Computational protocol: Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

Similar protocols

Protocol publication

[…] Preparation of hRyR21–606 protein, crystallization, data collection and processing have been described in detail in Borko et al. (2013). Substitution mutants were prepared using the QuikChange protocol (Stratagene). Crystals formed in hanging drops from a solution consisting of 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, 7 mM β-mercapto­ethanol, 1 mM CHAPS, 0.1% betaine mixed with reservoir solution (100 mM HEPES pH 6.9, 200 mM ammonium formate, 21% PEG 3350) in a 2:1 ratio.Data were collected to 2.39 Å resolution at 100 K at the BESSY II synchrotron-radiation source, Berlin, Germany using 15% ethylene glycol as cryoprotectant. Data collection was performed using a MAR345dtb image plate. Data were processed with iMosflm (Powell et al., 2013) and were scaled by SCALA (Evans, 2011). The crystals belonged to space group P42212, with unit-cell parameters a = 75.45, b = 75.45, c = 248.84 Å, α = β = γ = 90° and one molecule in the asymmetric unit (Borko et al., 2013). The structure was solved by molecular replacement with MOLREP (Vagin & Teplyakov, 2010) using oRyR11–559 (PDB entry 2xoa; Tung et al., 2010) as a search model. The structure was built using Buccaneer (Emsley & Cowtan, 2004) and refined with REFMAC 5.6.0117 (Murshudov et al., 2011) including TLS refinement (Winn et al., 2001). Three TLS groups (10–223, 224–408 and 409–544) were used. Refinement of the structure was altered by inspecting the map, correcting the amino-acid sequence and manually building the parts that were not built by Buccaneer. Water molecules were automatically built by Coot (Emsley & Cowtan, 2004) into electron densities at a ≥1σ level that were at hydrogen-bonding distances from protein atoms or other water molecules. Data-collection and refinement statistics of the hRyR21–606 structure are summarized in Table 1. [...] The sample was prepared according to Borko et al. (2013). The sample quality was tested by dynamic light scattering (DLS; Zetasizer Nano-S) and size-exclusion chromatography (SEC; GE ÄKTA FPLC, Superose 12 HR10/30) at 5–10°C. Prior to SAXS data collection the protein sample was diluted at ratios of 1:1, 1:2, 1:3, 1:4 and 1:5 with the sample buffer without detergents. The protein concentration during the measurement was in the range 2–10 mg ml−1. SAXS data were collected on beamline X33, DESY, Hamburg (see Supporting Information for beamline characteristics). The SAXS data were processed with programs from the ATSAS 2.5 package (Supplementary Fig. S1). Initial processing and analysis were performed with PRIMUS (Konarev et al., 2003). The radius of gyration (R g) was estimated by Guinier approximation and from the pair-distribution function P(r). Information about the degree of protein disorder was obtained from the Kratky plot. The particle volume was calculated using the Porod invariant and the molecular weight was estimated from the Porod volume. Ab initio modelling was performed with GASBOR (Svergun et al., 2001) using ten repetitions with one scattering curve, with χ2 between 0.9 and 1. The normalized spatial discrepancy (NSD) of all models was compared using DAMSEL (Volkov & Svergun, 2003). The average NSD was 1.50 ± 0.07. […]

Pipeline specifications

Software tools iMosflm, CCP4, Molrep, Buccaneer, Coot, ATSAS, GASBOR
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus, Homo sapiens
Chemicals Amino Acids, Calcium, Ryanodine