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- EMDB-0454: Cryo-EM structure of the T. thermophilus Csm complex bound to tar... -

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Basic information

Entry
Database: EMDB / ID: EMD-0454
TitleCryo-EM structure of the T. thermophilus Csm complex bound to target ssRNA
Map dataRNA-protein complex
Sample
  • Complex: Thermus thermophilus Csm complex bound to target ssRNA
    • RNA: RNA
KeywordsCsm / RNA substrate / RNA BINDING PROTEIN
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLiu JJ / 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: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6o1o
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0454.png

Downloads & links

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Map

FileDownload / File: emd_0454.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA-protein complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 380 pix.
= 410.4 Å		1.08 Å/pix.
x 380 pix.
= 410.4 Å		1.08 Å/pix.
x 380 pix.
= 410.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9 / Movie #1: 0.9
Minimum - Maximum-1.861513 - 4.2819433
Average (Standard dev.)0.0014707397 (±0.074400425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-190-190-190
Dimensions380380380
Spacing380380380
CellA=B=C: 410.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z380380380
origin x/y/z0.0000.0000.000
length x/y/z410.400410.400410.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-190-190-190
NC/NR/NS380380380
D min/max/mean-1.8624.2820.001

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Supplemental data

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Sample components

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Entire : Thermus thermophilus Csm complex bound to target ssRNA

EntireName: Thermus thermophilus Csm complex bound to target ssRNA
Components
  • Complex: Thermus thermophilus Csm complex bound to target ssRNA
    • RNA: RNA

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Supramolecule #1: Thermus thermophilus Csm complex bound to target ssRNA

SupramoleculeName: Thermus thermophilus Csm complex bound to target ssRNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermus thermophilus (bacteria)

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Macromolecule #1: RNA

MacromoleculeName: RNA / type: rna / ID: 1
Source (natural)Organism: Thermus thermophilus (bacteria)
SequenceString:
GGAUAUGCGC CUUGACGUGG UCGUCCCCGG GCGCCUUAUC UACGGUAUCA

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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

BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: DIRECT ELECTRON DE-16 (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 87321
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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