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- EMDB-21367: Cryo-EM structure of Cas13(crRNA) -

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Basic information

Entry
Database: EMDB / ID: EMD-21367
TitleCryo-EM structure of Cas13(crRNA)
Map dataCas13(crRNA)
Sample
  • Complex: Cas13-crRNA complex
    • Protein or peptide: CRISPR-associated endoribonuclease Cas13a
    • RNA: RNA (51-MER)
KeywordsCRISPR-Cas system / Cas13 / IMMUNE SYSTEM / TypeVI
Function / homologyendonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesListeria seeligeri (bacteria) / Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954) (bacteria) / Listeria seeligeri serovar 1/2b str. SLCC3954 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJia N / Meeske AJ
CitationJournal: Science / Year: 2020
Title: A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity.
Authors: Alexander J Meeske / Ning Jia / Alice K Cassel / Albina Kozlova / Jingqiu Liao / Martin Wiedmann / Dinshaw J Patel / Luciano A Marraffini /
Abstract: The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. ...The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.
History
DepositionFeb 7, 2020-
Header (metadata) releaseMar 4, 2020-
Map releaseJun 10, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vrc
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21367.png

Downloads & links

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Map

FileDownload / File: emd_21367.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas13(crRNA)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.111940406 - 0.2286021
Average (Standard dev.)0.00024842937 (±0.004725863)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 211.68001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z196196196
origin x/y/z0.0000.0000.000
length x/y/z211.680211.680211.680
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS196196196
D min/max/mean-0.1120.2290.000

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Supplemental data

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Sample components

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Entire : Cas13-crRNA complex

EntireName: Cas13-crRNA complex
Components
  • Complex: Cas13-crRNA complex
    • Protein or peptide: CRISPR-associated endoribonuclease Cas13a
    • RNA: RNA (51-MER)

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Supramolecule #1: Cas13-crRNA complex

SupramoleculeName: Cas13-crRNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Listeria seeligeri (bacteria)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: CRISPR-associated endoribonuclease Cas13a

MacromoleculeName: CRISPR-associated endoribonuclease Cas13a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954) (bacteria)
Strain: ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954
Molecular weightTheoretical: 133.295297 KDa
Recombinant expressionOrganism: Listeria seeligeri (bacteria)
SequenceString: MWISIKTLIH HLGVLFFCDY MYNRREKKII EVKTMRITKV EVDRKKVLIS RDKNGGKLVY ENEMQDNTEQ IMHHKKSSFY KSVVNKTIC RPEQKQMKKL VHGLLQENSQ EKIKVSDVTK LNISNFLNHR FKKSLYYFPE NSPDKSEEYR IEINLSQLLE D SLKKQQGT ...String:
MWISIKTLIH HLGVLFFCDY MYNRREKKII EVKTMRITKV EVDRKKVLIS RDKNGGKLVY ENEMQDNTEQ IMHHKKSSFY KSVVNKTIC RPEQKQMKKL VHGLLQENSQ EKIKVSDVTK LNISNFLNHR FKKSLYYFPE NSPDKSEEYR IEINLSQLLE D SLKKQQGT FICWESFSKD MELYINWAEN YISSKTKLIK KSIRNNRIQS TESRSGQLMD RYMKDILNKN KPFDIQSVSE KY QLEKLTS ALKATFKEAK KNDKEINYKL KSTLQNHERQ IIEELKENSE LNQFNIEIRK HLETYFPIKK TNRKVGDIRN LEI GEIQKI VNHRLKNKIV QRILQEGKLA SYEIESTVNS NSLQKIKIEE AFALKFINAC LFASNNLRNM VYPVCKKDIL MIGE FKNSF KEIKHKKFIR QWSQFFSQEI TVDDIELASW GLRGAIAPIR NEIIHLKKHS WKKFFNNPTF KVKKSKIING KTKDV TSEF LYKETLFKDY FYSELDSVPE LIINKMESSK ILDYYSSDQL NQVFTIPNFE LSLLTSAVPF APSFKRVYLK GFDYQN QDE AQPDYNLKLN IYNEKAFNSE AFQAQYSLFK MVYYQVFLPQ FTTNNDLFKS SVDFILTLNK ERKGYAKAFQ DIRKMNK DE KPSEYMSYIQ SQLMLYQKKQ EEKEKINHFE KFINQVFIKG FNSFIEKNRL TYICHPTKNT VPENDNIEIP FHTDMDDS N IAFWLMCKLL DAKQLSELRN EMIKFSCSLQ STEEISTFTK AREVIGLALL NGEKGCNDWK ELFDDKEAWK KNMSLYVSE ELLQSLPYTQ EDGQTPVINR SIDLVKKYGT ETILEKLFSS SDDYKVSAKD IAKLHEYDVT EKIAQQESLH KQWIEKPGLA RDSAWTKKY QNVINDISNY QWAKTKVELT QVRHLHQLTI DLLSRLAGYM SIADRDFQFS SNYILERENS EYRVTSWILL S ENKNKNKY NDYELYNLKN ASIKVSSKND PQLKVDLKQL RLTLEYLELF DNRLKEKRNN ISHFNYLNGQ LGNSILELFD DA RDVLSYD RKLKNAVSKS LKEILSSHGM EVTFKPLYQT NHHLKIDKLQ PKKIHHLGEK STVSSNQVSN EYCQLVRTLL TMK HHHHHH

UniProtKB: CRISPR-associated endoribonuclease Cas13a

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Macromolecule #2: RNA (51-MER)

MacromoleculeName: RNA (51-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Listeria seeligeri serovar 1/2b str. SLCC3954 (bacteria)
Molecular weightTheoretical: 19.049383 KDa
SequenceString:
GACUACCUCU AUAUGAAAGA GGACUAAAAC CAUAUUUCCA AACUCCACUU UGACUACACC

GENBANK: GENBANK: FN557490.1

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Component - Formula: Tris / Details: 20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.23 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: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 190543
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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