Plasmids, DNAs, and RNAs
A DNA fragment encoding B. subtilis HelD was PCR-amplified from strain MH5636 (Supplementary Table 1). The PCR product was inserted into expression vector pGEX-6p-1 via BamHI and XhoI restriction sites, in frame with a region encoding an N-terminal GST-tag. DNA fragments encoding B. subtilis σA, δ or δNTD were PCR-amplified from strain MH5636 and inserted into a pETM-11 vector (EMBL, Heidelberg) via NcoI/HindIII or NcoI/XhoI restriction sites, respectively, in frame with a region encoding an N-terminal His6-tag. DNA and RNA oligomers used for the assembly of transcription complexes were purchased from Eurofins and IBA Lifesciences, respectively.
Protein production and purification
B. subtilis strains MH5636, LK782 (ΔhelD) or LK1032 (ΔhelDΔrpoE; Supplementary Table 1) were used to produce stationary phase RNAP, RNAPΔHelD or RNAPΔδΔHelD, respectively. In these strains, the chromosomally-encoded β′ subunit carries a C-terminal His10-tag. Strains were grown in TB medium at 37 °C to an OD600 of 1.0 and were then shifted to 18 °C and grown to an OD600 of about 11. All purification steps were performed at 4 °C. Cells were harvested by centrifugation, resuspended in buffer A (50 mM Na2HPO4, 300 mM NaCl, 3 mM 2-mercaptoethanol, 5% [v/v] glycerol, pH 7.9), and lysed by sonication. The lysate was cleared by centrifugation. RNAP variants were captured on Ni2+-NTA affinity resin (Macherey-Nagel), washed with buffer A supplemented with 25 mM imidazole, and eluted with buffer A supplemented with 250 mM imidazole. The eluate was dialyzed overnight against 50 mM Na2HPO4, 300 mM NaCl, 3 mM DTT, 5% [v/v] glycerol, pH 7.9, loaded on a 5 ml HiTrap Heparin HP column (GE Healthcare), washed with buffer B (50 mM TRIS-HCl, 100 mM NaCl, 3 mM DTT, 0.1 mM EDTA, 5% [v/v] glycerol, pH 7.9) and eluted with a linear gradient to buffer B with 700 mM NaCl. Fractions containing RNAPs were pooled and further purified by SEC on a HiLoad Superdex 200 Increase 16/600 column (GE Healthcare) in 20 mM TRIS-HCl, 150 mM NaCl, 0.5 mM DTT, 5% (v/v) glycerol, pH 8.0. The final samples were concentrated to approximately 16 mg/ml. RNAP produced from strain MH5636 was directly used for EM sample preparation. Other RNAP preparations were aliquoted, flash-frozen in liquid N2, and stored at −80 °C.
Recombinant B. subtilis GST-HelD was produced in E. coli Rosetta(DE3) cells, His6-δ, His6-δNTD, and His6-σA were produced in E. coli BL21(DE3)-RIL cells. Cells were grown in auto-inducing media59 at 37 °C to an OD600 of 1.0 and further incubated at 20 °C overnight. All purification steps were performed at 4 °C. GST-HelD cells were harvested by centrifugation, resuspended in buffer C (50 mM TRIS-HCl, 500 mM NaCl, 1 mM 2-mercaptoethanol, 10% [v/v] glycerol, pH 7.9), and lysed by sonication. The lysate was cleared by centrifugation, GST-HelD was captured on glutathione resin (Macherey-Nagel), washed with buffer C, and eluted with 50 mM TRIS-HCl, 300 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 20 mM reduced glutathione, pH 7.9. Eluted fractions were dialyzed against buffer D (20 mM TRIS-HCl, 200 mM NaCl, 1 mM DTT, 5% [v/v] glycerol, pH 7.9) in the presence of GST-tagged PreScission protease. HelD was separated from uncleaved protein, GST, and GST-PreScission by a second passage through glutathione resin. The flowthrough was further purified by SEC on a HiLoad Superdex 200 Increase 16/600 column equilibrated in buffer D. Fractions containing HelD were concentrated to approximately 15 mg/ml, aliquoted, flash-frozen in liquid N2, and stored at −80 °C.
