PMC:2728203 / 12451-15925 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"Structure of the Nab2 N-terminal domain\nThe structure of the N-terminal domain of Nab2 was established using both NMR and X-ray crystallography. A fragment corresponding to residues 1–105 of Nab2 was produced in large quantities by bacterial expression and purified using conventional ion-exchange and gel-filtration chromatographic methods (see Materials and Methods). This material was exceptionally soluble, which facilitated the collection of NMR spectra, from which a model was generated using conventional methods. The final ensemble of 45 calculated structures of the Nab2 N-terminal domain is shown in Fig. 2a. Of the total of 50 structures calculated in the final round, these 45 correspond to a well-defined plateau region in the energy and energy-ordered root-mean-square deviation (rmsd) profiles (Fig. 2a), indicating that they form a suitable set for reporting structural statistics (Table 1).31 Residues Met1–Gln3 and Gly100–Ala105 were unstructured in solution, as evidenced by the lack of any medium- or long-range nuclear Overhauser enhancement (NOE) connectivities for these residues and by the sharpness and near random-coil chemical shift values of their NMR resonances. The remainder of the domain was well ordered, although there was slightly increased disorder in the loops, especially those between helix 1 and helix 2 (Pro22–Asp27) and, to a lesser extent, between helix 3 and helix 4 (Asp57–Ser60). The backbone rmsd over residues 4–99 was 0.47 ± 0.13 Å.\nAs illustrated in Fig. 2b, the solution structure of the N-terminal domain of Nab2 was based on a bundle of five α-helices (H1–H5). Unlike the highly compact bundle formed by helices H1–H4, each of which made multiple contacts with at least two of the other helices, helix H5 was less tightly associated with the rest of the structure. The only NOE constraints from helix H5 to other parts of the structure were all contacts to helix H1 (Ala84 contacts Asn9, Val12, and Ile13; Ile87 contacts Ile13 and Glu16; Ile91 contacts Glu16 and Ala19; Asn95 contacts Ala19 and Gly20). These contacts were sufficient to constrain the position of helix H5 with comparable precision to those of the other helices, but they were much fewer in number than the constraints involving any of the other helices. Further evidence for the relatively loose attachment of helix H5 to the rest of the structure came from the observation that NMR samples were slowly proteolyzed from the C-terminus. Over a period of several weeks, many signals assigned to residues of helix H5 were progressively lost from the spectra, whereas the signals assigned to helices H1–H4 were largely unaffected. The signals for protons in helix H5 were generally weaker than other components of the structure and decreased with time, consistent with its being gradually proteolyzed.\nWe also obtained P43212 symmetry crystals of the Nab2 N-terminal domain that diffracted past 1.8 Å resolution in-house (Table 2). We used the solution NMR structure of this domain as a model for molecular replacement and, after trying a number of different models containing different fragments of the structure, were able to obtain a unique solution using residues 7–82 (corresponding to helices H1–H4). After solvent flattening, the electron density enabled residues 6–94 to be built to produce a structural model that had an R-factor of 20.7% (Rfree = 26.4%) and excellent geometry (Table 2) after iterative cycles of refinement and rebuilding (Fig. 3).","divisions":[{"label":"title","span":{"begin":0,"end":39}},{"label":"p","span":{"begin":40,"end":1481}},{"label":"p","span":{"begin":1482,"end":2817}}],"tracks":[{"project":"2_test","denotations":[{"id":"18190927-20686883-62517986","span":{"begin":907,"end":909},"obj":"20686883"}],"attributes":[{"subj":"18190927-20686883-62517986","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#c393ec","default":true}]}]}}