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- EMDB-9742: Cryo-EM structure of AcrVA5-acetylated MbCas12a in complex with crRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-9742
TitleCryo-EM structure of AcrVA5-acetylated MbCas12a in complex with crRNA
Map data
Sample
  • Complex: cpf1-crRNA
    • Protein or peptide: nuclease
    • RNA: RNA (59-MER)
Function / homologyCRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain / Type V CRISPR-associated protein Cpf1
Function and homology information
Biological speciesMoraxella bovoculi (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDong L / Li N / Guan X / Zhu Y / Gao N / Huang Z
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China31825008 China
National Natural Science Foundation of China31422014 China
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: An anti-CRISPR protein disables type V Cas12a by acetylation.
Authors: Liyong Dong / Xiaoyu Guan / Ningning Li / Fan Zhang / Yuwei Zhu / Kuan Ren / Ling Yu / Fengxia Zhou / Zhifu Han / Ning Gao / Zhiwei Huang /
Abstract: Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained ...Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.
History
DepositionDec 2, 2018-
Header (metadata) releaseApr 10, 2019-
Map releaseApr 10, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.08
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  • Surface view with fitted model
  • Atomic models: PDB-6iv6
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9742.png

Downloads & links

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Map

FileDownload / File: emd_9742.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0493 / Movie #1: 0.08
Minimum - Maximum-0.33200693 - 0.5582311
Average (Standard dev.)0.0007311323 (±0.013228383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z212.480212.480212.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3320.5580.001

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Supplemental data

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Sample components

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Entire : cpf1-crRNA

EntireName: cpf1-crRNA
Components
  • Complex: cpf1-crRNA
    • Protein or peptide: nuclease
    • RNA: RNA (59-MER)

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Supramolecule #1: cpf1-crRNA

SupramoleculeName: cpf1-crRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Moraxella bovoculi (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: nuclease

MacromoleculeName: nuclease / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Moraxella bovoculi (bacteria)
Molecular weightTheoretical: 145.253391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLFQDFTHLY PLSKTVRFEL KPIG(ALY)TLEHI HAKNFLNQDE TMADMYQKVK AILDDYHRDF IADMMGEVKL TKLAEF YDV YLKFRKNPKD DGLQKQLKDL QAVLRKEIVK PIGNGGKYKA GYDRLFGAKL FKDGKELGDL AKFVIAQEGE SSP (ALY)LAHLA ...String:
MLFQDFTHLY PLSKTVRFEL KPIG(ALY)TLEHI HAKNFLNQDE TMADMYQKVK AILDDYHRDF IADMMGEVKL TKLAEF YDV YLKFRKNPKD DGLQKQLKDL QAVLRKEIVK PIGNGGKYKA GYDRLFGAKL FKDGKELGDL AKFVIAQEGE SSP (ALY)LAHLA HFEKFSTYFT GFHDNRKNMY SDEDKHTAIA YRLIHENLPR FIDNLQILAT IKQKHSALYD QIINELTASG LDVSLASHL DGYHKLLTQE GITAYNTLLG GISGEAGSRK IQGINELINS HHNQHCHKSE RIAKLRPLHK QILSDGMGVS F LPSKFADD SEVCQAVNEF YRHYADVFAK VQSLFDGFDD YQKDGIYVEY KNLNELSKQA FGDFALLGRV LDGYYVDVVN PE FNERFAK AKTDNAKAKL TKEKDKFIKG VHSLASLEQA IEHYTARHDD ESVQAGKLGQ YFKHGLAGVD NPIQKIHNNH STI KGFLER ERPAGERALP KIKSDKSPEI RQLKELLDNA LNVAHFAKLL TTKTTLHNQD GNFYGEFGAL YDELAKIATL YNKV RDYLS QKPFSTEKYK LNFGNPTLLN GWDLNKEKDN FGVILQKDGC YYLALLDKAH KKVFDNAPNT GKSVYQKMIY KLLPG PNKM LP(ALY)VFFAKSN LDYYNPSAEL LDKYAQGTHK KGDNFNLKDC HALIDFFKAG INKHPEWQHF GFKFSPTSSY QD LSDFYRE VEPQGYQVKF VDINADYINE LVEQGQLYLF QIYNKDFSPK AHGKPNLHTL YFKALFSEDN LVNPIYKLNG EAE IFYRKA SLDMNETTIH RAGEVLENKN PDNPK(ALY)RQFV YDIIKDKRYT QDKFMLHVPI TMNFGVQGMT IKEFNKKVNQ SIQQYDEVN VIGIDRGERH LLYLTVINSK GEILEQRSLN DITTASANGT QMTTPYHKIL DKREIERLNA RVGWGEIETI K ELKSGYLS HVVHQISQLM LKYNAIVVLE DLNFGFKRGC FKVEKQIYQN FENALIKKLN HLVLKDKADD EIGSYKNALQ LT NNFTDLK SIGKQTGFLF YVPAWNTSKI DPETGFVDLL KPRYENIAQS QAFFGKFDKI CYNADRGYFE FHIDYAKFND KAK NSRQIW KICSHGDKRY VYDKTANQNK GATIGVNVND ELKSLFTRYH INDKQPNLVM DICQNNDKEF HKSLMYLLKT LLAL RYSNA SSDEDFILSP VANDEGVFFN SALADDTQPQ NADANGAYHI ALKGLWLLNE LKNSDDLNKV KLAIDNQTWL NFAQN R

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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: 18.673896 KDa
SequenceString:
GUCUAACGAC CUUUUAAAUU UCUACUGUUU GUAGAUCUGA UGGUCCAUGU CUGUUACUC

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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: 8
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 81.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: making initial model from cisTEM
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93000

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