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- EMDB-30767: Cryo-EM structure of LshCas13a-crRNA-anti-tag RNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-30767
TitleCryo-EM structure of LshCas13a-crRNA-anti-tag RNA complex
Map data
Sample
  • Complex: LshCas13a-crRNA-anti-tag RNA complex
    • Complex: LshCas13a
      • Protein or peptide: CRISPR/Cas system Cas13a
    • Complex: Lsh crRNA
      • RNA: CRISPR RNA
    • Complex: Anti-tag target RNA
      • RNA: Anti-tag target RNA
KeywordsType VI-A CRISPR-Cas system / Cas13a / anti-tag RNA / inhibition / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex
Function / homologyendonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / CRISPR/Cas system Cas13a / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesLeptotrichia shahii (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsWang B / Zhang T
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971135 China
CitationJournal: Mol Cell / Year: 2021
Title: Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.
Authors: Beibei Wang / Tianlong Zhang / Jun Yin / You Yu / Wenhao Xu / Jianping Ding / Dinshaw J Patel / Hui Yang /
Abstract: Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, ...Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13a in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.
History
DepositionDec 5, 2020-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
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: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7dmq
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30767.png

Downloads & links

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Map

FileDownload / File: emd_30767.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8613 Å
Density
Contour LevelBy AUTHOR: 0.0038 / Movie #1: 0.007
Minimum - Maximum-0.04132527 - 0.07069397
Average (Standard dev.)0.000085222084 (±0.0013997545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 220.4928 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.861300781250.861300781250.86130078125
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z220.493220.493220.493
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0410.0710.000

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Supplemental data

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Sample components

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Entire : LshCas13a-crRNA-anti-tag RNA complex

EntireName: LshCas13a-crRNA-anti-tag RNA complex
Components
  • Complex: LshCas13a-crRNA-anti-tag RNA complex
    • Complex: LshCas13a
      • Protein or peptide: CRISPR/Cas system Cas13a
    • Complex: Lsh crRNA
      • RNA: CRISPR RNA
    • Complex: Anti-tag target RNA
      • RNA: Anti-tag target RNA

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Supramolecule #1: LshCas13a-crRNA-anti-tag RNA complex

SupramoleculeName: LshCas13a-crRNA-anti-tag RNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Leptotrichia shahii (bacteria)

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Supramolecule #2: LshCas13a

SupramoleculeName: LshCas13a / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: Lsh crRNA

SupramoleculeName: Lsh crRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Supramolecule #4: Anti-tag target RNA

SupramoleculeName: Anti-tag target RNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: CRISPR/Cas system Cas13a

MacromoleculeName: CRISPR/Cas system Cas13a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Leptotrichia shahii (bacteria)
Molecular weightTheoretical: 166.697641 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSMGNLFGHK RWYEVRDKKD FKIKRKVKVK RNYDGNKYIL NINENNNKEK IDNNKFIRKY INYKKNDNIL KEFTRKFHAG NILFKLKGK EGIIRIENND DFLETEEVVL YIEAYGKSEK LKALGITKKK IIDEAIRQGI TKDDKKIEIK RQENEEEIEI D IRDEYTNK ...String:
GSMGNLFGHK RWYEVRDKKD FKIKRKVKVK RNYDGNKYIL NINENNNKEK IDNNKFIRKY INYKKNDNIL KEFTRKFHAG NILFKLKGK EGIIRIENND DFLETEEVVL YIEAYGKSEK LKALGITKKK IIDEAIRQGI TKDDKKIEIK RQENEEEIEI D IRDEYTNK TLNDCSIILR IIENDELETK KSIYEIFKNI NMSLYKIIEK IIENETEKVF ENRYYEEHLR EKLLKDDKID VI LTNFMEI REKIKSNLEI LGFVKFYLNV GGDKKKSKNK KMLVEKILNI NVDLTVEDIA DFVIKELEFW NITKRIEKVK KVN NEFLEK RRNRTYIKSY VLLDKHEKFK IERENKKDKI VKFFVENIKN NSIKEKIEKI LAEFKIDELI KKLEKELKKG NCDT EIFGI FKKHYKVNFD SKKFSKKSDE EKELYKIIYR YLKGRIEKIL VNEQKVRLKK MEKIEIEKIL NESILSEKIL KRVKQ YTLE HIMYLGKLRH NDIDMTTVNT DDFSRLHAKE ELDLELITFF ASTNMELNKI FSRENINNDE NIDFFGGDRE KNYVLD KKI LNSKIKIIRD LDFIDNKNNI TNNFIRKFTK IGTNERNRIL HAISKERDLQ GTQDDYNKVI NIIQNLKISD EEVSKAL NL DVVFKDKKNI ITKINDIKIS EENNNDIKYL PSFSKVLPEI LNLYRNNPKN EPFDTIETEK IVLNALIYVN KELYKKLI L EDDLEENESK NIFLQELKKT LGNIDEIDEN IIENYYKNAQ ISASKGNNKA IKKYQKKVIE CYIGYLRKNY EELFDFSDF KMNIQEIKKQ IKDINDNKTY ERITVKTSDK TIVINDDFEY IISIFALLNS NAVINKIRNR FFATSVWLNT SEYQNIIDIL DEIMQLNTL RNECITENWN LNLEEFIQKM KEIEKDFDDF KIQTKKEIFN NYYEDIKNNI LTEFKDDING CDVLEKKLEK I VIFDDETK FEIDKKSNIL QDEQRKLSNI NKKDLKKKVD QYIKDKDQEI KSKILCRIIF NSDFLKKYKK EIDNLIEDME SE NENKFQE IYYPKERKNE LYIYKKNLFL NIGNPNFDKI YGLISNDIKM ADAKFLFNID GKNIRKNKIS EIDAILKNLN DKL NGYSKE YKEKYIKKLK ENDDFFAKNI QNKNYKSFEK DYNRVSEYKK IRDLVEFNYL NKIESYLIDI NWKLAIQMAR FERD MHYIV NGLRELGIIK LSGYNTGISR AYPKRNGSDG FYTTTAYYKF FDEESYKKFE KICYGFGIDL SENSEINKPE NESIR NYIS HFYIVRNPFA DYSIAEQIDR VSNLLSYSTR YNNSTYASVF EVFKKDVNLD YDELKKKFKL IGNNDILERL MKPKKV SVL ELESYNSDYI KNLIIELLTK IENTNDTL

UniProtKB: CRISPR/Cas system Cas13a

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Macromolecule #2: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Leptotrichia shahii (bacteria)
Molecular weightTheoretical: 18.565096 KDa
SequenceString:
GGCCACCCCA AUAUCGAAGG GGACUAAAAC UAGAUUGCUG UUCUACCAAG UAAUCCAU

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Macromolecule #3: Anti-tag target RNA

MacromoleculeName: Anti-tag target RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 11.884038 KDa
SequenceString:
GAUGGAUUAC UUGGUAGAAC AGCAAUCUAG UUUUAGU

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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: 7.2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 8.0 sec. / Average electron dose: 64.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 212861

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7dmq:
Cryo-EM structure of LshCas13a-crRNA-anti-tag RNA complex

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