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- EMDB-14571: Negative stain 3D structure of the plastid-encoded RNA polymerase... -

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Basic information

Entry
Database: EMDB / ID: EMD-14571
TitleNegative stain 3D structure of the plastid-encoded RNA polymerase from Sinapis alba
Map dataNegative stain 3D map of Sinapsis alba PEP
Sample
  • Complex: RNA polymerase of Sinapsis alba
Biological speciesSinapis alba (white mustard)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsEffantin G / Fenel D
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-17-CE11-0031 France
CitationJournal: Int J Mol Sci / Year: 2022
Title: Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from .
Authors: Rémi Ruedas / Soumiya Sankari Muthukumar / Sylvie Kieffer-Jaquinod / François-Xavier Gillet / Daphna Fenel / Grégory Effantin / Thomas Pfannschmidt / Yohann Couté / Robert Blanvillain / David Cobessi /
Abstract: RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription ...RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.
History
DepositionMar 21, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14571.png

Downloads & links

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Map

FileDownload / File: emd_14571.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain 3D map of Sinapsis alba PEP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 200 pix.
= 440. Å		2.2 Å/pix.
x 200 pix.
= 440. Å		2.2 Å/pix.
x 200 pix.
= 440. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.04337342 - 0.1426762
Average (Standard dev.)-0.0010479807 (±0.013015185)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map1 of Sinapsis alba PEP

Fileemd_14571_half_map_1.map
Annotationhalf map1 of Sinapsis alba PEP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 of Sinapsis alba PEP

Fileemd_14571_half_map_2.map
Annotationhalf map 2 of Sinapsis alba PEP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RNA polymerase of Sinapsis alba

EntireName: RNA polymerase of Sinapsis alba
Components
  • Complex: RNA polymerase of Sinapsis alba

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Supramolecule #1: RNA polymerase of Sinapsis alba

SupramoleculeName: RNA polymerase of Sinapsis alba / type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)Organism: Sinapis alba (white mustard)
Molecular weightTheoretical: 1 MDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
StainingType: NEGATIVE / Material: SST

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: GATAN ORIUS SC1000 (4k x 2.7k) / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated defocus min: 1.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 17567
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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