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- EMDB-22341: CryoEM structure of Streptococcus thermophilus SHP pheromone rece... -

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Basic information

Entry
Database: EMDB / ID: EMD-22341
TitleCryoEM structure of Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
Map dataSharpened map generated by cryoSPARC
Sample
  • Complex: Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
    • Protein or peptide: Positive transcriptional regulator MutR family
    • Protein or peptide: SHP3
KeywordsDNA binding transcription factor / quorum sensing / RRNPP / pheromone binding / DNA BINDING PROTEIN
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 (strain ATCC BAA-250 / LMG 18311) (bacteria) / Streptococcus thermophilus CNRZ1066 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsPetrou VI / Capodagli GC
Funding support United States, 2 items
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, 2020-
Header (metadata) releaseOct 7, 2020-
Map releaseOct 7, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ji0
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22341.png

Downloads & links

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Map

FileDownload / File: emd_22341.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map generated by cryoSPARC
Voxel sizeX=Y=Z: 1.038 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum-2.095046 - 4.850014
Average (Standard dev.)0.00014376034 (±0.09949575)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 265.728 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0381.0381.038
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z265.728265.728265.728
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.0954.8500.000

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Supplemental data

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Mask #1

Fileemd_22341_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map generated by cryoSPARC

Fileemd_22341_additional_1.map
AnnotationUnsharpened map generated by cryoSPARC
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half-map 2 generated by cryoSPARC

Fileemd_22341_half_map_1.map
AnnotationHalf-map 2 generated by cryoSPARC
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half-map 1 generated by cryoSPARC

Fileemd_22341_half_map_2.map
AnnotationHalf-map 1 generated by cryoSPARC
Projections & Slices
AxesZYX

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Sample components

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Entire : Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex...

EntireName: Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
Components
  • Complex: Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
    • Protein or peptide: Positive transcriptional regulator MutR family
    • Protein or peptide: SHP3

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Supramolecule #1: Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex...

SupramoleculeName: Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus thermophilus (bacteria) / Organ: CNRZ1066

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Macromolecule #1: Positive transcriptional regulator MutR family

MacromoleculeName: Positive transcriptional regulator MutR family / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (strain ATCC BAA-250 / LMG 18311) (bacteria)
Strain: ATCC BAA-250 / LMG 18311
Molecular weightTheoretical: 33.242078 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKSKLGSTLR KVRNGKQISI CSVADEHLSK SQISRFERGE SEISCIRLIN ILDKLHITLD EFLILHDEDY TKTESFANLV QYIRKQYSL QNINNIQSLL SDSSNYTLDP FEKTMVKSIL HTMDSSIIPS DDELLQLADY LFKVEKWGYY EIILLGNCVR T IDYNSVFL ...String:
MKSKLGSTLR KVRNGKQISI CSVADEHLSK SQISRFERGE SEISCIRLIN ILDKLHITLD EFLILHDEDY TKTESFANLV QYIRKQYSL QNINNIQSLL SDSSNYTLDP FEKTMVKSIL HTMDSSIIPS DDELLQLADY LFKVEKWGYY EIILLGNCVR T IDYNSVFL LTKEMLNNYI YSSLNKTNKR IVTQLAINCL ILSIDMEEFT NCFYLIDEIK ALLDNELNFY EQTVFLYATG YF EFKRWQS TSGIEKMKQA IQVLDILGED NLKLHYTIHF DKLINNK

UniProtKB: Positive transcriptional regulator MutR family

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Macromolecule #2: SHP3

MacromoleculeName: SHP3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus CNRZ1066 (bacteria)
Molecular weightTheoretical: 798.969 Da
Recombinant expressionOrganism: synthetic construct (others)
SequenceString:
DIIIIVGG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium chloride
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 3 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 1464 / Average exposure time: 8.0 sec. / Average electron dose: 47.13 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 389743
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6w1e

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.13.2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.13.2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.13.2) / Number images used: 44069
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6w1e


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7ji0:
CryoEM structure of Streptococcus thermophilus SHP pheromone receptor Rgg3 in complex with SHP3

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