2018-02-14 OBS RCSB RCSB RCSB 2016-11-04 2017-03-15 2018-02-07 2018-02-14 2018-02-14 Tonggu L Zhan X Wang L Tonggu L Zhan X Wang L To Be Published 0353 EMD-8468 associated EM volume large conductance voltage- and calcium-activated potassium (BK) channels large conductance voltage- and calcium-activated potassium (BK) channels 0

full-length human Slo protein (GI:507922)

Homo sapiens Homo sapiens hek293 pcdna3.1 500 singleParticle particle 0.2 7.2 20.0 C8H18N2O4S hepes 150.0 KCl potassium chloride Quantifoil R2/2 COPPER 400 CARBON HOLEY ARRAY 20.0

hexane wash

ETHANE 40 298 HOMEMADE PLUNGER

Two-dimensional (2D) streptavidin crystals were grown at room temperature on perforated carbon grids. After the 2D streptavidin crystal was washed with crystal buffer (150mM NaCl, 50mM HEPES, pH 7.3) once and buffer A (20 mM HEPES, pH 7.2, 150 mM KC) twice, the crystal was incubated with BK proteoliposome suspensions for 10-40 min to allow binding. The sample was blotted at room temperature and immediately fast-frozen in liquid ethane cooled by liquid nitrogen..

BK proteins were reconstituted into POPC liposomes. The proteoliposomes were tethered onto the streptavidin crystal grown on a quantifoil grid

FEI TECNAI F20 FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 200 100.0 2.0 0.5 4.0 GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER NITROGEN GATAN K2 SUMMIT (4k x 4k) SUPER-RESOLUTION 0.2 1.0 1 88041

EM boxer and homemade matlab program

EMD-5114 C4 FOURIER SPACE 8.1 FSC 0.143 CUT-OFF 88041 OTHER

Employ constraints based on the spherical geometry of the proteoliposomes as previously described (Wang and Sigworth, 2009)

PROJECTION MATCHING

RSC method written in matlab