Computational protocol: RING tetramerization is required for nuclear body biogenesis and PML sumoylation

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Protocol publication

[…] The diffraction data were recorded at the wavelength of Zn anomalous dispersion peak (1.2824 Å) and subsequently processed, integrated and scaled using MOSFLM/SCALA. The statistics of the data collection are shown in Supplementary Table . Single wavelength dispersion (SAD) method implemented in CRANK2 was used to phase PML RING. Eight Zn2+ positions were determined by CRUNCH2 using data between 20 and 1.6 Å. An interpretable map was obtained by solvent flattening using program SOLOMON. The autotracing program ARP/wARP was then used to produce a σA-weighted 2Fo-Fc map for further manual model building. REFMAC5 was used for structural refinement. Intermittent manual building implemented in COOT was used to correct and improve the initial models produced by ARP/wARP. The B-factors were refined with TLS corrections (4 TLS group, 84 parameters). The final model of PML RING tetramer contains 187 residues and 224 water molecules. Ramachandran statistics of PML RING calculated by PROCHECK indicate that 99.4% of the atoms are in the most favored region, and 0.6% are in the allowed regions. The detailed structure refinement statistics are reported in Table . [...] The recombinant PML RING49–104 was purified and concentrated to 1, 3 and 5 mg ml−1 in 20 mM Tris, pH 8.0, 100 mM NaCl, with/without 1 mM DTT, respectively. The X-ray scattering experiment was carried out at Beamline station BL19U2 (National Facility for Protein Science Shanghai, NCPSS, China) and scattered X-ray intensities were collected by using a Pilatus 1 M detector (DECTRIS Ltd). The measurements were carried out with 1 s exposure time and repeat for 20 times to avoid possible sample radiation damage. The collected data were processed with ATSAS software package. The details of SAXS data collection and processing are shown in Supplementary Table . The guinier region of experimental group are linear, indicating the measured sample is homogeneous in solution. Crystal data fitting was done using the OLIGOMER algorism implemented in CRYSOL. In the analysis of crystal fitting (0.01 ≤ q ≤ 0.25 Å−1), the crystallographic tetramer, crystallographic dimers and crystallographic and NMR monomers were used to fit the merged experimental SAXS data derived from three protein concentrations, leading to the determination of each fraction of monomer/dimer/tetramer in the solutions. […]

Pipeline specifications

Software tools CCP4, Crank, ARP/wARP, REFMAC5, PROCHECK, ATSAS, CRYSOL
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma
Diseases Leukemia, Promyelocytic, Acute
Chemicals Arsenic