The arrangement of the multiple catalytic subunits in the CSC and, as a result, the shape and size of cellulose fibrils varies between organisms. Terrestrial plants and their Charophyte algal relatives have a rosette-type CSC consisting of six proteinaceous globules in a circle, as shown by freeze fracture transmission electron microscopy of the transmembrane helices of CESA as they cross the plasma membrane. The entire rosette CSC is about 25 nm diameter, inclusive of six 7 nm globules as measured where the TMH of CESA cross the membrane. The exact number of CESA subunits in a rosette CSC and the correlated number of glucan chains in a native cellulose GDC-0199 Bcl-2 inhibitor fibril are still debated. Genetic analysis shows that cellulose synthesis requires three distinct CESA subunits, which form a high molecular weight complex of unknown stoichiometry. Traditional models propose 36 CESAs within one rosette CSC, although fewer may actually be present. Typically, para-crystalline cellulose I fibrils contain at least 18–36 glucan chains, so the six-chain structure modeled in this work can be considered a protofibril. Here, we use the term ‘fibril’ for newly formed native cellulose to be consistent with the use of ‘protofibril’ for the in silico assembly of six glucan chains. Another important knowledge gap concerns the assembly of the crystalline fibrils. How do the multiple glucan chains organize topologically within the crystalline domains of cellulose fibrils? Does this occur spontaneously or does it require a specific guidance mechanism? Do protofibrils initially arise from each one of the six globules of the rosette CSC before formation of the larger composite cellulose fibril and, if so, what are the forces involved? Does fibril formation require a strict coordination of the activity of the multiple catalytic subunits? How can we reconcile the stiffness of the cellulose fibril with the bending that must occur as it aligns itself horizontally with the innermost layer of the cell wall?. In this study we used molecular modeling and FF-TEM to obtain more insight into the initial stages of cellulose fibril formation. Although molecular modeling of cellulose is an increasingly active field, we are not aware of any published study addressing the initial stages of cellulose fibril organization after the exit of the glucan chains from the CSC embedded in the plasma membrane. In one 
relevant prior study, the entire CSC was modeled as six connected spheres each producing one glucan chain. Although not a molecular description, this model makes predictions about the motion of the rosette CSC and the deposition of bundled glucan chains onto the cell wall. The authors showed that the polymerization and crystallization of the glucan chains can provide the driving force for the rosette CSC movement and that the chain stiffness and membrane elasticity can act as force transmitters, findings that correspond to the experimentally demonstrated movement of GFP-tagged CESAs within the plant plasma membrane. Here, we carried out molecular dynamics simulations on ALK5 Inhibitor II groups of six atomistic glucan chains, each originating from a position that approximates a site of glucan extrusion within one globule of the rosette CSC. The computational simulations revealed the initial formation of an uncrystallized aggregate of chains, from which a protofibril arose spontaneously through a ratchet mechanism.