His6-δ or His6-δNTD cells were harvested by centrifugation, resuspended in 50 mM TRIS-HCl, 500 mM NaCl, 0.5 mM 2-mercaptoethanol 5% [v/v] glycerol, pH 6.0, and lysed by sonication. The lysate was cleared by centrifugation, His6-δ/His6-δNTD was captured on Ni2+-NTA resin, washed with 50 mM TRIS-HCl, 300 mM NaCl, 0.5 mM 2-mercaptoethanol, 10 mM imidazole, 5% (v/v) glycerol, pH 6.0, and eluted with 20 mM TRIS-HCl, 150 mM NaCl, 0.5 mM 2-mercaptoethanol, 400 mM imidazole, 5% (v/v) glycerol, pH 6.0. For the assembly of complexes for cryoEM analysis, eluted His6-δ was supplemented with His-tagged TEV protease (1:40 [w/w]), dialyzed against buffer E (20 mM TRIS-HCl, 150 mM NaCl, 1 mM DTT, 5% (v/v) glycerol, pH 6.0) overnight and passed through fresh Ni2+-NTA resin to remove the uncleaved His6-δ, the cleaved His6-tag, and His-tagged TEV protease. Proteins were further purified by SEC on a Superdex 75 Increase 10/300 column (GE Healthcare) in buffer E. Fractions containing His6-δ, δ or His6-δNTD were concentrated to ~4 mg/ml (His6-δ, His6-δNTD) and 23 mg/ml (δ), aliquoted, flash-frozen in liquid N2 and stored at −80 °C.
σA cells were harvested by centrifugation, resuspended in buffer F (20 mM TRIS-HCl, 500 mM NaCl, 1 mM 2-mercaptoethanol, 5% [v/v] glycerol, pH 7.5) supplemented with 20 mM imidazole, and lysed by sonication. The lysate was cleared by centrifugation, His6-σA was captured on Ni2+-NTA resin, washed with buffer F supplemented with 50 mM imidazole, and eluted with buffer F supplemented with 400 mM imidazole. Eluted His6-σA was supplemented with His-tagged TEV protease (1:40 [w/w]), dialyzed against buffer F supplemented with 1 mM EDTA overnight, and passed through fresh Ni2+-NTA resin to remove uncleaved His6-σA, cleaved His6-tag and His-tagged TEV protease. The target protein was further purified by SEC on a Superdex 75 Increase 16/600 column (GE Healthcare) in 25 mM TRIS-HCl, 300 mM NaCl, 0.1 mM DTT, 5% (v/v) glycerol, pH 7.5. Fractions containing σA were concentrated to approximately 39 mg/ml, aliquoted, flash-frozen in liquid N2, and stored at −80 °C.
Sulfo-SDA predominantly establishes lysine-X crosslinks through a primary amine-reactive moiety on one side and a UV-activatable moiety on the other (theoretical crosslinking limit 25 Å). Sulfo-SDA was prepared at 3 mg/ml in 20 mM HEPES-NaOH, 5 mM Mg(OAc)2, 300 mM NaCl, 5 mM DTT, 5% (v/v) glycerol, pH 8.0 immediately prior to addition of RNAPΔδΔHelD, RNAPΔδΔHelD-δ, RNAPΔδΔHelD-HelD, or RNAPΔδΔHelD-δ-HelD (protein:sulfo-SDA 1:3 [w/w]). Samples were incubated on ice for two hours and then irradiated in a thin film using 365 nm UV (UVP CL‐1000 UV Crosslinker, UVP Inc.) for 20 min on ice (5 cm distance from UV-A lamp). The cross-linked samples were separated by 4–12% BIS-TRIS NuPAGE, gel bands corresponding to crosslinked monomeric complexes were excised and digested in-gel60. The resulting peptides were desalted using C18 StageTips61.
