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- EMDB-12701: CryoEM structure of the transcription termination factor Rho from... -

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Basic information

Entry
Database: EMDB / ID: EMD-12701
TitleCryoEM structure of the transcription termination factor Rho from Mycrobacterium Tuberculosis
Map data
Sample
  • Complex: Transcription termination factor Rho from Mycrobacterium Tuberculosis
    • Protein or peptide: Transcription termination factor Rho from Mycrobacterium Tuberculosis
Function / homology
Function and homology information


ATP-dependent activity, acting on RNA / helicase activity / DNA-templated transcription termination / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / hydrolase 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transcription termination factor Rho
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsMartin K / Saridakis E / Vishwakarma R / Lai Kee Him J / Simon I / Cohen-Gonsaud M / Coste F / Margeat E / Boudvillain M / Bron P
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-15-CE11-0024 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
DepositionApr 1, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateFeb 23, 2022-
Current statusFeb 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0344
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0344
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7oqh
  • Surface level: 0.0344
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12701.png

Downloads & links

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Map

FileDownload / File: emd_12701.map.gz / Format: CCP4 / Size: 39.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.49376 Å
Density
Contour LevelBy AUTHOR: 0.0344 / Movie #1: 0.0344
Minimum - Maximum-0.14178915 - 0.23906106
Average (Standard dev.)6.82482e-06 (±0.0052359276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions218218218
Spacing218218218
CellA=B=C: 325.63968 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.49376146788991.49376146788991.4937614678899
M x/y/z218218218
origin x/y/z0.0000.0000.000
length x/y/z325.640325.640325.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS218218218
D min/max/mean-0.1420.2390.000

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Supplemental data

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Mask #1

Fileemd_12701_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Additional map: #1

Fileemd_12701_additional_1.map
Projections & Slices
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Half map: #2

Fileemd_12701_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_12701_half_map_2.map
Projections & Slices
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Sample components

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Entire : Transcription termination factor Rho from Mycrobacterium Tuberculosis

EntireName: Transcription termination factor Rho from Mycrobacterium Tuberculosis
Components
  • Complex: Transcription termination factor Rho from Mycrobacterium Tuberculosis
    • Protein or peptide: Transcription termination factor Rho from Mycrobacterium Tuberculosis

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Supramolecule #1: Transcription termination factor Rho from Mycrobacterium Tuberculosis

SupramoleculeName: Transcription termination factor Rho from Mycrobacterium Tuberculosis
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant cell: Rosette-DE3 / Recombinant plasmid: pET28b

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Macromolecule #1: Transcription termination factor Rho from Mycrobacterium Tuberculosis

MacromoleculeName: Transcription termination factor Rho from Mycrobacterium Tuberculosis
type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: VVQPVAGILD VLDNYAFVRT SGYLPGPHDV YVSMNMVRKN GMRRGDAVTG AVRVPKEQKF NPLVRLDSIN GGSVEDAKKR PEFGKLTPLY PNQRLRLATS TERLTTRVID LIMPIGKGQR ALIVSPPKAG KTTILQDIAN AITRNNPECH LMVVLVDERP EEVTDMQRSV ...String:
VVQPVAGILD VLDNYAFVRT SGYLPGPHDV YVSMNMVRKN GMRRGDAVTG AVRVPKEQKF NPLVRLDSIN GGSVEDAKKR PEFGKLTPLY PNQRLRLATS TERLTTRVID LIMPIGKGQR ALIVSPPKAG KTTILQDIAN AITRNNPECH LMVVLVDERP EEVTDMQRSV KGEVIASTFD RPPSDHTSVA ELAIERAKRL VEQGKDVVVL LDSITRLGRA YNNASPASGR ILSGGVDSTA LYPPKRFLGA ARNIEEGGSL TIIATAMVET GSTGDTVIFE EFKGTGNAEL KLDRKIAERR VFPAVDVNPS GTRKDELLLS PDEFAIVHKL RRVLSGLDSH QAIDLLMSQL RKTKNNYEFL VQVS

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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

Concentration0.5 mg/mL
BufferpH: 7.9
Details: 20 mM Tris-Cl 5 % [v/v] glycerol 0.2 mM EDTA 0.2 M KCl 0.2 mM DTT pH 7.9
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
DetailsRho (0.5mg/mL) 10uM dC20 oligonucleotide 1mM ATP.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 165000
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: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 10888 / Average exposure time: 8.0 sec. / Average electron dose: 48.0 e/Å2
Details: The scheme of image recording was made with 7 images per position plus 1 middle, and as a series of 40 frames per movie.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2217252
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1pvo

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 986385
DetailsK2 Summitdirect electron detector configured in counting mode, mounted on a Gatan Quantum 967 LS energy filter using a 20 eV slit width in zero loss mode
FSC plot (resolution estimation)

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