A crystallographic study of human NONO (p54(nrb)): overcoming pathological problems with purification, data collection and noncrystallographic symmetry.
Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia.
Gavin J
GJ
Knott
Australian Synchrotron, Victoria, Australia.
Santosh
S
Panjikar
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, England.
Andrea
A
Thorn
School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA 6009, Australia.
Archa H
AH
Fox
Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, England.
Maria R
MR
Conte
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
Mihwa
M
Lee
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia.
Charles S
CS
Bond
D003461:Crystallography / Q000379:methods; D004268:DNA-Binding Proteins; D006801:Humans; D008958:Models, Molecular; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry*; D050810:Octamer Transcription Factors / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D055503:Protein Multimerization; D016601:RNA-Binding Proteins / Q000737:chemistry*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction / Q000379:methods*
D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D016601:RNA-Binding Proteins; D011392:Proline
L statistic; NONO (p54nrb); cumulative intensity distribution; l-proline; noncrystallographic symmetry