Computational protocol: Ligand Activation of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC

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Protocol publication

[…] The purified protein was crystallized in sitting drops at 4°C. Protein containing additional 0.5 mg/ml E. coli polar lipids (Avanti Polar Lipids, Inc.) was mixed in a 1∶1 ratio with reservoir solution (100 mM ADA pH 6.5, 200 mM NaLiSO4, and 10% (w/v) PEG4000). Bromopropylamine was soaked into the crystals by incubation with solutions containing 5 mM of the ligand. The crystals were cryoprotected by transfer into solutions containing 30% glycerol. All datasets were collected on frozen crystals on the X06SA beamline at the Swiss Light Source (SLS) of the Paul Scherrer Institut (PSI) on a PILATUS detector (Dectris). The data were indexed, integrated, and scaled with XDS and further processed with CCP4 programs . The structure of the F246A mutant at 3.3 Å was determined by Molecular Replacement using the structure of ELIC as the search model. The model was rebuilt in O and refined maintaining strong 10-fold NCS constraints in PHENIX . The structures of the WT protein in complex with bromopropylamine at 4.0 Å was improved by rigid body refinement in CNS . R and Rfree were monitored throughout. Rfree was calculated by selecting 5% of the reflection data in thin slices that were selected for the initial dataset of ELIC and that were omitted in refinement (). […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, CNS
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma
Chemicals Acetylcholine, gamma-Aminobutyric Acid