the samscl gene was inserted into a pet15b vector with a stop codon at glu 95 introduced by site - directed mutagenesis to generate the samscl - c26 mutant .
the channel was overexpressed in bl21-gold cells and purified in the detergent ldao by immobilized metal affinity and gel filtration chromatographies .
crosslinking was performed using n , n - dicyclohexylcarbodiimide at room temperature for 30 min-1 h and the products were analyzed by sds - polyacrylamide gel electrophoresis .
the mjf465 ( mscl , mscs , msck ) ( de3 ) strain was used for the osmotic downshock assay .
cells hosting ecmscl and an empty vector were included as positive and negative control sets , respectively .
for the osmotic shock , the culture was diluted 1:500 into sterile double - distilled h2o . for the mock shock , lb
mscl protein was reconstituted into giant azolectin liposomes via the dehydration / rehydration method and assayed by patch clamp electrophysiology .
patches containing at least 1 - 10 channels were excised and currents measured at various pressures .
recording and pipette solutions both contained 200 mm kcl , 40 mm mgcl2 , 5 mm hepes , ph 7.2 .
 the ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals , gravitropism in plants , and bacterial osmoregulation1 , 2 . in bacteria , 
 mechanosensation is mediated by the mechanosensitive channels of large ( mscl ) , small ( mscs ) , potassium - dependent ( msck ) , and mini ( mscm ) conductances . 
 these channels act as  emergency relief valves  protecting bacteria from lysis upon acute osmotic downshock3 . among them , mscl has been intensively studied since the original identification and characterization 15 years ago by kung and co - workers4 . 
 mscl is reversibly and directly gated by changes in membrane tension . in the open state , 
 mscl forms a nonselective 3 ns - conductance channel which gates at tensions close to the lytic limit of the bacterial membrane . 
 an earlier crystal structure at 3.5  resolution of a pentameric mscl from mycobacterium tuberculosis ( tbmscl ) represents a closed - state or nonconducting conformation5 , 6 . 
 mscl has a complex gating behaviour ; it exhibits several intermediates between the closed and open states , including one putative nonconductive expanded state and at least three sub - conducting states7 . 
 although our understanding of the closed5 , 6 and open8 - 10 states of mscl has been increasing , little is known about the structures of the intermediate states despite their importance in elucidating the complete gating process of mscl . 
 here we present the crystal structure of a truncation mutant ( 95 - 120 ) of mscl from staphylococcus aureus ( samscl - c26 ) at 3.8  resolution . 
 strikingly , samscl - c26 forms a tetrameric channel with both transmembrane ( tm ) helices tilted away from the membrane normal at angles close to that inferred for the open state9 , likely corresponding to a nonconductive but partially expanded intermediate state . 
Methods Summary
Supplementary Material