+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20132 | |||||||||
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Title | CryoEM structure of the LbCas12a-crRNA-AcrVA4-DNA complex | |||||||||
Map data | LbCas12a-crRNA-AcrVA4-DNA | |||||||||
Sample |
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Keywords | UNKNOWN FUNCTION-DNA-RNA complex | |||||||||
Function / homology | CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain / Uncharacterized protein / Cpf1 Function and homology information | |||||||||
Biological species | Lachnospiraceae bacterium ND2006 (bacteria) / Moraxella bovoculi (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
Authors | Chang L / Li Z | |||||||||
Citation | Journal: Cell Host Microbe / Year: 2019 Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins. Authors: Heng Zhang / Zhuang Li / Courtney M Daczkowski / Clinton Gabel / Andrew D Mesecar / Leifu Chang / Abstract: CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are ...CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_20132.map.gz | 1.8 MB | EMDB map data format | |
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Header (meta data) | emd-20132-v30.xml emd-20132.xml | 14.5 KB 14.5 KB | Display Display | EMDB header |
Images | emd_20132.png | 83.2 KB | ||
Filedesc metadata | emd-20132.cif.gz | 6.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20132 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20132 | HTTPS FTP |
-Related structure data
Related structure data | 6omvMC 0445C 0446C 0447C 0449C 9398C 6nm9C 6nmaC 6nmcC 6nmdC 6nmeC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_20132.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | LbCas12a-crRNA-AcrVA4-DNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.066 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : DNA complex
Entire | Name: DNA complex |
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Components |
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-Supramolecule #1: DNA complex
Supramolecule | Name: DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: Lachnospiraceae bacterium ND2006 (bacteria) |
-Macromolecule #1: DNA (5'-D(P*TP*AP*AP*TP*TP*TP*CP*CP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
Macromolecule | Name: DNA (5'-D(P*TP*AP*AP*TP*TP*TP*CP*CP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3') type: dna / ID: 1 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Lachnospiraceae bacterium ND2006 (bacteria) |
Molecular weight | Theoretical: 5.21041 KDa |
Sequence | String: (DT)(DA)(DA)(DT)(DT)(DT)(DC)(DC)(DT)(DA) (DA)(DA)(DG)(DG)(DA)(DC)(DG) |
-Macromolecule #2: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*GP*GP*A)-3')
Macromolecule | Name: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*GP*GP*A)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Lachnospiraceae bacterium ND2006 (bacteria) |
Molecular weight | Theoretical: 3.65339 KDa |
Sequence | String: (DC)(DG)(DT)(DC)(DC)(DT)(DT)(DT)(DA)(DG) (DG)(DA) |
-Macromolecule #3: AcrVA4
Macromolecule | Name: AcrVA4 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Moraxella bovoculi (bacteria) |
