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- PDB-6p7n: Cryo-EM structure of LbCas12a-crRNA: AcrVA4 (2:2 complex) -

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Basic information

Entry
Database: PDB / ID: 6p7n
TitleCryo-EM structure of LbCas12a-crRNA: AcrVA4 (2:2 complex)
Components
  • Cas12a
  • anti-CRISPR VA4
  • mature crRNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR-Cas / anti-CRISPR / Cas12a / Cpf1 / LbCas12a / AcrVA4 / RNA BINDING PROTEIN-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 speciesMoraxella bovoculi (bacteria)
Lachnospiraceae bacterium ND2006 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsKnott, G.J. / Liu, J.J. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Elife / Year: 2019
Title: Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.
Authors: Gavin J Knott / Brady F Cress / Jun-Jie Liu / Brittney W Thornton / Rachel J Lew / Basem Al-Shayeb / Daniel J Rosenberg / Michal Hammel / Benjamin A Adler / Marco J Lobba / Michael Xu / Adam ...Authors: Gavin J Knott / Brady F Cress / Jun-Jie Liu / Brittney W Thornton / Rachel J Lew / Basem Al-Shayeb / Daniel J Rosenberg / Michal Hammel / Benjamin A Adler / Marco J Lobba / Michael Xu / Adam P Arkin / Christof Fellmann / Jennifer A Doudna /
Abstract: CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein ...CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.
#1: Journal: Acta Crystallogr D Biol Crystallogr / Year: 2010
Title: PHENIX: a comprehensive Python-based system for macromolecular structure solution.
Authors: Paul D Adams / Pavel V Afonine / Gábor Bunkóczi / Vincent B Chen / Ian W Davis / Nathaniel Echols / Jeffrey J Headd / Li-Wei Hung / Gary J Kapral / Ralf W Grosse-Kunstleve / Airlie J McCoy ...Authors: Paul D Adams / Pavel V Afonine / Gábor Bunkóczi / Vincent B Chen / Ian W Davis / Nathaniel Echols / Jeffrey J Headd / Li-Wei Hung / Gary J Kapral / Ralf W Grosse-Kunstleve / Airlie J McCoy / Nigel W Moriarty / Robert Oeffner / Randy J Read / David C Richardson / Jane S Richardson / Thomas C Terwilliger / Peter H Zwart /
Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many ...Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 6, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
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

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Structure visualization

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Assembly

Deposited unit
C: anti-CRISPR VA4
G: anti-CRISPR VA4
A: Cas12a
E: Cas12a
B: mature crRNA
F: mature crRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)369,33310
Polymers369,2356
Non-polymers974
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Data presented within associated publication., SAXS, Data presented within associated publication.
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19670 Å2
ΔGint-134 kcal/mol
Surface area111940 Å2

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Components

#1: Protein anti-CRISPR VA4 / AcrVA4


Mass: 27641.422 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 (E. coli) / References: UniProt: A0A0U2APF4
#2: Protein Cas12a


Mass: 144160.609 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Gene: lbcas12a / Production host: Escherichia coli (E. coli) / References: UniProt: A0A182DWE3*PLUS
#3: RNA chain mature crRNA


Mass: 12815.634 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
#4: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1LbCas12a-crRNA-AcrVA4 Complex (2:2, State I)COMPLEXHomodimeric AcrVA4 bound to two copies of the LbCas12a-crRNA complex.#1-#30MULTIPLE SOURCES
2LbCas12aCOMPLEX#21RECOMBINANT
3crRNACOMPLEX#31NATURAL
4AcrVA4COMPLEX#11RECOMBINANT
Molecular weightExperimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Lachnospiraceae bacterium ND2006 (bacteria)1410628
23Lachnospiraceae bacterium ND2006 (bacteria)1410628
34Moraxella bovoculi (bacteria)386891
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
24Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2200 mMpotassium chlorideKCl1
31 mMtris((2-carboxyethyl)phosphine)C9H15O6P1
40.1 % (v/v)glycerolC3H8O31
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 42 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.15_3459refinement
PHENIX1.15_3459refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79787 / Num. of class averages: 1 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001820860
ELECTRON MICROSCOPYf_angle_d0.523728310
ELECTRON MICROSCOPYf_chiral_restr0.04043096
ELECTRON MICROSCOPYf_plane_restr0.00363434
ELECTRON MICROSCOPYf_dihedral_angle_d26.222912520

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