Protein Expression and Purification Procedures
Vector: pFB-LIC-Bse (available from The Addgene Nonprofit Plasmid Repository)
Cell line: DH10Bac, Sf9 cells
Tags and additions: N-terminal, TEV protease cleavable-6-His- tag
Wild-type sequence (with tag):
The full length gene for human HCN4 was obtained as a gift from GSK. This gene was cloned into the pFB-LIC-Bse vector and baculoviruses were produced by transformation of DH10Bac cells. Spodoptera frugiperda (Sf9) insect cells in Sf-900 II SFM medium (Life Technologies) were infected with recombinant baculovirus and incubated for 72 h at 27°C in shaker flasks and then collected by centrifugation at 900Xg and the pellet flash frozen in liquid nitrogen.
Cell Lysis and detergent extraction of membrane protein
Extraction Buffer, (EXB): 50 mM HEPES (pH 7.5), 150 mM NaCl, 5% Glycerol, Roche complete EDTA free protease inhibitors.
The insect cell pellet was resuspended in 40 ml of EXB / 1L pellet in a 50 ml tube and thawed in tepid water, mixing constantly to keep the sample cold. Cells were lysed with a EmulsiFlex-C5 homogenizer (Avestin) (chilled at 4 °C, 2 passes). 5 mL of 10 % : 1 % (w:w) stock of DDM/CHS was added per Litre of cell culture and the volume was adjusted by the addition of EXB to a final volume of 50 mL/L of cell culture. The sample was rotated in the cold room for 1 h, then extracted membrane proteins were separated from insoluble membranes by centrifugation at 35,000 g for 45 min at 4 oC.
Column 1: Co2+ talon resin (0.5 ml volume in a gravity-flow column):
Wash Buffer (WB): 50 mM HEPES (pH 7.5), 20 mM imidazole (pH 8.0), 150 mM NaCl, 5 % Glycerol, 0.02% DDM, and 0.002 % CHS.
Elution Buffer (EB): 50 mM HEPES (pH 7.5), 300 mM imidazole (pH 8.0), 150 mM NaCl, 5 % Glycerol, 0.02% DDM, 0.002 % CHS.
Gel Filtration Buffer (GFB): 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.02 %DDM, 0.002 % CHS.
The detergent-extracted membrane protein fraction from each litre of cells was combined with 1 ml of pre-equilibrated slurry of 50 % Co2+ talon resin (pre-equilibrated with EXB after washing in H2O). The sample was rotated in the cold room for 1 h then collected in an econo column fitted with a filter. The residual talon resin was washed with 5 ml wash buffer and pipetted onto the econo column. The talon resin was washed with 20x talon resin volume of WB and the protein eluted with 2x Talon resin volume of elution buffer in 1 ml fractions. The peak fractions were combined and the imidazole removed using two PD10 columns per purification (pre-equilibrated with SEC buffer). Protein was eluted from the PD10 columns using SEC buffer.
TEV protease cleavage and reverse purification
TEV protease was added at a ratio of 10 : 1 (HCN4: TEV protease (w:w)). Samples were incubated on ice in the cold room overnight. The 6X His tagged TEV was separated from the tag cleaved HCN4 using cobalt affinity chromatography as before, with the flow through containing pure protein.
Column 2: Superose 6 Size Exclusion Chromatography
The protein sample was concentrated in a 100 kDa PES centrifugal concentrator (pre-equilibrated with SEC buffer), with mixing every 10 mins, to a final volume of 500 μL. After centrifugation at 21, 000 g for 30 min the sample was subjected to size exclusion chromatography on a Superose 6 column in SEC buffer. 3-4 0.5 ml peak fractions containing HCN4 protein were concentrated with a Sartorius 2 ml PES 100 kDa concentrator pre-equilibrated with SEC buffer, at 3,220 g to a final concentration of no more than 15 mg/ml. The protein was centrifuged at 21,000 g for 15 mins, then flash frozen in liquid nitrogen.
Thermostability assay using CPM dye fluorescence
This method is based on the protocol described in Alexandrov et al., 2008 (22). Concentrated protein was defrosted and diluted to 1 mg/ml before aliquoting into assay plate well with each well containing 2 µg HCN4 in a final volume of 25 µl. The remaining volume was made up with GFB with or without addition of ligand (5 mM) and 5 µl of 0.04 mg/ml CPM (7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin) dye (diluted from a 4 mg/ml stock made up in DMSO) was added fresh before a 30 s centrifugation at 800 X g of the assay plate. Fluorescence measurements were carried out in an Mx3005P™ Real-Time PCR System with ANS filter with initial temperature set to 25 ̊C and the plate heating to 95C at a rate of 1̊C per minute.
Cryo-EM grid preparation
Concentrated protein was defrosted and diluted to 5 mg/ml. When ligands were added they were left to incubate with the protein for 1 hour at this stage and then the sample was centrifuged at 21,000 X g for 30 minutes before plunge freezing onto cryo EM grids. Sample was applied to glow discharged holey carbon coated grids (1.2-μm hole size, 1.3 μm spacing, 300 or 400 mesh; Quantifoil) Grids were blotted at 100% humidity for 3- 5 s and plunge-frozen in liquid ethane cooled by liquid nitrogen with a vitrification apparatus (Vitrobot Mark IV, FEI).
Cryo EM data collection and processing
Cryo-EM data collection was carried out at the eBIC national facility on a Titan Krios operated at 300 kV at liquid nitrogen temperature. Data were acquired with EPU software on a direct electron detector (K2 Summit, Gatan) mounted behind an energy filter (GIF Quantum LS, Gatan) and operated in zero-loss mode (0–20 eV). Movies were recorded in electron-counting mode (total dose 48e−/Å2) with underfocus in a range from 1.1 to 2.5 μm. The calibrated magnification used was 37,037× (corresponding to a pixel size of 1.34Å).
All initial processing was carried out in RELION 2.0. Frames in each movie stack were aligned with MOTIONCOR2 and CTF parameters were estimated using CTFFIND 4.0. Dose weighted stacks were subjected to semi-automatic particle picking using Gautomatch.
For both datasets particles were semi automatically picked and put through several rounds of 2D class averaging to remove bad particles. These were then subjected to template based 3D classification with a low pass filtered (60 Å) model of HCN4 based on HCN1 used as a template. An initial 3D classification, run with no symmetry imposed, clearly indicated that HCN4 formed a tetrameric channel. Further 3D classification with C4 symmetry imposed identified a subset particles that were subsequently used for a first 3D 'gold-standard' refinement wherein two half sets of the data were refined independently. Both datasets yielded reconstructions to 3.4Å resolution (FSC=0.143). The optimised particle sets from RELION were refined in parallel using cryoSPARC which gave resolutions of 3.3 Å.
Structure building and refinement procedures
The unliganded structure was built manually in COOT based on the coordinates of the unliganded HCN1 channel (PDB: 5U6O). The structure was refined against the B-factor sharpened cryoSPARC map to 3.4Å resolution using REFMAC and phenix.real_space_refine. This refined model was initially fitted automatically to the 3.4Å cAMP reconstruction using morphed refinement in phenix.real_space_refine and subsequently manually corrected in COOT. For the unliganded complex, the final model comprises residues Met214-Leu708. In the cAMP complex, the C-terminus is resolved to Ile714 due to ordering of the C-terminal CNBD helix on nucleotide binding.