Molecular weight | Theoretical: 27.369162 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MYEIKLNDTL IHQTDDRVNA FVAYRYLLRR GDLPKCENIA RMYYDGKVIK TDVIDHDSVH SDEQAKVSNN DIIKMAISEL GVNNFKSLI KKQGYPFSNG HINSWFTDDP VKSKTMHNDE MYLVVQALIR ACIIKEIDLY TEQLYNIIKS LPYDKRPNVV Y SDQPLDPN ...String: MYEIKLNDTL IHQTDDRVNA FVAYRYLLRR GDLPKCENIA RMYYDGKVIK TDVIDHDSVH SDEQAKVSNN DIIKMAISEL GVNNFKSLI KKQGYPFSNG HINSWFTDDP VKSKTMHNDE MYLVVQALIR ACIIKEIDLY TEQLYNIIKS LPYDKRPNVV Y SDQPLDPN NLDLSEPELW AEQVGECMRY AHNDQPCFYI GSTKRELRVN YIVPVIGVRD EIERVMTLEE VRNLHK UniProtKB: Uncharacterized protein |
-Macromolecule #4: Cpf1
Macromolecule | Name: Cpf1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lachnospiraceae bacterium ND2006 (bacteria) |
Molecular weight | Theoretical: 143.750219 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MSKLEKFTNC YSLSKTLRFK AIPVGKTQEN IDNKRLLVED EKRAEDYKGV KKLLDRYYLS FINDVLHSIK LKNLNNYISL FRKKTRTEK ENKELENLEI NLRKEIAKAF KGNEGYKSLF KKDIIETILP EFLDDKDEIA LVNSFNGFTT AFTGFFDNRE N MFSEEAKS ...String: MSKLEKFTNC YSLSKTLRFK AIPVGKTQEN IDNKRLLVED EKRAEDYKGV KKLLDRYYLS FINDVLHSIK LKNLNNYISL FRKKTRTEK ENKELENLEI NLRKEIAKAF KGNEGYKSLF KKDIIETILP EFLDDKDEIA LVNSFNGFTT AFTGFFDNRE N MFSEEAKS TSIAFRCINE NLTRYISNMD IFEKVDAIFD KHEVQEIKEK ILNSDYDVED FFEGEFFNFV LTQEGIDVYN AI IGGFVTE SGEKIKGLNE YINLYNQKTK QKLPKFKPLY KQVLSDRESL SFYGEGYTSD EEVLEVFRNT LNKNSEIFSS IKK LEKLFK NFDEYSSAGI FVKNGPAIST ISKDIFGEWN VIRDKWNAEY DDIHLKKKAV VTEKYEDDRR KSFKKIGSFS LEQL QEYAD ADLSVVEKLK EIIIQKVDEI YKVYGSSEKL FDADFVLEKS LKKNDAVVAI MKDLLDSVKS FENYIKAFFG EGKET NRDE SFYGDFVLAY DILLKVDHIY DAIRNYVTQK PYSKDKFKLY FQNPQFMGGW DKDKETDYRA TILRYGSKYY LAIMDK KYA KCLQKIDKDD VNGNYEKINY KLLPGPNKML PKVFFSKKWM AYYNPSEDIQ KIYKNGTFKK GDMFNLNDCH KLIDFFK DS ISRYPKWSNA YDFNFSETEK YKDIAGFYRE VEEQGYKVSF ESASKKEVDK LVEEGKLYMF QIYNKDFSDK SHGTPNLH T MYFKLLFDEN NHGQIRLSGG AELFMRRASL KKEELVVHPA NSPIANKNPD NPKKTTTLSY DVYKDKRFSE DQYELHIPI AINKCPKNIF KINTEVRVLL KHDDNPYVIG IDRGERNLLY IVVVDGKGNI VEQYSLNEII NNFNGIRIKT DYHSLLDKKE KERFEARQN WTSIENIKEL KAGYISQVVH KICELVEKYD AVIALEDLNS GFKNSRVKVE KQVYQKFEKM LIDKLNYMVD K KSNPCATG GALKGYQITN KFESFKSMST QNGFIFYIPA WLTSKIDPST GFVNLLKTKY TSIADSKKFI SSFDRIMYVP EE DLFEFAL DYKNFSRTDA DYIKKWKLYS YGNRIRIFRN PKKNNVFDWE EVCLTSAYKE LFNKYGINYQ QGDIRALLCE QSD KAFYSS FMALMSLMLQ MRNSITGRTD VDFLISPVKN SDGIFYDSRN YEAQENAILP KNADANGAYN IARKVLWAIG QFKK AEDEK LDKVKIAISN KEWLEYAQTS VK UniProtKB: Cpf1 |
-Macromolecule #5: RNA
Macromolecule | Name: RNA / type: rna / ID: 5 / Number of copies: 1 |
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Source (natural) | Organism: Lachnospiraceae bacterium ND2006 (bacteria) |
Molecular weight | Theoretical: 12.879634 KDa |
Sequence | String: AAUUUCUACU AAGUGUAGAU GGAAAUUAGG UGCGCUUGGC |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #7: water
Macromolecule | Name: water / type: ligand / ID: 7 / Number of copies: 6 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Details: unspecified |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 130000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000 |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 35.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: OTHER |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 94720 |