the intracellular fragment of the human slo1 gene was subcloned into the pfastbac - hta vector and expressed in sf9 insect cells using a baculovirus system . the construct contains an n - terminal his - tag , tev protease cleavage site and an extra 32-amino acid stretch of sequence rmkqiedkleeilsklyhienelarikkllge from the mutant transcription factor gcn4 leucine zipper ( gcn4_li ) immediately before the n - terminus of the bk intracellular domain . expressed protein was first purified on co affinity resin followed by overnight tev cleavage at 4 c to remove the his - tag . protein was concentrated and then treated with lambda protein phosphatase before further purification on a gel filtration column . purified protein was concentrated to about 7 mg / ml and crystallized at 4 c using the sitting drop vapor diffusion method by mixing equal volumes of concentrated protein and well solution containing 3.5 m sodium formate and 0.1 m tris.hcl , ph 8.5 . the crystals were of space group i422 with cell dimensions of a = b=134.1  , c=231.6  , ===90 , and contained one subunit per asymmetric unit . the structure was determined by single wavelength anomalous dispersion ( sad ) using selenomethionine substituted protein crystals . model building was aided by the se sites and conserved structure features of rck domains . the final model was refined to 3.1  with rwork of 23.8% and rfree of 28.9% , and contained residues 330 to 616 , 684 to 833 , and 872 to 1059 . the density for the n - terminal gcn4_li coiled - coil is weak and is not modeled in the final structure .
high - conductance ca2 + -gated k+ ( bk ) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release1 - 4 . 
 this group of channels can be activated synergistically by both voltage and intracellular ca2 + , with the large c - terminal intracellular portion being responsible for ca2 + sensing5 - 13 . here 
 we present the crystal structure of the entire cytoplasmic region of the human bk channel in a ca2 + free state . 
 the structure reveals four intracellular subunits , each comprising two tandem rck domains , assembled into a gating ring similar to that seen in the mthk channel14 and likely representing its physiological assembly . 
 three ca2 + binding sites including the ca2 + bowl are mapped onto the structure based on mutagenesis data . 
 the ca2 + bowl , located within the second rck domain , forms an ef - hand like motif and is strategically positioned close to the assembly interface between two subunits . 
 the other two ca2 + ( or mg2 + ) binding sites , asp367 and glu374/glu399 , are located on the first rck domain . 
 the asp367 site has high ca2 + sensitivity and is positioned in the groove between the n- and c- terminal subdomains of rck1 , whereas the low affinity mg2 + -binding glu374/glu399 site is positioned on the upper plateau of the gating ring and close to the membrane . 
 our structure also contains the linker connecting the transmembrane and intracellular domains , allowing us to dock a voltage - gated k+ channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full bk channel .