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- PDB-6omv: CryoEM structure of the LbCas12a-crRNA-AcrVA4-DNA complex -

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Basic information

Entry
Database: PDB / ID: 6omv
TitleCryoEM structure of the LbCas12a-crRNA-AcrVA4-DNA complex
Components
  • AcrVA4
  • Cpf1
  • DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*GP*GP*A)-3')
  • DNA (5'-D(P*TP*AP*AP*TP*TP*TP*CP*CP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
  • RNA
KeywordsUNKNOWN FUNCTION/DNA/RNA / UNKNOWN FUNCTION-DNA-RNA complex
Function / homology
Function and homology information


: / CRISPR-associated endonuclease Cpf1 PI domain / : / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Uncharacterized protein / Cpf1
Similarity search - Component
Biological speciesMoraxella bovoculi (bacteria)
Lachnospiraceae bacterium ND2006 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsChang, L. / Li, Z. / Zhang, H.
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
DepositionApr 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-20132
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
E: DNA (5'-D(P*TP*AP*AP*TP*TP*TP*CP*CP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')
F: DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*GP*GP*A)-3')
A: AcrVA4
B: Cpf1
G: RNA
C: AcrVA4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)220,2818
Polymers220,2326
Non-polymers492
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules EF

#1: DNA chain DNA (5'-D(P*TP*AP*AP*TP*TP*TP*CP*CP*TP*AP*AP*AP*GP*GP*AP*CP*G)-3')


Mass: 5210.410 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
#2: DNA chain DNA (5'-D(*CP*GP*TP*CP*CP*TP*TP*TP*AP*GP*GP*A)-3')


Mass: 3653.390 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)

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Protein , 2 types, 3 molecules ACB

#3: Protein AcrVA4


Mass: 27369.162 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Moraxella bovoculi (bacteria) / Gene: AAX07_09545 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0U2APF4
#4: Protein Cpf1


Mass: 143750.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A182DWE3

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RNA chain , 1 types, 1 molecules G

#5: RNA chain RNA


Mass: 12879.634 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)

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Non-polymers , 2 types, 8 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: DNA complex / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 35 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94720 / Symmetry type: POINT

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