Computational protocol: Structural Insights Reveal the Dynamics of the Repeating r(CAG) Transcript Found in Huntington’s Disease (HD) and Spinocerebellar Ataxias (SCAs)

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Protocol publication

[…] A complete diffraction dataset at 2.3 Å resolution was collected using liquid nitrogen-immersed flash-frozen crystals. ADSC CCD detectors on beamline 9–1 at SSRL or beamline LS-CAT (21-ID) at the Advanced Photo Source (Argonne National Laboratory, Argonne, IL) under cryoconditions (100 K) were used to collect the data. The data were scaled and integrated using HKL2000 []. Initial phases for the crystal were determined by molecular replacement in PHASER [], a module in PHENIX interfaces []. A 19-bp standard form of RNA generated from COOT [] served as a phasing model. COOT was used for multiple rounds of manual fitting and rebuilding of the structure. The structure was further refined in the CCP4 program suite []. Data collection, processing and refinement statistics are listed in . [...] RNA duplexes with a single CAG motif, 5´ r(CCGCAGCGG)2, and three CAG motifs, 5´ r(GGGCCAGCAGCAGGUCC)2, were used for NMR spectroscopy. RNA samples were prepared by resuspending lyophilised RNA in 10 mM phosphate buffer, pH 7.2, 0.1 M NaCl, and 50 mM EDTA in 10% D2O. NMR spectra were recorded with a Bruker NMR spectrometer with field strengths of 400 and 500 MHz. One dimensional proton spectra were recorded at various temperatures. Spectral assignments were performed using NOESY (Nuclear Overhauser Effect SpectroscopY), DQF-COSY (Double Quantum Filtered Correlation SpectroscopY) and TOCSY (Total Correlation SpectroscopY).Two-dimensional (2D) NOESY was performed acquiring 4096 data points with 64 transients for each of the 296 FID signals. The spectra were recorded at 288, 298 and 308 K with mixing times of 300, 200 and 100 ms. A NOESY spectrum was recorded using an excitation exculpating pulse sequence to suppress residual water signals.SPARKY was used to visualise the spectra and calculate 1H-1H NOE distances. These distances were used to restrain the RNA duplex for restrained Molecular Dynamic simulation studies.The minimisation and restrained Molecular Dynamics (rMD) simulation were performed using Discovery Studio 3.5 (Accelerys Inc., USA). The RNA duplex was built using the Macromolecule module in Discovery Studio 3.5. After typing the duplex with a CHARMM force field [], the model was solvated with an explicit solvent model such that the nucleic acid atoms were surrounded by an orthorhombic periodic box of TIP3P [] water molecules extending to 20 Å. To effectively consider the long-range electrostatic interactions, minimisation was performed by the Particle Mesh Ewald (PME) method []. A total of 1000 steps using the steepest descent minimisation were performed using SHAKE [] with bonds containing hydrogen.After minimisation and optimisation, the RNA duplex was subjected to Standard Dynamic Cascade. Within this cascade, the system was again minimised with a steepest descent minimisation of 500 steps followed by a conjugate gradient minimisation of 500 steps. The system was then subjected to gradual heating from 50 to 300 K for 4 picoseconds. The molecule was equilibrated for 10 picoseconds with a time step of 2 femtoseconds at a constant temperature of 300 K. Equilibration was followed by production runs under a constant temperature (300 K) with PME treatment for electrostatic interactions, SHAKE for bonds containing hydrogen and a 2 femtosecond time step. Simulations were performed for 25 nanoseconds, and 100 conformations were generated. The output files were analysed with Discovery Studio 3.5 based on properties such as potential energy of the system, root mean square deviation (RMSD) and the presence of hydrogen bonds. […]

Pipeline specifications

Software tools PHENIX, Coot, CCP4, Sparky, CHARMM
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Homo sapiens
Diseases Huntington Disease, Machado-Joseph Disease, Neurodegenerative Diseases
Chemicals Adenine