Computational protocol: Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA

Similar protocols

Protocol publication

[…] Crystals were mounted on CryoLoops (Hampton Research) and soaked into a solution composed of the mother liquor supplemented with 30% methyl-2,4-pentanediol prior to flash-cooling using a CryoStream (Oxford Cryosystems). Initial diffraction experiments and data collection were carried out using an EIGER detector on the X06SA beamline, Swiss Light Source, Villigen, Switzerland. X-ray images were recorded with an EIGER detector using the fine-slicing method (Dauter, 1999) with 0.2° oscillations at 100 K, a wavelength of 1.0 Å and a crystal-to-detector distance of 386 mm (see Table 1 for data-collection details and statistics). X-ray diffraction data sets were collected to a resolution of 2.9 Å from native protein crystals. Data processing and scaling were accomplished with XDS (Kabsch, 2010) and AIMLESS (Evans & Murshudov, 2013) as implemented in autoPROC (Vonrhein et al., 2011). Based on the diffraction pattern, these crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, α = β = γ = 90° (Table 3). To determine the packing of the FnCpf1 ternary complex in the asymmetric unit of the crystal, we calculated the Matthews coefficient (Matthews, 1968), which yielded a V M of 2.35 Å3 Da−1, corresponding to a solvent content of 48%, with one complex in the asymmetric unit. We attempted to solve the structure of the ternary complex by the molecular-replacement method using LbCpf1 and AsCpf1 without success, suggesting that major conformational changes may occur in the ternary complex. Therefore, selenomethionine-derivatized FnCpf1 was produced to obtain experimental phases using the MAD phasing method. […]

Pipeline specifications

Software tools XDS, CCP4
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli, Francisella tularensis subsp. novicida
Chemicals Nucleotides