking / Mass: 603.160 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N5O17P4 / Feature type: SUBJECT OF INVESTIGATION
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: 2.69 Å3/Da / Density % sol: 54.2 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / Details: Peg 4000, Tris HCl pH 8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.01 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jul 22, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.01 Å / Relative weight: 1
ReflectionResolution: 2.95→88.461 Å / Num. obs: 22603 / % possible obs: 98.1 % / Redundancy: 2.9 % / Biso Wilson estimate: 90.75 Å2 / CC1/2: 0.997 / Rpim(I) all: 0.063 / Rsym value: 0.091 / Net I/σ(I): 7.3
Reflection shellResolution: 2.95→3.08 Å / Num. unique obs: 2028 / CC1/2: 0.338 / Rpim(I) all: 0.95 / Rsym value: 1.38

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX1.6.4_486refinement
XDSdata reduction
SCALAdata scaling
MOLREPphasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5U56

5u56


Resolution: 2.95→88.461 Å / SU ML: 0.48 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 33.6
RfactorNum. reflection% reflection
Rfree0.2898 1752 7.75 %
Rwork0.2254 --
obs0.2304 22603 98.05 %
Solvent computationShrinkage radii: 1.13 Å / VDW probe radii: 1.2 Å / Bsol: 61.972 Å2 / ksol: 0.291 e/Å3
Displacement parametersBiso max: 548.22 Å2 / Biso mean: 115.36 Å2 / Biso min: 9 Å2
Baniso -1Baniso -2Baniso -3
1-12.5347 Å20 Å2-0 Å2
2--3.9014 Å20 Å2
3----16.4361 Å2
Refinement stepCycle: final / Resolution: 2.95→88.461 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6517 0 74 0 6591
Biso mean--134 --
Num. residues----812
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0046706
X-RAY DIFFRACTIONf_angle_d0.8059083
X-RAY DIFFRACTIONf_chiral_restr0.0591056
X-RAY DIFFRACTIONf_plane_restr0.0031131
X-RAY DIFFRACTIONf_dihedral_angle_d16.4312584
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.95-3.05030.40741740.351206399
3.0503-3.17240.41271720.3284205399
3.1724-3.31680.4011740.3347206398
3.3168-3.49170.36311710.2976203597
3.4917-3.71050.37511760.2732209499
3.7105-3.99690.32451740.2204208299
3.9969-4.39920.24761740.1957205997
4.3992-5.03570.24611780.183212099
5.0357-6.34420.29151750.2277208697
6.3442-88.4610.24361840.193219696
Refinement TLS params.Method: refined / Origin x: -31.9861 Å / Origin y: -0.9611 Å / Origin z: -10.7611 Å
111213212223313233
T0.1283 Å20.0371 Å20.0538 Å2-0.1081 Å2-0.0076 Å2--0.1987 Å2
L0.1267 °2-0.2764 °20.0667 °2-0.0672 °20.4559 °2--1.7324 °2
S0.0959 Å °0.0019 Å °-0.1106 Å °0.1401 Å °-0.0337 Å °0.0011 Å °0.2778 Å °0.0355 Å °-0 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allC2 - 194
2X-RAY DIFFRACTION1allA2 - 201
3X-RAY DIFFRACTION1allD1 - 194
4X-RAY DIFFRACTION1allB0 - 201
5X-RAY DIFFRACTION1allR1
6X-RAY DIFFRACTION1allT1
7X-RAY DIFFRACTION1allE1 - 2
8X-RAY DIFFRACTION1allP90 - 108

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