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- EMDB-0455: Negative stain EM structure of T. thermophilus Csm with a transcr... -

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Basic information

Entry
Database: EMDB / ID: EMD-0455
TitleNegative stain EM structure of T. thermophilus Csm with a transcription elongation complex
Map dataCsm-RNAP
Sample
  • Complex: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
KeywordsCsm / RNA substrate / DNA / RNA BINDING PROTEIN / RNA polymerase
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 17.0 Å
AuthorsLiu J-J / Liu TY
Funding support United States, 4 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01GM051487 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM122334 United States
Life Sciences Research Foundation United States
CitationJournal: Nat Commun / Year: 2019
Title: Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.
Authors: Tina Y Liu / Jun-Jie Liu / Abhishek J Aditham / Eva Nogales / Jennifer A Doudna /
Abstract: Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and ...Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.
History
DepositionJan 12, 2019-
Header (metadata) releaseFeb 20, 2019-
Map releaseJul 10, 2019-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0455.png

Downloads & links

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Map

FileDownload / File: emd_0455.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCsm-RNAP
Voxel sizeX=Y=Z: 4.36 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.03912864 - 0.112419434
Average (Standard dev.)0.0001628129 (±0.006225997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 558.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z558.080558.080558.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0390.1120.000

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Supplemental data

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Sample components

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Entire : Complex of Thermus thermophilus Csm with the Thermus thermophilus...

EntireName: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
Components
  • Complex: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex

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Supramolecule #1: Complex of Thermus thermophilus Csm with the Thermus thermophilus...

SupramoleculeName: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
type: complex / ID: 1 / Parent: 0
Details: T. thermophilus Csm was recombinantly expressed in E. coli, and T. thermophilus RNA polymerase was isolated from the natural source.
Source (natural)Organism: Thermus thermophilus (bacteria)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
StainingType: NEGATIVE / Material: UA
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Average electron dose: 58.0 e/Å2

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 8351

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