10% of each sample were analyzed by LC-MS/MS without fractionation, the remaining 90% were fractionated using SEC on a Superdex Peptide 3.2/300 column (GE Healthcare) in 30% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid at a flow rate of 10 µl/min to enrich for crosslinked peptides62. The first six peptide-containing fractions (50 μl each) were collected, the solvent was removed using a vacuum concentrator and the fractions were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific), connected to an Ultimate 3000 RSLCnano system (Dionex, ThermoFisher Scientific).
The non-fractionated samples were injected onto a 50 cm EASY-Spray C18 LC column (ThermoFisher Scientific) operated at 50 °C. Peptides were separated using a linear gradient going from 2% mobile phase B (80% [v/v] acetonitrile, 0.1% [v/v] formic acid) to 40% mobile phase B in mobile phase A (0.1% [v/v] formic acid) at a flow rate of 0.3 μl/min over 110 min, followed by a linear increase from 40 to 95% mobile phase B in 11 min. Eluted peptides were ionized by an EASY-Spray source (ThermoFisher Scientific) and MS data were acquired in the data-dependent mode with the top-speed option. For each 3-s acquisition cycle, the full scan mass spectrum was recorded in the Orbitrap with a resolution of 120,000. The ions with a charge state from 3+ to 7+ were isolated and fragmented using higher-energy collisional dissociation (HCD) with 30% collision energy. The fragmentation spectra were then recorded in the Orbitrap with a resolution of 50,000. Dynamic exclusion was enabled with a single repeat count and 60 s exclusion duration.
SEC fractions were analyzed using an identical LC-MS/MS setup. Peptides were separated by applying a gradient ranging from 2 to 45% mobile phase B (optimized for each fraction) over 90 min, followed by ramping up mobile phase B to 55 and 95% within 2.5 min each. For each three-second data-dependent MS acquisition cycle, the full scan mass spectrum was recorded in the Orbitrap with a resolution of 120,000. The ions with a charge state from 3+ to 7+ were isolated and fragmented using HCD. For each isolated precursor, one of three collision energy settings (26%, 28%, or 30%) was selected for fragmentation using a data-dependent decision tree based on the m/z and charge of the precursor. The fragmentation spectra were recorded in the Orbitrap with a resolution of 50,000. Dynamic exclusion was enabled with a single repeat count and 60 s exclusion duration.
LC-MS/MS data generated from the four complexes were processed separately. MS2 peak lists were generated from the raw MS data files using the MSConvert module in ProteoWizard (version 3.0.11729). The default parameters were applied, except that Top MS/MS Peaks per 100 Da was set to 20 and the denoising function was enabled. Precursor and fragment m/z values were recalibrated. Identification of cross-linked peptides was carried out using xiSEARCH software (https://www.rappsilberlab.org/software/xisearch; version 1.7.4)63. For RNAPΔδΔHelD, peak lists were searched against the sequence and the reversed sequence of RNAP subunits (α, β, β′, and ε) and two co-purified proteins, σA and σB. For RNAPΔδΔHelD-δ, RNAPΔδΔHelD-HelD, and RNAPΔδΔHelD-δ-HelD samples, protein sequences of δ, HelD or both were included in the database. The following parameters were applied for the search: MS accuracy = 4 ppm; MS2 accuracy = 8 ppm; enzyme = trypsin (with full tryptic specificity); allowed number of missed cleavages = 2; missing monoisotopic peak = 2; crosslinker = sulfo-SDA (the reaction specificity for sulfo-SDA was assumed to be for lysine, serine, threonine, tyrosine, and protein N-termini on the NHS ester end, and any amino acid residue for the diazirine end); fixed modifications = carbamidomethylation on cysteine; variable modifications = oxidation on methionine and sulfo-SDA loop link. Identified crosslinked peptide candidates were filtered using xiFDR64. A false discovery rate of 5% on the residue-pair level was applied with the “boost between” option selected. Crosslinked residue pairs identified from the four complexes are summarized in Supplementary Table 4 and Supplementary Data 1.
