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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 data
SampleComplex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 17 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)F32GM122334 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01GM051487 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-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing 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
  • Download
  • 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.36 Å/pix.
x 128 pix.
= 558.08 Å
4.36 Å/pix.
x 128 pix.
= 558.08 Å
4.36 Å/pix.
x 128 pix.
= 558.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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
Details: T. thermophilus Csm was recombinantly expressed in E. coli, and T. thermophilus RNA polymerase was isolated from the natural source.
Number of components: 1

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Component #1: protein, Complex of Thermus thermophilus Csm with the Thermus the...

ProteinName: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
Details: T. thermophilus Csm was recombinantly expressed in E. coli, and T. thermophilus RNA polymerase was isolated from the natural source.
Recombinant expression: No
SourceSpecies: Thermus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 12
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 58 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI CETA (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 8351
3D reconstructionResolution: 17 Å / Resolution method: FSC 0.5 CUT-OFF

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