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[…] Data reduction was carried out using HKL2000 software (); all other software was collectively used through SBGrid (). The LAP1-VHH-BS1 structure was solved using molecular replacement. The MRage pipeline procedure from the PHENIX suite () was used for initial search model determination. The Phaser-MR tool was subsequently used for phasing and initial refinement. In the initial map, with phases only provided by the VHH search model, the outlines of the major secondary structure elements of LAP1 were visible. With iterative model building and refinement, the model phases gradually improved and the electron density maps became better defined. The final model was refined against native data extending to 1.6 Å. The data were cut judged by the CC1/2 value for the highest resolution shell, and visual inspection of the 2Fo-Fc map (). Model building was carried out with Coot () and refinement was done with phenix refine from the PHENIX suite. [...] Multiple sequence alignments of Torsin and LAP1 were performed using MUSCLE () and visualized with Jalview (). Since differentiating between LAP1 and LULL1 is difficult based on primary sequence, we refer to the sequences as LAP1 for simplicity. The species nomenclature used for the alignments is as follows: Homo sapiens (hs), Ornithorhynchus anatinus (oa), Gallus gallus (gg), Takifugu rubripes (tr), Danio rerio (dr), Branchiostoma floridae (bf), Strongylocentrotus purpuratus (stp), Ciona savignyi (cs), Ciona intestinalis (ci), Nematostella vectensis (nv), Caenorhabditis elegans (ce), and Drosophila melanogaster (dm). [...] The structure of human TorsinA was modeled using HHpred in combination with Modeler through the Bioinformatics Toolkit platform (). The D2 domain of ClpA (PDB entry 1R6B; ) was picked as the closest template structure. Models were generated with another four closely related structures. While there are a few uncertain loops and some discrepancies in modeling the C-terminal domain, the model of the nucleotide binding site is nearly identically in all cases.The structure of the heterohexameric TorsinA-LAP1 was modeled based on the hexameric D2 ring within the MecA-ClpC assembly (3PXI; ), by alternately superimposing the LAP1 structure and the TorsinA model onto neighboring ClpC-D2 domains within the ring. […]

Pipeline specifications

Software tools PHENIX, Coot, MUSCLE, Jalview, HHPred
Applications Protein structure analysis, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Movement Disorders, Dystonic Disorders