W0309: Low resolution SAXS model of beta-mannosidase from T. Reesei enhanced by X-ray crystallography

Low Resolution SAXS Model of (-mannosidase from T. Reesei Enhanced by X-ray Crystallography. Aparicio, R.^a, Fischer, H.^b; Craievich, A.F.^b; Golubev, A.M.^c, Neustroev, K.N.^c, Eneiskaya, E.V.^c, Kulminskaya, A.A.^c, Savel'ev, A.N.^d, and Polikarpov, I.^e,f, ^aInstituto de Física Gleb Wataghin, UNICAMP, Campinas, SP,Brazil; ^bInstituto de Física, USP, São Paulo, SP, Brazil, ^cPetersburg Nuclear Physics Institute, St. Petersburg, Russia, ^dSt.Petersburg Technical University, St.Petersburg, Russia; ^eInstituto de Física, USP, São Carlos, SP, Brazil, ^fLaboratório Nacional de Luz Sincrotron/LNLS, Campinas, SP, Brazil.

(-mannosidase (EC 3.2.1.25) catalyzes hydrolysis of non-reducing residues of (-D-mannose in (-D-mannosides and participates in the lysosomal degradation of N-glycoproteins. In man and ruminants, a deficiency of (-mannosidase leads to an autosomal recessive inherited lysosomal storage disease (Watts & Gibbs, 1986) called (-mannosidosis. To obtain structural information and better understand the mechanism of enzymatic action, (-mannosidase from Trichoderma reesei, a 105 KDa glycoprotein, crystallographic studies were initiated (Aparicio et. al., 2000). Electron density maps extending to 2.5 A were obtained by the Single-wavelength Anomalous Dispersion (SAD) method. A number of problems including non-isomorphism and anisotropy, related to protein glycosylation and crystal disorder, have had strong influence on the quality of maps, making them poorly interpretable. As an attempt to obtain low-resolution shape of the molecule we performed Small-angle X-ray Scattering (SAXS) experiments. The low resolution models derived from the SAXS data were successfully used for Phased Molecular Replacement. The higher resolution of crystallographic data has been used to enhance the informational content of the original SAXS model, leading to more accurate, reliable shape of the protein. In this work, we describe the results obtained, including technical details of the experimental procedures and present the low resolution shape and the putative fold of (-mannosidase from T. reesei.