Computational protocol: Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix

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Protocol publication

[…] Native diffraction data were collected under cryocooled conditions (100 K) at the Advanced Photon Source, Argonne National Laboratory on beamline 24-ID-C; iridium diffraction data were collected under cryocooled conditions (100 K) at the National Synchrotron Light Source, Brookhaven National Laboratory on beamline X-25 at iridium’s peak (1.1053 Å), inflection (1.106 Å) and remote (1.1046 Å) wavelengths. All crystals belonged to the P3221 space group and all data were processed using XDS. Non-isomorphic crystals (cross-crystal R values of approximately 20%–30%) prevented the merging of data from different crystals. The ShelXC/D program located the initial heavy-atom sites by MAD, yielding experimental maps that were interpretable for two of the three RNA molecules (A and B) in the asymmetric unit (). Resulting anomalous difference Fourier maps confirmed the presence of iridium hexamine bound to RNA with at least three strong iridium hexamine-binding sites for each RNA molecule. Using the geometric configuration of the three strong iridium-binding sites, we identified molecule C in the weak density (). Initial electron density maps were further improved by refining heavy-atom locations using MLPHARE, sharpening amplitudes by B = −60 Å2 (ref.), and performing multidomain electron density averaging using DMMULTI in the CCP4 suite followed by multicrystal averaging of the Ir and native data sets (). Using these experimental maps, a model for all three RNA molecules was built through iterative cycles using Coot.The model was refined against the sharpened amplitudes (B = −60 Å2) of the native data at 3.1 Å resolution using Refmac5 with translation/liberation/screw motions included in the final step,. Our final model contains three molecules with the following nucleotides: nts 1–76 for molecule A, nts 1–55 and 59–76 for molecule B and nts 1–54 and 60–76 for molecule C. Due to crystal packing, molecules A and B were more ordered (average B factor of 30.2 Å2 for A and 47.9 Å2 for B) than molecule C (average B factor 88.9 Å2). Electron density for ions and water molecules was observed in the final refined maps. The identities of these ions and water molecules were not verified; therefore, they were deposited as water molecules in the final PDB file. Figures were created using PyMOL. […]

Pipeline specifications

Software tools XDS, SHELX, CCP4, Coot, REFMAC5, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Human gammaherpesvirus 8, Homo sapiens
Diseases Neoplasms
Chemicals Nucleotides