CryoEM sample preparation, data collection, and processing
Equimolar amounts of tDNA, ntDNA, and RNA were mixed in buffer G (20 mM TRIS-HOAc, 5 mM Mg[OAc]2, 100 mM KOAc, 2 mM DTT, 5% [v/v] glycerol, pH 8.0) and annealed by heating to 95 °C for 5 min and subsequent cooling to 25 °C at 1 °C/min. The annealed scaffold was incubated with B. subtilis RNAP in a 1.3:1 molar ratio in buffer H (20 mM TRIS-HOAc, 5 mM Mg[OAc]2, 300 mM KOAc, 2 mM DTT, 5% [v/v] glycerol, pH 8.0) for 10 min on ice, then for 10 min at 32 °C. Equimolar amounts (to RNAP) of δ and HelD were added stepwise, followed by incubation for 10 min at 32 °C after each addition. The mixture was subjected to SEC on a Superdex 200 Increase 3.2/300 column (GE Healthcare) in buffer H. Fractions containing RNAP, δ, and HelD were pooled and concentrated to approximately 5 mg/ml.
Immediately before preparation of the grids, the sample was supplemented with 0.15% (w/v) n-octylglucoside (critical micellar concentration 0.6% [w/v]). 3.8 µl of the final mixture were spotted on plasma-treated Quantifoil R1/2 holey carbon grids at 10 °C/100% humidity and plunged into liquid ethane using an FEI Vitrobot Mark IV. Image acquisition was conducted on an FEI Titan Krios G3i (300 kV) transmission electron microscope (TEM) with a Falcon 3EC camera at a nominal magnification of 92,000x in counting mode using EPU software (ThermoFisher Scientific) with a calibrated pixel size of 0.832 Å. A total electron dose of 40 e−/Å2 was accumulated over an exposure time of 36 s. Movie alignment was done with MotionCor265 using 5 × 5 patches followed by ctf estimation with Gctf66.
All following image analysis steps were done with cryoSPARC67. Class averages of manually selected particles were used to generate an initial template for reference-based particle picking from 9127 micrographs. Particle images were extracted with a box size of 440 and binned to 110 for initial analysis. Ab initio reconstruction using a small subset of particles was conducted to generate an initial 3D reference for 3D heterogeneous refinement. The dataset was iteratively classified into two well-resolved populations representing monomeric and dimeric RNAP-δ-HelD. Selected particles were re-extracted with a box of 220 and again classified in 3D to further clean the dataset. Finally, selected particle images were re-extracted with a box of 280 (1.3 Å/px) and subjected to local refinement using a generously enlarged soft-mask for monomeric or dimeric RNAP-δ-HelD. Local refinement of the dimer particles using the monomeric mask was conducted as a control to trace differences of RNAP-δ-HelD in the authentic monomer and dimer structures. After per-particle CTF correction, non-uniform refinement was applied to generate the final reconstructions.
Negative stain EM analysis
RNAP-δ-HelD complex was prepared as for cryoEM analysis, diluted to 25 µg/ml in buffer H and supplemented with 0.15% n-octylglucoside or buffer immediately before grid preparation. 4 µl of the samples were added to glow-discharged Formvar/carbon grids (S162, Plano GmbH), left to settle for 40 s and manually blotted with Whatman paper No. 1, followed by addition of 4 µl of 1% (w/v) uranyl acetate staining solution. After 40 s incubation, the grids were manually blotted and dried at ambient temperature overnight. Samples were imaged on an FEI Talos L120C TEM, operated at 120 kV, equipped with an FEI CETA 16 M CCD camera at a nominal magnification of 57,000x. The calibrated pixel size was 2.53 Å/px. Images were acquired manually in low dose mode using TEM Imaging & Analysis (TIA) software, supplied by the manufacturer, accumulating a total electron dose of 50 e−/Å2. Image analysis was done with cryoSPARC. After CTF estimation, manually selected particle images were used as a reference for template-based particle picking. Particle images were extracted with a box size of 160 px and resampled to 80 px. A mask of 220 Å diameter was applied during 2D classification.
