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- EMDB-0705: Structure of LbCas12a-crRNA complex bound to AcrVA4 (form B complex) -

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Basic information

Entry
Database: EMDB / ID: EMD-0705
TitleStructure of LbCas12a-crRNA complex bound to AcrVA4 (form B complex)
Map data
Sample
  • Complex: LbCas12a-crRNA complex bound to AcrVA4 (form B complex)
    • Protein or peptide: LbCas12a
    • Protein or peptide: AcrVA4
    • RNA: crRNA
  • Ligand: MAGNESIUM ION
  • Ligand: water
Function / homologyUncharacterized protein
Function and homology information
Biological speciesLachnospiraceae bacterium (bacteria) / Moraxella bovoculi (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsPeng R / Li Z / Xu Y / He S / Peng Q / Shi Y / Gao GF
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4.
Authors: Ruchao Peng / Zhiteng Li / Ying Xu / Shaoshuai He / Qi Peng / Lian-Ao Wu / Ying Wu / Jianxun Qi / Peiyi Wang / Yi Shi / George F Gao /
Abstract: Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the ...Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.
History
DepositionJul 30, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 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: 0.009
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6klb
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6klb
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0705.png

Downloads & links

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Map

FileDownload / File: emd_0705.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.009
Minimum - Maximum-0.03583638 - 0.061164357
Average (Standard dev.)0.00004954892 (±0.00087795313)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0360.0610.000

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Supplemental data

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Sample components

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Entire : LbCas12a-crRNA complex bound to AcrVA4 (form B complex)

EntireName: LbCas12a-crRNA complex bound to AcrVA4 (form B complex)
Components
  • Complex: LbCas12a-crRNA complex bound to AcrVA4 (form B complex)
    • Protein or peptide: LbCas12a
    • Protein or peptide: AcrVA4
    • RNA: crRNA
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: LbCas12a-crRNA complex bound to AcrVA4 (form B complex)

SupramoleculeName: LbCas12a-crRNA complex bound to AcrVA4 (form B complex)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Lachnospiraceae bacterium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
Molecular weightExperimental: 300 KDa

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Macromolecule #1: LbCas12a

MacromoleculeName: LbCas12a / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lachnospiraceae bacterium (bacteria)
Molecular weightTheoretical: 143.888359 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
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 VKH

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Macromolecule #2: AcrVA4

MacromoleculeName: AcrVA4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Moraxella bovoculi (bacteria)
Molecular weightTheoretical: 27.369162 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
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

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Macromolecule #3: crRNA

MacromoleculeName: crRNA / type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.48202 KDa
SequenceString:
AAUUUCUACU AAGUGUAGAU CGGUCUCGCA AAGAAUGGAU AU

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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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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 11 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration1.0 mg/mL
BufferpH: 7.5
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 508000
CTF correctionSoftware - Name: Gctf (ver. 0.50)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5id6

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 60000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6kl9

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6klb:
Structure of LbCas12a-crRNA complex bound to AcrVA4 (form B complex)

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