Computational protocol: Centromeric binding and activity of Protein Phosphatase 4

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Protocol publication

[…] Images were acquired on a Zeiss Axiovert 200 M microscope (objective × 100/numerical aperture 1.4) with a Cool-SNAP HQ camera (Photometrics) using MAG Biosystems Software—Metamorph (Molecular Devices). Images showing Flfl, Flfl::FLAG or FLAG::Flfl1–168 localization were acquired using a Zeiss LSM 510 Meta Confocal Microscope (× 100 objective) using LSM510 software (release version 4.2 SP1, Carl Zeiss MicroImaging) and processed using ImageJ (NIH, USA). Line scans were done using ImageJ and values were imported to MS Excel, which was used to prepare plots. [...] Crystals of native protein–peptide complex were grown in sitting drops by the vapour diffusion method. Solution of protein at 22 mg ml−1 was incubated overnight with peptide (PDESSADVVFKKPLAPAPR) in a ratio of 1:1.3. Crystals of complex were grown by mixing 0.2 μl of this solution with a 1:1 dilution of ½ reservoir buffer (3.31 M Ammonium Sulfate, 9.5% Glycerol) and ½ Silver Bullet screen (Hampton Research; 0.2% w/v 4-Aminobenzoic, 0.2% w/v Azelaic acid, 0.2% w/v o-Sulfobenzoic acid monoammonium salt, 0.2% w/v p-Coumaric acid, 0.2% w/v Sodium 4-aminosalicylate dihydrate and 20 mM HEPES sodium pH 6.8) with distilled water. Crystals grew in space group P61 (unit cell dimensions: a=b=67.75 Å, c=53.37 Å) to a size of 0.4 × 0.2 × 0.1 mm. Under the assumption of one protein molecule per asymmetric unit, a VM value of 2.46 Å3 Da−1 corresponding to a solvent content of 49.9% was calculated. Crystals used for data collection were soaked in 3.4 M sodium malonate for a period of 2 min, transferred to loops and immersed in liquid nitrogen. X-ray diffraction data were collected at beamline ID29 (see ref. ) of the European Synchrotron Radiation Facility. Data used to solve the structure were collected from a single crystal flash frozen at −180 °C. All data were processed and scaled using XDS. Data collection statistics are shown in .The structure of Flfl1–122 was solved by molecular replacement using the programme Mr Bump and two structures of RanBD and Spred-1 domains (PDB i.d. 1XOD, 1RRP) with a sequence identity of 20% as search models in a Phaser ensemble. The structure was iteratively rebuilt using Coot and refined with Refmac5 (see ref. ) and phenix.refine. The peptide sequence was docked after refinement for Flfl1–122 alone had converged, and anisotropic B-factor parameterization was employed for all non-water atoms. The structure was validated using tools from the Molprobity suite, which showed 99% of residues to be in the favoured Ramachandran region and gave an overall clash score of 0.52. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster