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- EMDB-9398: CryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-9398
TitleCryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer
Map dataLbCas12a-crRNA-AcrVA4 dimer
Sample
  • Complex: protein complex 5
    • Protein or peptide: AcrVA4
    • Protein or peptide: Cpf1Cas12a
    • RNA: RNA (25-MER)
  • Ligand: MAGNESIUM ION
KeywordsUNKNOWN FUNCTION-RNA complex
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 / Uncharacterized protein / Cpf1
Function and homology information
Biological speciesLachnospiraceae bacterium ND2006 (bacteria) / Moraxella bovoculi (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsChang L / Li Z
CitationJournal: 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
DepositionJan 10, 2019-
Header (metadata) releaseJan 23, 2019-
Map releaseJun 12, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0177
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0177
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nm9
  • Surface level: 0.0177
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9398.png

Downloads & links

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Map

FileDownload / File: emd_9398.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLbCas12a-crRNA-AcrVA4 dimer
Voxel sizeX=Y=Z: 1.066 Å
Density
Contour LevelBy AUTHOR: 0.0177 / Movie #1: 0.0177
Minimum - Maximum-0.06602688 - 0.11155322
Average (Standard dev.)0.00028914717 (±0.0031515923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 255.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0661.0661.066
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z255.840255.840255.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0660.1120.000

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Supplemental data

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Sample components

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Entire : protein complex 5

EntireName: protein complex 5
Components
  • Complex: protein complex 5
    • Protein or peptide: AcrVA4
    • Protein or peptide: Cpf1Cas12a
    • RNA: RNA (25-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: protein complex 5

SupramoleculeName: protein complex 5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)

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Macromolecule #1: AcrVA4

MacromoleculeName: AcrVA4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Moraxella bovoculi (bacteria)
Molecular weightTheoretical: 27.369162 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: Cpf1

MacromoleculeName: Cpf1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 143.750219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #3: RNA (25-MER)

MacromoleculeName: RNA (25-MER) / type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 12.879634 KDa
SequenceString:
AAUUUCUACU AAGUGUAGAU GGAAAUUAGG UGCGCUUGGC

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration1 mg/mL
BufferpH: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 35.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number images used: 47609

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