Model building and refinement
The final cryoEM map for the dimeric RNAP-δ-HelD complex was used for initial model building. Coordinates of M. smegmatis RNAP α, β, and β′ subunits (PDB ID 5VI8)68 were docked into the cryoEM map using Coot69. Modeling of δ was based on the NMR structure of B. subtilis δ (PDB ID 2M4K)70. Modeling of ε was supported by the structure of YkzG from Geobacillus stearothermophilus (PDB ID 4NJC)26. Model building of HelD was supported by the structure of UvrD helicase from E. coli (PDB ID 3LFU)71 as well as the C-terminal domain of a putative DNA helicase from Lactobacillus plantarun (PDB ID 3DMN). The subunits were manually rebuilt into the cryoEM map. The model was completed and manually adjusted residue-by-residue, supported by real-space refinement in Coot. The manually built model was refined against the cryoEM map using the real-space refinement protocol in PHENIX72. Model building of the monomeric complex was done in the same way but starting with a model of half of the dimeric complex. The structures were evaluated with Molprobity73. Structure figures were prepared using PyMOL (Version 1.8 Schrödinger, LLC).
Structures were compared by global superposition of complex structures or by superposition of selected subunits in complexes using the “secondary structure matching” algorithm implemented in Coot or the “align” algorithm implemented in PyMOL.
Size exclusion chromatography/multi-angle light scattering
SEC/MALS analysis was performed on an HPLC system (Agilent) coupled to mini DAWN TREOS multi-angle light scattering and RefractoMax 520 refractive index detectors (Wyatt Technology). RNAP-δ-HelD complex was assembled as for cryoEM. 60 μl of the sample at 1–1.3 mg/ml were chromatographed on a Superose 6 Increase 10/300 column (GE Healthcare) in buffer H or buffer H plus 0.15% (w/v) n-octylglucoside, supplemented with 0.02% (w/v) NaN3, at 18 °C with a flow rate of 0.6 ml/min. Data were analyzed with the ASTRA 6.1 software (Wyatt Technology) using monomeric bovine serum albumin (Sigma-Aldrich) as a reference.
HelD interactions with δ or δNTD were analyzed by analytical SEC. 21 µM HelD and 42 µM δ or δNTD were mixed in 20 mM HEPES-NaOH, 50 mM NaCl, 1 mM DTT, pH 7.5, and incubated for 10 min at room temperature. 50 µl of the samples were loaded on a Superdex 200 Increase PC 3.2 column (GE Healthcare) and chromatographed at 4 °C with a flow rate of 40 µl/min. Fractions were analyzed by 12.5% SDS-PAGE.
Nucleic acid displacement assays
Equimolar amounts of 5′-[32P]-labeled ntDNA and unlabeled tDNA capable of forming an artificial bubble, or additionally an RNA 9-mer with complementarity to the tDNA in the bubble (Supplementary Table 1), were mixed in buffer G and annealed by heating to 95 °C for 5 min and subsequent cooling to 25 °C at 1 °C/min. The labeled DNA duplex or DNA/RNA scaffold and RNAPΔδΔHelD (10 nM and 1 µM final concentrations, respectively) were incubated in buffer G for 10 min at 4 °C, followed by an additional 10 min incubation at 32 °C. Subsequently, (i) buffer, (ii) HelD (1 µM final concentration); (iii) δ (1 µM final concentration), (iv) combinations of HelD (1 µM final concentration) and δ (titrated final concentration; Fig. 3g) or (v) HelD and δNTD (1 µM final concentration each) were added, and the samples were further incubated for 10 min at 32 °C. Samples were loaded on a 4% native PAGE gel and electrophoresed in 0.5X TBE buffer. Radiolabeled bands were visualized using a Storm phospohorimager and quantified using ImageQuant software (GE Healthcare).
