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- EMDB-22246: Cryo-EM structure of EcmrR-DNA complex in EcmrR-RPitc-4nt -

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Basic information

Entry
Database: EMDB / ID: EMD-22246
TitleCryo-EM structure of EcmrR-DNA complex in EcmrR-RPitc-4nt
Map data
Sample
  • Complex: EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcribing complex with 4-nt RNA transcript (EcmrR-RPitc-4nt)
    • DNA: synthetic non-template strand DNA (54-MER)
    • DNA: synthetic template strand DNA (54-MER)
    • Protein or peptide: MerR family transcriptional regulator EcmrR
  • Ligand: CHAPSOCHAPS detergent
  • Ligand: TETRAPHENYLANTIMONIUM ION
KeywordsTranscriptional factor / TRANSCRIPTION / promoter / multidrug recognition
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYang Y / Liu C
CitationJournal: Nat Commun / Year: 2021
Title: Structural visualization of transcription activated by a multidrug-sensing MerR family regulator.
Authors: Yang Yang / Chang Liu / Wei Zhou / Wei Shi / Ming Chen / Baoyue Zhang / David G Schatz / Yangbo Hu / Bin Liu /
Abstract: Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of ...Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.
History
DepositionJun 28, 2020-
Header (metadata) releaseApr 7, 2021-
Map releaseApr 7, 2021-
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: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xlk
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22246.png

Downloads & links

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Map

FileDownload / File: emd_22246.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.333 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.10012259 - 0.2017577
Average (Standard dev.)-0.00014982795 (±0.0041654115)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 341.248 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3331.3331.333
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z341.248341.248341.248
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1000.202-0.000

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Supplemental data

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Sample components

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Entire : EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcrib...

EntireName: EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcribing complex with 4-nt RNA transcript (EcmrR-RPitc-4nt)
Components
  • Complex: EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcribing complex with 4-nt RNA transcript (EcmrR-RPitc-4nt)
    • DNA: synthetic non-template strand DNA (54-MER)
    • DNA: synthetic template strand DNA (54-MER)
    • Protein or peptide: MerR family transcriptional regulator EcmrR
  • Ligand: CHAPSOCHAPS detergent
  • Ligand: TETRAPHENYLANTIMONIUM ION

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Supramolecule #1: EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcrib...

SupramoleculeName: EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcribing complex with 4-nt RNA transcript (EcmrR-RPitc-4nt)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: synthetic non-template strand DNA (54-MER)

MacromoleculeName: synthetic non-template strand DNA (54-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 16.718658 KDa
SequenceString: (DG)(DC)(DC)(DT)(DT)(DG)(DA)(DC)(DC)(DC) (DT)(DC)(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DG) (DG)(DG)(DA)(DG)(DG)(DG)(DT)(DT)(DT) (DA)(DG)(DA)(DT)(DT)(DG)(DT)(DG)(DT)(DG) (DC) (DA)(DG)(DT)(DC)(DT)(DG) ...String:
(DG)(DC)(DC)(DT)(DT)(DG)(DA)(DC)(DC)(DC) (DT)(DC)(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DG) (DG)(DG)(DA)(DG)(DG)(DG)(DT)(DT)(DT) (DA)(DG)(DA)(DT)(DT)(DG)(DT)(DG)(DT)(DG) (DC) (DA)(DG)(DT)(DC)(DT)(DG)(DA)(DC) (DG)(DC)(DG)(DG)(DC)(DG)

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Macromolecule #2: synthetic template strand DNA (54-MER)

MacromoleculeName: synthetic template strand DNA (54-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 16.634643 KDa
SequenceString: (DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(DG)(DT)(DA)(DG)(DG) (DA)(DA)(DT)(DC)(DT)(DA)(DA)(DA)(DC) (DC)(DC)(DT)(DC)(DC)(DC)(DC)(DT)(DT)(DA) (DG) (DG)(DG)(DG)(DA)(DG)(DG) ...String:
(DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(DG)(DT)(DA)(DG)(DG) (DA)(DA)(DT)(DC)(DT)(DA)(DA)(DA)(DC) (DC)(DC)(DT)(DC)(DC)(DC)(DC)(DT)(DT)(DA) (DG) (DG)(DG)(DG)(DA)(DG)(DG)(DG)(DT) (DC)(DA)(DA)(DG)(DG)(DC)

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Macromolecule #3: MerR family transcriptional regulator EcmrR

MacromoleculeName: MerR family transcriptional regulator EcmrR / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 31.394191 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SQIGLFSKIC RVTIKTLHYY NKIGLLVPAY INPDNGYRFY TSDQLMKFHQ IASLRQLGFT ITEIVTLTQD ENSCHIIERR RLEIQKQIR DMADMLSRIN HYLQHKKKER IMLYQAALKE IPECIVYSKR FIVPDFSSYI KLIPPIGQEV MKANPGLTLT T PAYCFTLY ...String:
SQIGLFSKIC RVTIKTLHYY NKIGLLVPAY INPDNGYRFY TSDQLMKFHQ IASLRQLGFT ITEIVTLTQD ENSCHIIERR RLEIQKQIR DMADMLSRIN HYLQHKKKER IMLYQAALKE IPECIVYSKR FIVPDFSSYI KLIPPIGQEV MKANPGLTLT T PAYCFTLY HDKEYKEKNM DVEFCEAVND FGKNEGNIIF QVIPAITAVT VIHKGPYDSL RNAYIYLMQW VEDNGYLLTN SP RESYIDG IWNKQDSAEW MTEIQFPVEK V

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Macromolecule #4: CHAPSO

MacromoleculeName: CHAPSO / type: ligand / ID: 4 / Number of copies: 2 / Formula: 1N7
Molecular weightTheoretical: 631.884 Da
Chemical component information

ChemComp-1N7:
CHAPSO / detergent*YM / CHAPS detergent

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Macromolecule #5: TETRAPHENYLANTIMONIUM ION

MacromoleculeName: TETRAPHENYLANTIMONIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: 118
Molecular weightTheoretical: 430.176 Da
Chemical component information

ChemComp-118:
TETRAPHENYLANTIMONIUM ION

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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

Concentration0.92 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
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: SUPER-RESOLUTION / Digitization - Frames/image: 1-44 / Average exposure time: 13.2 sec. / Average electron dose: 50.46 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 372776
Startup modelType of model: NONE
Details: Ab initio models were generated from a set of particles directly in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Number images used: 80448

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Atomic model buiding 1

Initial modelPDB ID:

6wl5


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6xlk:
Cryo-EM structure of EcmrR-DNA complex in EcmrR-RPitc-4nt

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