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- PDB-7oqh: CryoEM structure of the transcription termination factor Rho from... -

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Basic information

Entry
Database: PDB / ID: 7oqh
TitleCryoEM structure of the transcription termination factor Rho from Mycobacterium tuberculosis
ComponentsTranscription termination factor Rho
KeywordsTRANSCRIPTION / transcription termination factor / RNA helicase / ATP-dependent / molecular motor
Function / homology
Function and homology information


ATP-dependent activity, acting on RNA / DNA-templated transcription termination / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ATP hydrolysis activity / RNA binding / ATP binding
Similarity search - Function
Transcription termination factor Rho / Rho termination factor, N-terminal / Rho termination factor, RNA-binding domain / Transcription termination factor Rho, ATP binding domain / Rho termination factor, RNA-binding domain / Rho termination factor, N-terminal domain / Rho RNA-binding domain profile. / Rho termination factor, N-terminal domain / Rho termination factor, N-terminal domain superfamily / Cold shock domain ...Transcription termination factor Rho / Rho termination factor, N-terminal / Rho termination factor, RNA-binding domain / Transcription termination factor Rho, ATP binding domain / Rho termination factor, RNA-binding domain / Rho termination factor, N-terminal domain / Rho RNA-binding domain profile. / Rho termination factor, N-terminal domain / Rho termination factor, N-terminal domain superfamily / Cold shock domain / Cold shock protein domain / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Transcription termination factor Rho
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsSaridakis, E. / Vishwakarma, R. / Lai Kee Him, J. / Martin, K. / Simon, I. / Cohen-Gonsaud, M. / Coste, F. / Bron, P. / Margeat, E. / Boudvillain, M.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-15-CE11-0024 France
European Union (EU)665790 France
CitationJournal: Commun Biol / Year: 2022
Title: Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism.
Authors: Emmanuel Saridakis / Rishi Vishwakarma / Josephine Lai-Kee-Him / Kevin Martin / Isabelle Simon / Martin Cohen-Gonsaud / Franck Coste / Patrick Bron / Emmanuel Margeat / Marc Boudvillain /
Abstract: The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis ...The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [Rho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of Rho at 3.3 Å resolution. The Rho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of Rho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of Rho and provides a framework for future antibiotic development.
History
DepositionJun 3, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Data collection / Database references / Category: citation / citation_author / em_imaging
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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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  • EMDB-12701
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Assembly

Deposited unit
A: Transcription termination factor Rho
B: Transcription termination factor Rho
C: Transcription termination factor Rho
D: Transcription termination factor Rho
E: Transcription termination factor Rho
F: Transcription termination factor Rho
hetero molecules


Theoretical massNumber of molelcules
Total (without water)407,34117
Polymers404,1766
Non-polymers3,16511
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15160 Å2
ΔGint-58 kcal/mol
Surface area91020 Å2

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Components

#1: Protein
Transcription termination factor Rho / ATP-dependent helicase Rho


Mass: 67362.703 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Production host: Escherichia coli (E. coli)
References: UniProt: A0A045J3H4, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: Transcription termination factor Rho from Mycrobacterium Tuberculosis
Type: COMPLEX
Details: The cryo-EM sample was prepared by mixing M tuberculosis Rho (0.6mg mL-1, as per nano-drop, A280) with 10uM dC20 oligonucleotide and 1mM ATP in a buffer (10mM Tris-HCl 150mM 5mM MgCl2 pH 7.6) ...Details: The cryo-EM sample was prepared by mixing M tuberculosis Rho (0.6mg mL-1, as per nano-drop, A280) with 10uM dC20 oligonucleotide and 1mM ATP in a buffer (10mM Tris-HCl 150mM 5mM MgCl2 pH 7.6) by incubating 10min at 22 oC
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Cell: Rosetta-DE3 / Plasmid: pET28b
Buffer solutionpH: 7.9
Buffer component
IDConc.NameBuffer-ID
120 mMTris-Cl1
25 %glycerol1
30.2 mMEDTA1
40.2 mMDTT1
50.2 MKCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 22 % / Chamber temperature: 295.15 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 FIELD / Calibrated magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 10888

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1RELION3.0.8particle selection
2SerialEMimage acquisition
4RELION3.0.8CTF correction
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2217252
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 986385 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingPDB-ID: 1PV4

1pv4


Accession code: 1PV4 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 36.54 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003817076
ELECTRON MICROSCOPYf_angle_d0.615823159
ELECTRON MICROSCOPYf_chiral_restr0.04412709
ELECTRON MICROSCOPYf_plane_restr0.00513003
ELECTRON MICROSCOPYf_dihedral_angle_d4.74992388

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