HelD release assays
Equimolar amounts of HelD and stationary phase RNAP were mixed in buffer I (20 mM TRIS-HCl, 300 mM NaCl, 2 mM DTT, 5% (v/v) glycerol, pH 8.0), incubated for 10 min on ice and then for 10 min at 32 °C. The sample was chromatographed on a HiLoad Superdex 200 Increase 10/300 column (GE Healthcare) in buffer I. Fractions were analyzed by 12.5% SDS-PAGE, fractions containing RNAP-HelD complex were collected and concentrated to ~3 mg/ml (6.7 µM). 80 µl of this complex were mixed with buffer I, 5 mM Mg2+-ATPγS/AMPPNP/ATP/ADP/AMP, 6.7 µM σA or σA plus Mg2+-ATPγS in buffer I. 90 µl of the samples were loaded on a Superdex 200 Increase PC 3.2 column (GE Healthcare) and chromatographed at 4 °C with a flow rate of 40 µl/min. Fractions were analyzed by 12.5% SDS-PAGE.
Structures of the glucocorticoid-bound adhesion receptor GPR97–Go complex
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.
HEK293 cells were obtained from the Cell Resource Center of Shanghai Institute for Biological Sciences (Chinese Academy of Sciences). Spodoptera frugiperda (Sf9) cells were purchased from Expression Systems (cat. 94-001S). Y-1 cells were originally obtained from the American Type Culture Collection (ATCC). The cells were grown in monolayer culture in RPMI 1640 with 10% FBS (Gibco) at 37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air.
Constructs of GPR97 and miniGo heterotrimer
For protein production in insect cells, the human GPR97 (residues 21–549) with the autoproteolysis motif mutation (H248/A and T250/A) was sub-cloned into the pFastBac1 vector. The native signal peptide was replaced with the haemagglutinin signal peptide (HA) to enhance receptor expression, followed by a Flag tag DYKDDDK (China peptide) to facilitate complex purification. An engineered human Gαo1 with Gαo1 H domain deletion, named miniGαo1 was cloned into pFastBac1 according to published literature29. Human Gβ1 with the C-terminal hexa-histidine tag and human Gγ2 were subcloned into the pFastBacDual vector. scFv16 was cloned into pfastBac1 with the C-terminal hexa-histidine tag and the N-terminal GP67 signal peptide. To examine the activities of GPR97, the GPR97-FL-WT (wild-type full-length GPR97), GPR97-FL-AA (GPR97 GPS site mutation, H248/A and T250/A), GPR97β (GPR97 with the NTF removed, residues 250–549) and GPR97-β-T (GPR97β with the N-terminal tethered Stachel sequence removed, residues 265–549) were sub-cloned into the pcDNA3.1 plasmid. The GPR97 mutations E298A, R299A, F345A, F353A, H362A, L363A, Y364A, V370A, F371A, Y406A, W421A, W490A, A493G, I494A, L498A and N510A were generated using the Quikchange mutagenesis kit (Stratagene). The G protein BRET probes were constructed according to previous publications42,43. Human G protein subunits (Gαq, Gβ1 and Gγ2) were sub-cloned into the pcDNA3.1 expression vectors. The Gαq-RlucII subunit was generated by amplifying and inserting the coding sequence of RlucII into Gαq between residue L97 and K98. The Gqo probe, in which the six amino acids of the C-terminal of Gαq-RlucII were substituted with those from Gαo1, was constructed by PCR amplification using synthesized oligonucleotides encoding swapped C-terminal sequences. The GFP10–Gγ2 plasmid was generated by fusing the GFP10 coding sequence in frame at the N terminus to Gγ2. All of the constructs and mutations were verified by DNA sequencing.
