2024-03-20 REL RCSB

human BK channel reconstituted into liposomes

RCSB RCSB 2018-12-13 2019-06-26 2020-06-17 2024-03-20 National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) R01GM096458 United States Wang L Tonggu L the large conductance voltage- and calcium-activated potassium (BK) channel, Ca2+ sensor, gating ring, random spherically constrained (RSC) single-particle cryo-EM, MEMBRANE PROTEIN Tonggu L Wang L To Be Published 0353 6nd0 FULLOVERLAP human BK channel reconstituted into liposomes human BK channel reconstituted into liposomes 0 1 Homo sapiens 0.5 Calcium-activated potassium channel subunit alpha-1 Homo sapiens 0.099256867 4 Homo sapiens LEVO LKTSNSIKLVNLLSIFISTWLTAAGFIHLVENSGDPWENFQNNQALTYWECVYLLMVTMSTVGYGDVYAKTTLGRLFMVF FILGGLAMFASYVPEIIELIGNRKKYGGSYSAVSGRKHIVVCGHITLESVSNFLKDFLHKDRDDVNVEIVFLHNISPNLE LEALFKRHFTQVEFYQGSVLNPHDLARVKIESADACLILANKYCADPDAEDASNIMRVISIKNYHPKIRIITQMLQYHNK AHLLNIPSWNWKEGDDAICLAELKLGFIAQSCLAQGLSTMLANLFSMRSFIKIEEDTWQKYYLEGVSNEMYTEYLSSAFV GLSFPTVCELCFVKLKLLMIAIEYKSANRESRILINPGNHLKIQEGTLGFFIASDAKEVKRAFFYCKACHDDITDPKRIK KCGCKRPKMSIYKRMRRACCFDCGRSERDCSCMSGRVRGNVDTLERAFPLSSVSVNDCSTSFRAFEDEQPSTLSPKKKQR NGGMRNSPNTSPKLMRHDPLLIPGNDQIDNMDSNVKKYDSTGMFHWCAPKEIEKVILTRSEAAMTVLSGHVVVCIFGDVS SALIGLRNLVMPLRASNFHYHELKHIVFVGSIEYLKREWETLHNFPKVSILPGTPLSRADLRAVNINLCDMCVILSANQN NIDDTSLQDKECILASLNIKSMQFDDSIGVLQANSQGFTPPGMDRSSPDNSPVHGMLRQPSITTGVNIPIITELVNDTNV QFLDQDDDDDPDTELYLTQPFACGTAFAVSVLDSLMSATYFNDNILTLIRTLVTGGATPELEALIAEENALRGGYSTPQT LANRDRCRVAQLALLDGPFADLGDGGCYGDLFCKALKTYNMLCFGIYRLRDAHLSTPSQCTKRYVITNPPYEFELVPTDL IFCL Q12791 singleParticle particle 1.0 7.3 12.0 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 90.0 KCl Potassium chloride 1.2 EDTA Ethylenediaminetetraacetic acid C-flat-2/2 COPPER 400 CARBON HOLEY 20.0 GLOW DISCHARGE 20 AIR 0.133 ETHANE 100 295 FEI VITROBOT MARK IV

BK proteoliposomes were applied onto a glow-discharged perforated carbon grid (CFlat R2/2, EMS), and incubated for 5-10 min In an FEI Vitrobot Mark IV (FEI). After incubation, the sample was blotted from the side and buffer containing 10 mM HEPES, pH 7.3, 75 mM KCl, and 1 mM EDTA was applied onto the TEM grid to further swell proteoliposomes. Then grids were blotted for 7 s with force 0 and fast frozen in liquid ethane cooled by liquid nitrogen..

Swelled BK proteoliposomes yielded a concentration of 2 mM lipid and 1mg/ml protein.

FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 0.852 4.0 130000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN 90.8 103.5 GIF Bioquantum 20

The Coma free alignment was performed using Auto-CTF software from FEI.

GATAN K2 SUMMIT (4k x 4k) SUPER-RESOLUTION 3838 3710 1-43 3 3089 8.6 60.0

Images were recorded in an automated fashion on a Gatan K2 Summit (Gatan) detector set to super-resolution mode with a super-resolution pixel size of 0.525 A using Leginon. The dose rate on the camera was set to be ~8 counts per physical pixel per second. The total exposure time was 8.6 s (0.2 s/frame), leading to a total accumulate dose of 60 electrons per A2 on the specimen.

1

Dose-fractionated super-resolution image stacks were motion-corrected with MotionCorr2. Each frame in the image stack was divided into 5x5 patches for anisotropic image motion correction and dose weighting was carried out to calculate the motion-corrected image.

275295

Approximately 4,800 particles from 350 micrographs were interactively selected and classified using RELION. Ten representative classes were selected as the templates for automated particle picking using RELION. The automated picked particles were screened using homemade MATLAB (MathWorks) programs to exclude particles more than 80 A away from any liposome, which resulted in 275,295 particles from 2,786 micrographs.

5TJI C2 3.5 FSC 0.143 CUT-OFF RELION 2.1 122456 RANDOM ASSIGNMENT RELION 2.1 MAXIMUM LIKELIHOOD RELION 2.1 100 2750.0 RELION 2.1 FLEXIBLE FIT REAL