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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: EMD-6777
TitleElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
Map dataElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
Sample
  • Complex: LbuCas13a-crRNA binary complex
    • Protein or peptide: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
    • RNA: RNA (59-MER)
KeywordsCas13a / CRISPR / RNA BINDING PROTEIN-RNA COMPLEX
Function / homology: / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesLeptotrichia buccalis (bacteria) / Leptotrichia buccalis (strain ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b) (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang X / Wang Y
Funding support China, 3 items
OrganizationGrant numberCountry
the Chinese Ministry of Science and Technology2014CB910102 China
the Natural Science Foundation of China91440201, 31571335, 31400640, 31570874 China
the Strategic Priority Research Program of the Chinese Academy of SciencesXDB08010203 China
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.
History
DepositionJun 30, 2017-
Header (metadata) releaseSep 13, 2017-
Map releaseSep 13, 2017-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • 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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6777.png

Downloads & links

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Map

FileDownload / File: emd_6777.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.12865293 - 0.20330638
Average (Standard dev.)0.00039493514 (±0.0066677476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 164.0 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: LbuCas13a-crRNA binary complex
    • Protein or peptide: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
    • RNA: RNA (59-MER)

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Supramolecule #1: LbuCas13a-crRNA binary complex

SupramoleculeName: LbuCas13a-crRNA binary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Leptotrichia buccalis (bacteria) / Strain: ATCC 14201

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Macromolecule #1: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a

MacromoleculeName: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 138.555156 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL SLKNGKKEN IDREYSETDI LESDVRDKKN FAVLKKIYLN ENVNSEELEV FRNDIKKKLN KINSLKYSFE KNKANYQKIN E NNIEKVEG ...String:
MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL SLKNGKKEN IDREYSETDI LESDVRDKKN FAVLKKIYLN ENVNSEELEV FRNDIKKKLN KINSLKYSFE KNKANYQKIN E NNIEKVEG KSKRNIIYDY YRESAKRDAY VSNVKEAFDK LYKEEDIAKL VLEIENLTKL EKYKIREFYH EIIGRKNDKE NF AKIIYEE IQNVNNMKEL IEKVPDMSEL KKSQVFYKYY LDKEELNDKN IKYAFCHFVE IEMSQLLKNY VYKRLSNISN DKI KRIFEY QNLKKLIENK LLNKLDTYVR NCGKYNYYLQ DGEIATSDFI ARNRQNEAFL RNIIGVSSVA YFSLRNILET ENEN DITGR MRGKTVKNNK GEEKYVSGEV DKIYNENKKN EVKENLKMFY SYDFNMDNKN EIEDFFANID EAISSIRHGI VHFNL ELEG KDIFAFKNIA PSEISKKMFQ NEINEKKLKL KIFRQLNSAN VFRYLEKYKI LNYLKRTRFE FVNKNIPFVP SFTKLY SRI DDLKNSLGIY WKTPKTNDDN KTKEIIDAQI YLLKNIYYGE FLNYFMSNNG NFFEISKEII ELNKNDKRNL KTGFYKL QK FEDIQEKIPK EYLANIQSLY MINAGNQDEE EKDTYIDFIQ KIFLKGFMTY LANNGRLSLI YIGSDEETNT SLAEKKQE F DKFLKKYEQN NNIKIPYEIN EFLREIKLGN ILKYTERLNM FYLILKLLNH KELTNLKGSL EKYQSANKEE AFSDQLELI NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK FDTNKIYFDG ENIIKHRAFY NIKKYGMLNL LEKIADKAGY KISIEELKK YSNKKNEIEK NHKMQENLHR KYARPRKDEK FTDEDYESYK QAIENIEEYT HLKNKVEFNE LNLLQGLLLR I LHRLVGYT SIWERDLRFR LKGEFPENQY IEEIFNFENK KNVKYKGGQI VEKYIKFYKE LHQNDEVKIN KYSSANIKVL KQ EKKDLYI ANYIAAFNYI PHAEISLLEV LENLRKLLSY DRKLKNAVMK SVVDILKEYG FVATFKIGAD KKIGIQTLES EKI VHLKNL KKKKLMTDRN SEELCKLVKI MFEYKMEEKK SEN

UniProtKB: CRISPR-associated endoribonuclease Cas13a

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Macromolecule #2: RNA (59-MER)

MacromoleculeName: RNA (59-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.869348 KDa
SequenceString:
GGACCACCCC AAAAAUGAAG GGGACUAAAA CACAAAUCUA UCUGAAUAAA CUCUUCUUC

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 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: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 238758
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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