High titre recombinant baculoviruses were generated using Bac-to-Bac Baculovirus Expression System. In brief, 2 μg of recombinant bacmid and 2 μl X-tremGENE HP transfection reagent (Roche) in 100 μl Opti-MEM medium (Gibco) were mixed and incubated for 20 min at room temperature. The transfection solution was added to 2.5 ml Sf9 cells with a density of 1 × 106 per ml in a 24-well plate. The infected cells were cultured in a shaker at 27 °C for 4 days. P0 virus was collected and then amplified to generate P1 virus. The viral titres were determined by flow cytometric analysis of cells stained with gp64-PE antibody (1:200 dilution; 12-6991-82, Thermo Fisher). Then, Sf9 cells were infected with viruses encoding GPR97-FL-AA, miniGαo, Gβγ, and with or without scFv16, respectively, at equal multiplicity of infection. The infected cells were cultured at 27 °C, 110 rpm for 48 h before collection. Cells were finally collected by centrifugation and the cell pellets were stored at −80 °C.
GPR97–Go complex formation and purification
Cell pellets transfected with virus encompassing the GPR97-FL-AA, miniGo trimer and scFv16 (only existed in cell pellets for purifying the cortisol–GPR97-FL-AA–Go–scFv16 complex) were resuspended in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 10 mM MgCl2 and 5 mM CaCl2 supplemented with Protease Inhibitor Cocktail (B14001, Bimake) and 100 μM TCEP (Thermo Fisher Scientific). The complex was formed for 2 h at room temperature by adding 10 μM BCM (HY-B1540, MedChemExpress) or cortisol (HY-N0583, MedChemExpress), 25 mU/ml apyrase (Sigma), and then solubilized by 0.5% (w/v) lauryl maltose neopentylglycol (LMNG; Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS; Anatrace) for 2 h at 4 °C. Supernatant was collected by centrifugation at 30,000 rpm for 40 min, and the solubilized complex was incubated with nickel resin for 2 h at 4 °C. The resin was collected and washed with 20 column volumes of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 25 mM imidazole, 0.01% (w/v) LMNG, 0.01% GDN (Anatrace), 0.004% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The complex was eluted with 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 200 mM imidazole, 0.01% (w/v) LMNG, 0.01% GDN, 0.004% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The elution of nickel resin was applied to M1 anti-Flag resin (Sigma) for 2 h and washed with 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 5 mM CaCl2, 0.01% (w/v) LMNG, 0.01% GDN, 0.004% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The GPR97–Go complex was eluted in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 0.01% (w/v) LMNG, 0.01% GDN, 0.004% (w/v) CHS, 10 μM BCM (or cortisol), 100 μM TCEP, 5 mM EGTA and 0.2 mg/ml Flag peptide. The complex was concentrated and then injected onto Superdex 200 increase 10/300 GL column equilibrated in the buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.00075% (w/v) LMNG, 0.00025% GDN, 0.0002% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The complex fractions were collected and concentrated individually for EM experiments.
Cryo-EM grid preparation and data collection
For the preparation of cryo-EM grids, 3 μl of purified BCM-bound and cortisol-bound GPR97–Go complex at approximately 20 mg/ml was applied onto a glow-discharged holey carbon grid (Quantifoil R1.2/1.3). Grids were plunge-frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM imaging was performed on a Titan Krios at 300 kV accelerating voltage in the Center of Cryo-Electron Microscopy, Zhejiang University. Micrographs were recorded using a Gatan K2 Summit direct electron detector in counting mode with a nominal magnification of ×29,000, which corresponds to a pixel size of 1.014 Å. Movies were obtained using serialEM at a dose rate of about 7.8 electrons per Å2 per second with a defocus ranging from −0.5 to −2.5 μm. The total exposure time was 8 s and intermediate frames were recorded in 0.2-s intervals, resulting in an accumulated dose of 62 electrons per Å2 and a total of 40 frames per micrograph. A total of 2,707 and 5,871 movies were collected for the BCM-bound and cortisol-bound GPR97–Go complex, respectively.
