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- EMDB-10685: Multibody refinement of RNA polymerase body of pseudouridimycin-s... -

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Basic information

Entry
Database: EMDB / ID: EMD-10685
TitleMultibody refinement of RNA polymerase body of pseudouridimycin-stalled Mycoplasma pneumoniae in-cell expressome
Map dataRNA polymerase body of PUM-induced stalled expressome, multibody refinement
Sample
  • Cell: wild-type Mycoplasma pneumoniae M129 cells were treated with 0.4 mg/ml pseudouridimycin (PUM), 15-20 minutes prior to blotting and vitrification.
Biological speciesMycoplasma pneumoniae M129 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 8.1 Å
AuthorsMahamid J / Xue L
Funding support Germany, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)760067 Germany
CitationJournal: Science / Year: 2020
Title: In-cell architecture of an actively transcribing-translating expressome.
Authors: Francis J O'Reilly / Liang Xue / Andrea Graziadei / Ludwig Sinn / Swantje Lenz / Dimitry Tegunov / Cedric Blötz / Neil Singh / Wim J H Hagen / Patrick Cramer / Jörg Stülke / Julia Mahamid ...Authors: Francis J O'Reilly / Liang Xue / Andrea Graziadei / Ludwig Sinn / Swantje Lenz / Dimitry Tegunov / Cedric Blötz / Neil Singh / Wim J H Hagen / Patrick Cramer / Jörg Stülke / Julia Mahamid / Juri Rappsilber /
Abstract: Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the ...Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.
History
DepositionFeb 20, 2020-
Header (metadata) releaseAug 5, 2020-
Map releaseAug 5, 2020-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10685.png

Downloads & links

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Map

FileDownload / File: emd_10685.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA polymerase body of PUM-induced stalled expressome, multibody refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 200 pix.
= 600. Å		3 Å/pix.
x 200 pix.
= 600. Å		3 Å/pix.
x 200 pix.
= 600. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-1.0929404 - 2.0502367
Average (Standard dev.)0.0014752932 (±0.0403494)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 600.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z600.000600.000600.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-1.0932.0500.001

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Supplemental data

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Mask #1

Fileemd_10685_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RNA polymerase body of PUM-induced stalled expressome, multibody...

Fileemd_10685_half_map_1.map
AnnotationRNA polymerase body of PUM-induced stalled expressome, multibody refinement, half 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RNA polymerase body of PUM-induced stalled expressome, multibody...

Fileemd_10685_half_map_2.map
AnnotationRNA polymerase body of PUM-induced stalled expressome, multibody refinement, half 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : wild-type Mycoplasma pneumoniae M129 cells were treated with 0.4 ...

EntireName: wild-type Mycoplasma pneumoniae M129 cells were treated with 0.4 mg/ml pseudouridimycin (PUM), 15-20 minutes prior to blotting and vitrification.
Components
  • Cell: wild-type Mycoplasma pneumoniae M129 cells were treated with 0.4 mg/ml pseudouridimycin (PUM), 15-20 minutes prior to blotting and vitrification.

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Supramolecule #1: wild-type Mycoplasma pneumoniae M129 cells were treated with 0.4 ...

SupramoleculeName: wild-type Mycoplasma pneumoniae M129 cells were treated with 0.4 mg/ml pseudouridimycin (PUM), 15-20 minutes prior to blotting and vitrification.
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mycoplasma pneumoniae M129 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Details: modified Hayflick medium as described in Halbedel, Hames, and Stulke 2004, with 0.4 mg/ml pseudouridimycin (PUM)
GridModel: Quantifoil R2/1 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 45 % / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number subtomograms used: 8920
ExtractionNumber tomograms: 83 / Number images used: 14679 / Software - Name: Warp (ver. 1.0.6)
CTF correctionSoftware - Name: Warp (ver. 1.0.6)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
FSC plot (resolution estimation)

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