While it is clear that a six-lobed membrane spanning cellulose synthesis complex (CSC) contains multiple cellulose synthase (CESA) glycosyltransferases for cellulose microfibril formation, the number of CESAs in the CSC remains unknown. CLSF members applied a combination of methods including improved FF-TEM, 2D image classification and averaging, analysis of small angle X-ray scattering data, negative stain TEM, and computational modeling to study the low resolution structure of rosettes from the nonvascular plant <i>Physcomitrella patens </i>and of the cytosolic or catalytic domain of CESA1 from<i> Arabidopsis thaliana</i>. The inner membrane portion of CESA in rosette lobes were frequently triangular, and their areas fit well with trimers of CESA. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM showed that the cytosolic domain of AtCESA1 is triangular and of a size that fits well to modeling of recent SAXS data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC, supporting a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 or fewer glucan chains. Here we report progress on 3D reconstruction of the AtCESA1 catalytic domain from negative stain and cryoEM images of single particles, structural evidence for non-redox mediated dimerization of NT-domains of AtCESAs 1 and 3, and GhCESA1, and TEM results showing that a single, heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

Progress toward structural understanding of cellulose synthesis by plants

253rd National Meeting (2017)

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