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1), probably by causing its conformational change (1–4).

The C-terminal region of an active TRA-2A could bind to FEM-3, thus inhibiting the active FEM complex comprising FEM-1, -2, and -3.

All diffraction data were processed with HKL2000 (10).

The eluant was concentrated using an Ultrafree 5000 molecular weight cutoff filter unit (Millipore) and further purified using a Superdex-200 (GE Healthcare) column equilibrated with a buffer containing 10 m of 0.8.

Recombinant His-tagged protein was purified by nickel affinity column chromatography and ion exchange chromatography and was further subjected to gel filtration chromatography (Superdex-200 column) in buffer containing 10 m DTT.

Regarding the final step, TRA-1, which acts as a transcription repressor of male-specific genes in somatic cells, is regulated by proteasomal degradation executed by a CUL-2-based ubiquitin ligase with FEM-1 as the substrate recognition subunit and FEM-2 and FEM-3 as cofactors (5).

The three genes play a central role in the entire sex determination pathway.

CC1/2 was used to determine the high resolution limit of the diffraction data (11).

The heavy atom positions in the iodine-soaked mutant crystal were determined using SHELXD (12).Biochemical studies suggested that the N-terminal domain does not directly regulate the dephosphorylation activity of FEM-2, but instead functions as a scaffold motif in the assembly of FEM-1/2/3 complex, the central component in the sex determination pathway.Furthermore, mutagenesis analysis identified regions in the N-terminal domain of FEM-2 involved in binding to FEM-1 and FEM-3.Different from the canonical PP2Cs, FEM-2 consists of an additional N-terminal domain (NTD) apart from its C-terminal catalytic domain.Interestingly, genetic studies have indicated indispensable roles for both of these two domains of FEM-2 in promoting male development, but the underlying mechanism remains unknown.Heavy atom parameters were then refined, and initial phases were generated in the program PHASER (13) with the single-wavelength anomalous dispersion experimental phasing module.

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