Sexual stages of Plasmodium such as zygote, ookinete, and young oocysts express 25？kDa surface protein P25, which along with P28 proteins protect the parasite from harmful environment inside mosquito midgut. Vaccines against these proteins induce antibodies in vertebrate host capable to inhibit parasite development in mosquito midgut and thus preventing the transmission of parasite from mosquito to other human host. Transmission-blocking vaccines help reduce malaria burden. The purpose of this study was in silico structural characterization of P25 family proteins and to predict their phylogenetic relationships with other proteins. Results indicate that members of P25 family have four EGF domains arranged in triangular fashion with major variations lying in the loop regions. All 22 cysteines are conserved forming 11 disulphide bonds. The C-loop of EGF domain IV in P25 proteins is smaller in comparison to P28 proteins. B loop of EGF domain II showed maximum RMSD variations followed by loops of EGF domain III. P25 proteins are tile-like triangular flat proteins that protect the parasite inside mosquito midgut. Obtained structures will help in understanding the biology of the parasite inside the mosquito midgut. These structures may also help in designing transmission-blocking vaccine against malaria in absence of experimentally determined structures. 1. Introduction During sexual stages of the Plasmodium parasite, many surface proteins are synthesized de novo. P25 proteins are the major surface proteins of Plasmodium ookinetes, having molecular weight of 25？kDa. P25 proteins are present on ookinete surface of all known Plasmodium species [1–3]. Many proteins have been identified as promising vaccine candidates against malaria including two proteins of P25 family [4]. P25 proteins start expressing immediately after fertilization and continue to be expressed on zygote, ookinete, and young oocyst stages of Plasmodium [5]. These proteins are present in abundance and are evenly distributed over the entire ookinete surface [6–8]. Gene knockout experiments suggest that these proteins along with P28 proteins are essential for the survival of parasite inside mosquito midgut [9]. Structurally, all P25 proteins contain a signal sequence, four epidermal growth factor (EGF) domains, and a C-terminal glycosylphosphatidylinositol (GPI) anchor [10, 11]. EGF domains are known to be present especially in surface proteins where they participate in recognition and adhesion-like processes [12], which indicates that they play important roles in host parasite interactions. P25
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