Computational protocol: Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein

Similar protocols

Protocol publication

[…] Diffraction data were collected at BM14, ESRF, Grenoble and processed to a resolution of 1.75Å using DENZO/SCALEPACK []. Merging statistics are summarized in Table . The structure, including two molecules of mthSBDS protein in the asymmetric unit, was determined by MOLREP [] from the CCP4 suite [] using the structure of the afSBDS protein (Protein Data Bank code 1P9Q) as search model. Although mthSBDS protein is closely homologous to the afSBDS protein (49% sequence identity), it was not possible to find a molecular replacement solution when the whole protein was used as the search model. Therefore, we dissected the reference model [PDB:1P9Q] into three domains (Domain I: residues 1–89; Domain II: residues 90–162 and Domain III: residues 163–231) and performed molecular replacement searches using these separate domains. The best solution was obtained for domain I of one of the two molecules (hereafter designated as molecule A) in the asymmetric unit. The solution for this domain was improved by rigid body refinement and was fixed during further molecular replacement searches. The second best solution was obtained for domain II of molecule B. At this stage rigid body refinement was applied to both domain solutions and was followed by 10 cycles of all-atoms restrained refinement using REFMAC []. At the next step, both refined domains were fixed, and the next best molecular replacement solution found was for domain II of molecule A. This procedure of refinement/molecular replacement search was iterated until solutions for all three domains of both molecules were found. The final molecular replacement model was further rebuilt and improved by use of ARP/WARP followed by cycles of manual model rebuilding with COOT [] and restrained refinement, including TLS option in REFMAC.For the purpose of projecting the normal modes onto the observed displacements between molecules A and B in the crystal of mthSBDS and between mthSBDS molecule A (mthSBDS-A) and the structure of afSBDS, the domains of mthSBDS-A were superimposed on the respective domains of the other molecules using the least-squares procedure in LSQKAB []. The domains were then joined together and the resulting chimeric structure superimposed back onto mthSBDS-A. This procedure ensures the presence of the same set of atoms in the two conformations analyzed by ElNémo.The atomic coordinates and structure factors have been deposited in the Protein Data Bank [PDB:2WBM]. [...] The gel mobility shift assay was used to quantitatively measure the affinity of mthSBDS-RNA binding. In this assay, protein concentration ranged from 10-4 M to 10-12 M and the 32P-radio-labelled RNA was added in 1% v/v quantities of total transcribed RNA sample. The shifted RNA species were excised, eluted and reverse transcribed to generate cDNA sequences of the selected RNAs. The DNAs were cloned into pGEM-T Easy vector (Promega) and sequenced (York Bioscience). Consensus sequence was generated using CLUSTALW (EBI) and RNA secondary structure was predicted using MFOLD []. [...] Sequences were aligned with MUSCLE [] and the alignment was represented using ESPRIPT []. The program CHIMERA [] was used for the visual inspection of structure properties and the generation of molecular representation figures. Electrostatic potential analyses were carried out with APBS []. […]

Pipeline specifications

Software tools Clustal W, Mfold, MUSCLE
Applications RNA structure analysis, Nucleotide sequence alignment
Organisms Homo sapiens, Bacteria, Methanothermobacter thermautotrophicus
Diseases Bone Marrow Diseases, Exocrine Pancreatic Insufficiency, Genetic Diseases, Inborn