Sephadex

Preparation of cDNA followed the procedure described in Mortazavi et al.^{2 (link)}, with minor modifications as described below. Prior to fragmentation, a 7 uL aliquot (∼ 500 pgs total mass) containing known concentrations of 7 “spiked in” control transcripts from A. thaliana and the lambda phage genome were added to a 100 ng aliquot of mRNA from each time point. This mixture was then fragmented to an average length of 200 nts by metal ion/heat catalyzed hydrolysis. The hydrolysis was performed in a 25 uL volume at 94°C for 90 seconds. The 5X hydrolyis buffer components are: 200 mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate. After removal of hydrolysis ions by G50 Sephadex filtration (USA Scientific catalog # 1415-1602), the fragmented mRNA was random primed with hexamers and reverse-transcribed using the Super Script II cDNA synthesis kit (Invitrogen catalog # 11917010). After second strand synthesis, the cDNA went through end-repair and ligation reactions according to the Illumina ChIP-Seq genomic DNA preparation kit protocol (Illumina catalog # IP102-1001), using the paired end adapters and amplification primers (Illumina Catalog # PE102-1004). Ligation of the adapters adds 94 bases to the length of the cDNA molecules.

The cDNA library was size-fractionated on a 2% TAE low melt agarose gel (Lonza catalog # 50080), with a 100 bp ladder (Roche catalog # 14703220) run in adjacent lanes. Prior to loading on the gel, the ligated cDNA library was taken over a G50 Sephadex column to remove excess salts that interfere with loading the sample in the wells. After post-staining the gel in ethidium bromide, a narrow slice (∼2mm) of the cDNA lane centered at the 300 bp marker was cut. The slice was extracted using the QiaEx II kit (Qiagen catalog # 20021), and the extract was filtered over a Microcon YM-100 microconcentrator (Millipore catalog # 42409) to remove DNA fragments shorter than 100 bps. Filtration was performed by pipeting the extract into the upper chamber of a microconcentrator, and adding ultra pure water (Gibco catalog # 10977) to a volume of 500 uLs. The filter was spun at 500 X g until only 50 uLs remained in the upper chamber (about 20 minutes per spin) and then the upper chamber volume was replenished to 500 uLs. This procedure was repeated 6 times. The filtered sample was then recovered from the filter chamber according to the manufacturer's protocol. Fragment length distributions obtained after size selection were estimated from the spike-in sequences and are show in Supplementary Fig. 1.

Previously crystallized GPCRs show little density for the poorly ordered amino and carboxy terminal domains. Although these domains are not critical for maintaining high ligand affinity, these flexible regions may inhibit crystallogenesis^{7 (link)}. We therefore removed these regions in the receptor construct used for crystallography. Specifically, a TEV protease recognition site was introduced after reside G51 in the amino-terminus and the carboxy terminus was truncated after Q360. The short third intracellular loop of μOR, consisting of residues 264–269 was replaced with T4 lysozyme residues 1–161 in a manner described previously^{7 (link)}. In order to facilitate receptor purification, a FLAG M1 tag was added to the amino-terminus and an octa-histidine tag was appended to the carboxy terminus. Finally, a proline residue was introduced N-terminal to the octahistidine tag to allow efficient removal of C-terminal histidines by carboxypeptidase A. For these studies, we utilized the Mus musculus μOR sequence because it expressed at higher levels. The mouse and human μOR share 94% sequence identity and there are only four residues in the resolved part of the structure that differ between mouse and human μOR. These include residues 66, 137, 187, and 306, which are all in the extracellular or intracellular loops of μOR and do not make contacts in the ligand-binding pocket. The final crystallization construct (μOR-T4L) is shown in a representative snake diagram in Supplementary Fig. 1a.
We compared the pharmacological properties of μOR-T4L to those of the wild-type receptor (Supplementary Fig. 1b; see below for methods details). Both constructs showed identical affinity for the radiolabeled antagonist [³H]-diprenorphine ([³H]DPN).
The μOR-T4L construct was expressed in Sf9 cells using the baculovirus system. Culture media was supplemented with 10 μM naloxone to stabilize the receptor during expression. Cells were infected at a density of 4×10⁶ cells per mL and culture flasks were shaken at 27 °C for 48 hr. After harvesting, cells were lysed by osmotic shock in a buffer comprised of 10mM Tris-HCl pH 7.5, 1mM EDTA, 100 µM TCEP, 1 µM naloxone, and 2 mg/ml iodoacetamide to block reactive cysteines. Extraction of μOR-T4L from Sf9 membranes was done with a Dounce homogenizer in a solubilization buffer comprised of 0.5% dodecyl maltoside (DDM), 0.3% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.03% cholesterol hemisuccinate (CHS), 20 mM HEPES pH 7.5, 0.5 M NaCl, 30% v/v glycerol, 2 mg/ml iodoacetamide, 100 μM TCEP, and 1 µM naloxone. After centrifugation, nickel-NTA agarose was added to the supernatant, stirred for two hours, and then washed in batch with 100 × g spins for 5 min each with a washing buffer of 0.1% DDM, 0.03% CHAPS, 0.01% CHS, 20 mM HEPES pH 7.5 and 0.5 M NaCl. The resin was poured into a glass column and bound receptor was eluted in washing buffer supplemented with 300 mM imidazole.
We utilized anti-FLAG M1 affinity resin to further purify μOR-T4L and to exchange the ligand to the covalent antagonist β-funaltrexamine (β-FNA). Nickel-resin eluate was loaded onto anti-FLAG M1 resin and washed extensively in the presence of 10 µM β-FNA. The detergent DDM was then gradually exchanged over 1 hr into a buffer with 0.01% lauryl maltose neopentyl glycol (MNG) and the NaCl concentration was lowered to 100 mM. Receptor was eluted from the anti-FLAG M1 affinity resin with 0.2 mg/mL FLAG peptide and 5 mM EDTA in the presence of 1 µM β-FNA. To remove the amino terminus of μOR-T4L, TEV protease was added at 1:3 w/w (TEV:μOR-T4L) and incubated at room temperature for 1 hr. Receptor was then treated with carboxypeptidase A (1:100 w/w) and incubated overnight at 4°C to remove the octa-histidine tag. The final purification step separated TEV and carboxypeptidase A from receptor by size exclusion chromatography (SEC) on a Sephadex S200 column (GE Healthcare) in a buffer of 0.01% MNG, 0.001% CHS, 100 mM NaCl, 20 mM HEPES pH 7.5, and 1 µM β-FNA. After size exclusion, β-FNA was added to a final concentration of 10 µM. The resulting receptor preparation was pure and monodisperse (Supplementary Fig. 12).