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- PDB-7ji0: CryoEM structure of Streptococcus thermophilus SHP pheromone rece... -

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Basic information

Entry
Database: PDB / ID: 7ji0
TitleCryoEM structure of Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
Components
  • Positive transcriptional regulator MutR family
  • SHP3
KeywordsDNA BINDING PROTEIN / DNA binding transcription factor / quorum sensing / RRNPP / pheromone binding
Function / homologyHTH-type transcriptional regulator Rgg, C-terminal domain / Transcription activator MutR, C-terminal / Cro/C1-type HTH domain profile. / Cro/C1-type helix-turn-helix domain / Lambda repressor-like, DNA-binding domain superfamily / Tetratricopeptide-like helical domain superfamily / DNA binding / Positive transcriptional regulator MutR family
Function and homology information
Biological speciesStreptococcus thermophilus (bacteria)
Streptococcus thermophilus CNRZ1066 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsPetrou, V.I. / Capodagli, G.C. / Kaelber, J.T. / Neiditch, M.B.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI125452 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM123228 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structure-function studies of Rgg binding to pheromones and target promoters reveal a model of transcription factor interplay.
Authors: Glenn C Capodagli / Kaitlyn M Tylor / Jason T Kaelber / Vasileios I Petrou / Michael J Federle / Matthew B Neiditch /
Abstract: Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in species, controlling virulence, ...Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Rgg2 and Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.
History
DepositionJul 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • EMDB-22341
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Assembly

Deposited unit
A: Positive transcriptional regulator MutR family
B: Positive transcriptional regulator MutR family
C: SHP3
D: SHP3


Theoretical massNumber of molelcules
Total (without water)68,0824
Polymers68,0824
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Positive transcriptional regulator MutR family


Mass: 33242.078 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (strain ATCC BAA-250 / LMG 18311) (bacteria)
Strain: ATCC BAA-250 / LMG 18311 / Gene: stu1044 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5M4D0
#2: Protein/peptide SHP3


Mass: 798.969 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus CNRZ1066 (bacteria)
Production host: synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Streptococcus thermophilus (bacteria) / Organ: CNRZ1066
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: 2.5 microliters Rgg3St-SHP3 was applied to glow-discharged UltraAuFoil (1.2/1.3) 300-mesh grids (Quantifoil), blotted with filter paper for 3-3.5 s, and flash-frozen by plunging in liquid ...Details: 2.5 microliters Rgg3St-SHP3 was applied to glow-discharged UltraAuFoil (1.2/1.3) 300-mesh grids (Quantifoil), blotted with filter paper for 3-3.5 s, and flash-frozen by plunging in liquid ethane cooled with liquid nitrogen. Grids were stored in liquid nitrogen.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 47.13 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1464
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.13.2particle selection
2EPU1.ximage acquisition
4cryoSPARC2.13.2CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC2.13.2initial Euler assignment
11cryoSPARC2.13.2final Euler assignment
13cryoSPARC2.13.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 389743
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44069 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6W1E

6w1e


Accession code: 6W1E / Source name: PDB / Type: experimental model

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