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

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Entry
Database: EMDB / ID: EMD-0454
TitleCryo-EM structure of the T. thermophilus Csm complex bound to target ssRNA
Map data
SampleThermus thermophilus Csm complex bound to target ssRNA:
nucleic-acidNucleic acid
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLiu JJ / Liu TY
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.
Validation ReportPDB-ID: 6o1o

SummaryFull reportAbout validation report
DateDeposition: Jan 12, 2019 / Header (metadata) release: Feb 20, 2019 / Map release: Jul 10, 2019 / Update: Jul 10, 2019

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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

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)
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
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
Number of components: 2

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Component #1: protein, Thermus thermophilus Csm complex bound to target ssRNA

ProteinName: Thermus thermophilus Csm complex bound to target ssRNA
Recombinant expression: No
SourceSpecies: Thermus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: nucleic-acid, RNA

nucleic acidName: RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GGAUAUGCGC CUUGACGUGG UCGUCCCCGG GCGCCUUAUC UACGGUAUCA
SourceSpecies: Thermus thermophilus (bacteria)

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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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: DIRECT ELECTRON DE-16 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 87321
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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