Molecular symmetry‐constrained systematic search approach to structure solution of the coiled‐coil SRGAP2 F‐BARx domain. Issue 12 (5th December 2016)

• Record Type: Journal Article
• Authors: Sporny, Michael; Guez-Haddad, Julia; Waterman, David G.; Isupov, Michail N.; Opatowsky, Yarden
• Is Part Of: Acta crystallographica. Volume 72:Issue 12(2016)
• Page Start: 1241
• Page End: 1253
• Publication Date: 2016-12-05
• Subjects: exhaustive search -- SRGAP2 -- F‐BAR -- coiled coil -- molecular replacement; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; Molecular biology -- Periodicals; Molecular structure -- Periodicals; Biomolecules -- Structure -- Periodicals; Cytology -- Periodicals; Biomolecules -- Structure; Crystallography; Cytology; Molecular biology; Molecular structure; X-ray crystallography; Periodicals; 548
• DOI: 10.1107/S2059798316016697
• Languages: English
• ISSNs: 2059-7983

Abstract

Abstract : The F‐BARx domain of SRGAP2 was produced for crystallization by co‐expressing it with the carboxy domains of the protein. The F‐BARx crystal structure was determined by a molecular symmetry‐constrained systematic search, utilizing the conserved biological symmetry of the F‐BAR fold, an approach that is shown to be useful in solving other F‐BAR structures. Abstract : SRGAP2 (Slit–Robo GTPase‐activating protein 2) is a cytoplasmic protein found to be involved in neuronal branching, restriction of neuronal migration and restriction of the length and density of dendritic postsynaptic spines. The extended F‐BAR (F‐BARx) domain of SRGAP2 generates membrane protrusions when expressed in COS‐7 cells, while most F‐BARs induce the opposite effect: membrane invaginations. As a first step to understand this discrepancy, the F‐BARx domain of SRGAP2 was isolated and crystallized after co‐expression with the carboxy domains of the protein. Diffraction data were collected from two significantly non‐isomorphous crystals in the same monoclinic C 2 space group. A correct molecular‐replacment solution was obtained by applying a molecular symmetry‐constrained systematic search approach that took advantage of the conserved biological symmetry of the F‐BAR domains. It is shown that similar approaches can solve other F‐BAR structures that were previously determined by experimental phasing. Diffraction data were reprocessed with a high‐resolution cutoff of 2.2 Å, chosen using less strict statistical criteria. This has improved the outcome of multi‐crystal averaging and other density‐modification procedures.