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

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Basic information

Entry
Database: PDB / ID: 6xlk
TitleCryo-EM structure of EcmrR-DNA complex in EcmrR-RPitc-4nt
Components
  • MerR family transcriptional regulator EcmrR
  • synthetic non-template strand DNA (54-MER)
  • synthetic template strand DNA (54-MER)
KeywordsTRANSCRIPTION / Transcriptional factor / promoter / multidrug recognition
Function / homologyTETRAPHENYLANTIMONIUM ION / DNA / DNA (> 10)
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYang, Y. / Liu, C. / Liu, B.
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 7, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year
Revision 1.2May 26, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Mar 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-22246
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Assembly

Deposited unit
N: synthetic non-template strand DNA (54-MER)
T: synthetic template strand DNA (54-MER)
G: MerR family transcriptional regulator EcmrR
H: MerR family transcriptional regulator EcmrR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,2668
Polymers96,1424
Non-polymers2,1244
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11750 Å2
ΔGint-89 kcal/mol
Surface area33090 Å2

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Components

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


Mass: 16718.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#2: DNA chain synthetic template strand DNA (54-MER)


Mass: 16634.643 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: Protein MerR family transcriptional regulator EcmrR


Mass: 31394.191 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-1N7 / CHAPSO / 2-hydroxy-N,N-dimethyl-3-sulfo-N-(3-{[(3beta,5beta,7beta,12beta)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)propan-1-aminium / CHAPS detergent


Mass: 631.884 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H59N2O8S / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
#5: Chemical ChemComp-118 / TETRAPHENYLANTIMONIUM ION


Mass: 430.176 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H20Sb / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: EcmrR-spacer DNA complex in EcmrR-RNAP-promoter initial transcribing complex with 4-nt RNA transcript (EcmrR-RPitc-4nt)
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.92 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: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 13.2 sec. / Electron dose: 50.46 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 44 / Used frames/image: 1-44

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
7ISOLDEmodel fitting
12cryoSPARC2.143D reconstruction
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 372776
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80448 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6WL5
Pdb chain-ID: A / Accession code: 6WL5 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0085685
ELECTRON MICROSCOPYf_angle_d0.6847908
ELECTRON MICROSCOPYf_dihedral_angle_d15.0863201
ELECTRON MICROSCOPYf_chiral_restr0.047876
ELECTRON MICROSCOPYf_plane_restr0.004832

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