|Entry||Database: PDB / ID: 6p7n|
|Title||Cryo-EM structure of LbCas12a-crRNA: AcrVA4 (2:2 complex)|
|Keywords||RNA BINDING PROTEIN/RNA / CRISPR-Cas / anti-CRISPR / Cas12a / Cpf1 / LbCas12a / AcrVA4 / RNA BINDING PROTEIN-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||Moraxella bovoculi (bacteria)|
Lachnospiraceae bacterium ND2006 (bacteria)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å|
|Authors||Knott, G.J. / Liu, J.J. / Doudna, J.A.|
|Funding support|| United States, 1items |
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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
C: anti-CRISPR VA4
G: anti-CRISPR VA4
B: mature crRNA
F: mature crRNA
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
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|
Mass: 12815.634 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Molecular weight||Experimental value: YES|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: unspecified|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 42 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C1 (asymmetric)|
|3D reconstruction||Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79787 / Num. of class averages: 1 / Symmetry type: POINT|
|Refinement||Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2|
|Refine LS restraints|
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