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- EMDB-12260: Allostery through DNA drives phenotype switching -

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Basic information

Entry
Database: EMDB / ID: EMD-12260
TitleAllostery through DNA drives phenotype switching
Map data
Sample
  • Complex: ComK transcription factor bound to its comG promoter DNA.
    • DNA: AddAB promoter
    • DNA: AddAB promoter
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsRosenblum G / Elad N / Rozenberg H / Wiggers F / Jungwirth J / Hofmann H
Funding support Israel, 1 items
OrganizationGrant numberCountry
Israel Science Foundation1549/15 Israel
CitationJournal: Nat Commun / Year: 2021
Title: Allostery through DNA drives phenotype switching.
Authors: Gabriel Rosenblum / Nadav Elad / Haim Rozenberg / Felix Wiggers / Jakub Jungwirth / Hagen Hofmann /
Abstract: Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery ...Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.
History
DepositionJan 27, 2021-
Header (metadata) releaseApr 7, 2021-
Map releaseApr 7, 2021-
UpdateJun 2, 2021-
Current statusJun 2, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nbn
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nbn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12260.png

Downloads & links

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Map

FileDownload / File: emd_12260.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.718 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.10430596 - 0.69927657
Average (Standard dev.)-0.00044610817 (±0.012523906)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 481.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.7181.7181.718
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z481.040481.040481.040
α/β/γ90.00090.00090.000
start NX/NY/NZ7297100
NX/NY/NZ181127145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1040.699-0.000

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Supplemental data

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Mask #1

Fileemd_12260_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12260_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12260_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ComK transcription factor bound to its comG promoter DNA.

EntireName: ComK transcription factor bound to its comG promoter DNA.
Components
  • Complex: ComK transcription factor bound to its comG promoter DNA.
    • DNA: AddAB promoter
    • DNA: AddAB promoter

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Supramolecule #1: ComK transcription factor bound to its comG promoter DNA.

SupramoleculeName: ComK transcription factor bound to its comG promoter DNA.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria)
Recombinant expressionOrganism: Synthetic construct (others)

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Macromolecule #1: AddAB promoter

MacromoleculeName: AddAB promoter / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 24.230533 KDa
SequenceString: (DG)(DG)(DA)(DG)(DA)(DG)(DA)(DT)(DG)(DT) (DG)(DC)(DG)(DG)(DA)(DG)(DA)(DT)(DA)(DA) (DT)(DC)(DA)(DG)(DC)(DT)(DT)(DT)(DT) (DT)(DA)(DT)(DA)(DT)(DG)(DT)(DG)(DA)(DA) (DA) (DA)(DG)(DG)(DC)(DC)(DG) ...String:
(DG)(DG)(DA)(DG)(DA)(DG)(DA)(DT)(DG)(DT) (DG)(DC)(DG)(DG)(DA)(DG)(DA)(DT)(DA)(DA) (DT)(DC)(DA)(DG)(DC)(DT)(DT)(DT)(DT) (DT)(DA)(DT)(DA)(DT)(DG)(DT)(DG)(DA)(DA) (DA) (DA)(DG)(DG)(DC)(DC)(DG)(DT)(DT) (DT)(DT)(DT)(DA)(DC)(DC)(DA)(DA)(DT)(DA) (DG)(DA) (DT)(DC)(DA)(DG)(DA)(DT)(DT) (DG)(DG)(DT)(DC)(DA)(DT)(DT)(DT)(DT)(DC) (DG)

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Macromolecule #2: AddAB promoter

MacromoleculeName: AddAB promoter / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 23.866352 KDa
SequenceString: (DC)(DG)(DA)(DA)(DA)(DA)(DT)(DG)(DA)(DC) (DC)(DA)(DA)(DT)(DC)(DT)(DG)(DA)(DT)(DC) (DT)(DA)(DT)(DT)(DG)(DG)(DT)(DA)(DA) (DA)(DA)(DA)(DC)(DG)(DG)(DC)(DC)(DT)(DT) (DT) (DT)(DC)(DA)(DC)(DA)(DT) ...String:
(DC)(DG)(DA)(DA)(DA)(DA)(DT)(DG)(DA)(DC) (DC)(DA)(DA)(DT)(DC)(DT)(DG)(DA)(DT)(DC) (DT)(DA)(DT)(DT)(DG)(DG)(DT)(DA)(DA) (DA)(DA)(DA)(DC)(DG)(DG)(DC)(DC)(DT)(DT) (DT) (DT)(DC)(DA)(DC)(DA)(DT)(DA)(DT) (DA)(DA)(DA)(DA)(DA)(DG)(DC)(DT)(DG)(DA) (DT)(DT) (DA)(DT)(DC)(DT)(DC)(DC)(DG) (DC)(DA)(DC)(DA)(DT)(DC)(DT)(DC)(DT)(DC) (DC)

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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

Concentration0.24 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
50.0 mMC6H14N4O2Arginine
150.0 mMKClKCl
5.0 mMMgCl2MgCl2
3.0 mMC4H10O2S2Dithiothreitol
0.001 %C58H114O26Tween-20
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsMonodisperse complex with dsDNA and 4 ComK equivalents.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 58207 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average exposure time: 2.0 sec. / Average electron dose: 55.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 130000
CTF correctionSoftware - Name: cryoSPARC
Startup modelType of model: INSILICO MODEL
Details: Sequence adapted idealized B-DNA fiber like duplex produced using Coot.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 100695
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7nbn:
Allostery through DNA drives phenotype switching

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