Molecular symmetry-constrained systematic search approach to structure solution of the coiled-coil SRGAP2 F-BARx domain.

This is the final version of the article. Available from International Union of Crystallography via the DOI in this record.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 C2 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.This work was supported by funds from the ISF (Grant No. 1425/15 to YO) and BSF (Grant No. 2013310 to YO)

The structural basis for SARM1 inhibition and activation under energetic stress

This is the author accepted manuscript. The final version is available on open access from eLife Sciences Publications via the DOI in this recordSARM1 an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition, and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolution. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.IS

Structural History of Human SRGAP2 Proteins

This is the author accepted manuscript. The final version is available from Oxford University Press via the DOI in this record.We thank Adam Frost and Eckart Gundelfinger for valuable advice on the manuscript, Michaela Vogel, Lada Gevorkyan-Airapetov, Rinat Vasserman and Tomer Orevi for technical assistance, and Hadar Amartely and Mario Lebendiker for help with SEC-MALS experiments and analysis. Thanks to the staff of beamlines ID14, ID23, and ID29 of ESRF, and the staff of BESSY II BL14.1. This work was supported by funds from the ISF (Grants no. 182/10 and 1425/15 to Y.O.) and BSF (Grant no. 2013310, to Y.O. and Adam Frost) as well as by the DFG grants QU116/6-2 to B.Q. and KE685/4-2 to M.M.K

Molecular symmetry constrained systematic search approach to structure solution of the coiled coil SRGAP2 F BARx domain

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 monoclinicC2 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.</jats:p

Multiparameter analysis of AgNOR in thyroid lesions: comparison with PCNA expression

The aim of the study was to examine numerous features of argyrophilic proteins related to nucleolar organizer regions (AgNORs) in thyroid tumors, relate them to PCNA expression and evaluate which of these features might be useful in the diagnosis of thyroid lesions. Paraffin sections of 100 thyroid tumors were silver-stained and divided into 9 groups: nodular goiter (NG), simple adenoma (SA), microfollicular adenoma (MFA), follicular carcinoma (FC), follicular variant of papillary carcinoma (PC-F), classical variant of papillary carcinoma (PC-C), Hürthle cell adenoma (HA), Hürthle cell carcinoma (HC), and anaplastic carcinoma (AC). The slides were analyzed with the computerized system for image analysis. A weak correlation was found between PCNA expression and AgNOR size. AC differed significantly from all other examined groups in many features of AgNOR dots. Hürthle cell neoplasms were characterized by the presence of a usually single and relatively large dot. With respect to diagnosing follicular lesions, we found that the evaluation of the total area of dots in the nucleus seemed to be the most useful for discrimination: the assumption of 4.9 µm2, as a cut-off value, allowed a correct classification of 77% of FC cases. Computeraided morphometric analysis of AgNORs may be useful in the diagnostics of thyroid lesions