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- EMDB-6777: Electron cryo-microscopy structure of LbuCas13a-crRNA binary complex -

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Basic information

Entry
Database: EMDB / ID: 6777
TitleElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
Map dataElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
SampleLbuCas13a-crRNA binary complex
  • A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
  • nucleic-acidNucleic acid
Function / homologydefense response to virus / endonuclease activity / Acting on Ester Bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a
Function and homology information
SourceLeptotrichia buccalis (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsZhang X / Wang Y
CitationJournal: Cell / Year: 2017
Title: The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a.
Authors: Liang Liu / Xueyan Li / Jun Ma / Zongqiang Li / Lilan You / Jiuyu Wang / Min Wang / Xinzheng Zhang / Yanli Wang
Abstract: Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a ...Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
Validation ReportPDB-ID: 5xwy

SummaryFull reportAbout validation report
DateDeposition: Jun 30, 2017 / Header (metadata) release: Sep 13, 2017 / Map release: Sep 13, 2017 / Last update: Sep 13, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5xwy
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6777.map.gz (map file in CCP4 format, 32001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
200 pix
0.82 Å/pix.
= 164. Å
200 pix
0.82 Å/pix.
= 164. Å
200 pix
0.82 Å/pix.
= 164. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density
Contour Level:0.02 (by author), 0.02 (movie #1):
Minimum - Maximum-0.12865293 - 0.20330638
Average (Standard dev.)0.00039493514 (0.0066677476)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions200200200
Origin000
Limit199199199
Spacing200200200
CellA=B=C: 164 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z164.000164.000164.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1290.2030.000

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Supplemental data

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Sample components

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Entire LbuCas13a-crRNA binary complex

EntireName: LbuCas13a-crRNA binary complex / Number of components: 3

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Component #1: protein, LbuCas13a-crRNA binary complex

ProteinName: LbuCas13a-crRNA binary complex / Recombinant expression: No
SourceSpecies: Leptotrichia buccalis (bacteria) / Strain: ATCC 14201
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a

ProteinName: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
Recombinant expression: No
MassTheoretical: 138.555156 kDa
Source (engineered)Expression System: Leptotrichia buccalis (strain ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b) (bacteria)
Strain: ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b

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Component #3: nucleic-acid, RNA (59-MER)

Nucleic-acidName: RNA (59-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GGACCACCCC AAAAAUGAAG GGGACUAAAA CACAAAUCUA UCUGAAUAAA CUCUUCUUC
MassTheoretical: 18.869348 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
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: 60 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 238758
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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