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- EMDB-20781: Structural basis for control of antibiotic production by bacteria... -

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Basic information

Entry
Database: EMDB / ID: EMD-20781
TitleStructural basis for control of antibiotic production by bacterial hormones
Map dataMap file - Postprocess masked .mrc UCSF Chimera 1.11, visualization -Volume viewer step 1 -Contour level 0.0181
Sample
  • Complex: protein-DNA complex
    • Protein or peptide: protein MmfR
    • DNA: synthetic DNA
Biological speciesStreptomyces coelicolor A3(2) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBhukya H
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Royal SocietyBB/M022765/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/M017982/1 United Kingdom
CitationJournal: Nature / Year: 2021
Title: Molecular basis for control of antibiotic production by a bacterial hormone.
Authors: Shanshan Zhou / Hussain Bhukya / Nicolas Malet / Peter J Harrison / Dean Rea / Matthew J Belousoff / Hariprasad Venugopal / Paulina K Sydor / Kathryn M Styles / Lijiang Song / Max J Cryle / ...Authors: Shanshan Zhou / Hussain Bhukya / Nicolas Malet / Peter J Harrison / Dean Rea / Matthew J Belousoff / Hariprasad Venugopal / Paulina K Sydor / Kathryn M Styles / Lijiang Song / Max J Cryle / Lona M Alkhalaf / Vilmos Fülöp / Gregory L Challis / Christophe Corre /
Abstract: Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture. Diffusible hormones frequently control the production of ...Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.
History
DepositionSep 29, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseOct 14, 2020-
UpdateMar 3, 2021-
Current statusMar 3, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20781.png

Downloads & links

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Map

FileDownload / File: emd_20781.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap file - Postprocess masked .mrc UCSF Chimera 1.11, visualization -Volume viewer step 1 -Contour level 0.0181
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 180 pix.
= 196.56 Å		1.09 Å/pix.
x 180 pix.
= 196.56 Å		1.09 Å/pix.
x 180 pix.
= 196.56 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.092 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0181 / Movie #1: 0.018
Minimum - Maximum-0.045197695 - 0.109287724
Average (Standard dev.)0.00030176292 (±0.0032651832)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 196.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0921.0921.092
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z196.560196.560196.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0450.1090.000

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Supplemental data

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Mask #1

Fileemd_20781_msk_1.map
Projections & Slices
AxesZYX

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Half map: Half map file - run half1 class001 unfil.mrc UCSF Chimera 1.11,...

Fileemd_20781_half_map_1.map
AnnotationHalf map file - run_half1_class001_unfil.mrc UCSF Chimera 1.11, visualization -Volume viewer step 1 -Contour level 0.017
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map file - run half2 class001 unfil.mrc UCSF Chimera 1.11,...

Fileemd_20781_half_map_2.map
AnnotationHalf map file - run_half2_class001_unfil.mrc UCSF Chimera 1.11, visualization -Volume viewer step 1 -Contour level 0.017
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : protein-DNA complex

EntireName: protein-DNA complex
Components
  • Complex: protein-DNA complex
    • Protein or peptide: protein MmfR
    • DNA: synthetic DNA

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Supramolecule #1: protein-DNA complex

SupramoleculeName: protein-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: protein-DNA complex. two homodimers of protein bound to a double stranded DNA
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightExperimental: 114 KDa

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Macromolecule #1: protein MmfR

MacromoleculeName: protein MmfR / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria) / Strain: A3(2)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHGKP IPNPLLGLDS TENLYFQGID PFTMTSAQQP TPFAVRSNVP RGPHPQQERS IKTRAQILEA ASEIFASRGY RGASVKDVAE RVGMTKGAVY FHFPSKESLA IAVVEEHYAR WPAAMEEIRI QGFTPLETVE EMLHRAAQAF RDDPVMQAGA RLQSERAFID ...String:
MHHHHHHGKP IPNPLLGLDS TENLYFQGID PFTMTSAQQP TPFAVRSNVP RGPHPQQERS IKTRAQILEA ASEIFASRGY RGASVKDVAE RVGMTKGAVY FHFPSKESLA IAVVEEHYAR WPAAMEEIRI QGFTPLETVE EMLHRAAQAF RDDPVMQAGA RLQSERAFID AELPLPYVDW THLLEVPLQD AREAGQLRAG VDPAAAARSL VAAFFGMQHV SDNLHQRADI MERWQELREL MFFALRA

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Macromolecule #2: synthetic DNA

MacromoleculeName: synthetic DNA / type: dna / ID: 2 / Details: 5'-ATACCTGCGGGAAGGTATT-3' / Classification: DNA
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
SequenceString:
ATACCTGCGG GAAGGTATT

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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

Concentration0.08 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
200.0 mMNaClNaCl

Details: 20 mM tris pH 8.0 and 200 mM NaCl fresh and autoclaved
GridModel: Quantifoil R1.2/1.3 / Material: COPPER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Detailsprotein-DNA complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 2 / Average exposure time: 10.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: -0.0005 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4pxi

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta) / Number images used: 379033
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT / Overall B value: 214

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