Cryo-EM data processing
Dose-fractionated image stacks for the BCM–GPR97–Go complex were subjected to beam-induced motion correction using MotionCor2.144. Contrast transfer function (CTF) parameters for each non-dose-weighted micrograph were determined by Gctf45. Particle selection, 2D and 3D classifications of the BCM–GPR97–Go complex were performed on a binned data set with a pixel size of 2.028 Å using RELION-3.0-beta246.
For the BCM–GPR97–Go complex, semi-automated particle selection yielded 2,026,926 particle projections. The projections were subjected to reference-free 2D classification to discard particles in poorly defined classes, producing 911,519 particle projections for further processing. The map of the 5-HT1BR–miniGo complex (EMDB-4358)47 low-pass filtered to 40 Å was used as a reference model for maximum-likelihood-based 3D classification, resulting in one well-defined subset with 307,700 projections. Further 3D classifications focusing the alignment on the complex produced two good subsets that accounted for 166,116 particles, which were subsequently subjected to 3D refinement, CTF refinement and Bayesian polishing. The final refinement generated a map with an indicated global resolution of 3.1 Å at a Fourier shell correlation of 0.143.
For the cortisol–GPR97–Go complex, particle selection yielded 4,323,518 particle projections for reference-free 2D classification. The well-defined classes with 2,201,933 particle projections were selected for a further two rounds of 3D classification using the map of the BCM-bound complex as reference. One good subset that accounted for 335,552 particle projections was selected for a further two rounds of 3D classifications that focused the alignment on the complex, and produced one high-quality subset with 75,814 particle projections. The final particle projections were subsequently subjected to 3D refinement, CTF refinement and Bayesian polishing, which generates a map with a global resolution of 2.9 Å. Local resolution for both density maps was determined using the Bsoft package with half maps as input maps48.
Model building and refinement
For the structure of the BCM–GPR97–Go complex, the initial template of GPR97 was generated using the module ‘map to model’ in PHENIX44. The coordinate of the 5-HT1BR–Go complex (PDB ID: 6G79) was used to generate the initial models for Go (ref. 44). Models were docked into the EM density map using UCSF Chimera49, followed by iterative manual rebuilding in COOT50 according to side-chain densities. BCM and lipid coordinates and geometry restraints were generated using phenix.elbow. BCM was built to the model using the ‘LigandFit’ module in PHENIX. The placement of BCM shows a correlation coefficient of 0.81, indicating a good ligand fit to the density. The model was further subjected to real-space refinement using Rosetta51 and PHENIX44.
For the structure of the cortisol–GPR97–Go complex, the coordinates of GPR97 and Go from the BCM-bound complex and scFv16 from the human NTSR1–Gi1 complex (PDB ID: 6OS9) were used as initial model. Models were docked into the density map and then were manual rebuilt in COOT. The agonist cortisol was built to the model using the ‘LigandFit’ module as described, showing a good density fit with a correlation coefficient of 0.80. The model was further refined using Rosetta51 and PHENIX44. The final refinement statistics for both structures were validated using the module ‘comprehensive validation (cryo-EM)’ in PHENIX44. The goodness of the fit of the model to the map was performed for both structures using a global model-versus-map FSC (Extended Data Fig. 2). The refinement statistics are provided in Extended Data Table 1. Figures of the structures were generated using UCSF Chimera, UCSF ChimeraX52 and PyMOL53.
Molecular dynamics simulation of the BCM–GPR97 and cortisol–GPR97 complexes
On the basis of the favour binding poses of BCM and cortisol with the receptor GPR97, which was calculated by the LigandFit program of PHENIX, the GPR97–agonist complexes were substrate from the two GPR97–agonist–mGo complexes for molecular dynamics simulation. The orientations of receptors were calculated by the Orientations of Proteins in Membranes (OPM) database. Following this, the whole systems were prepared by the CHARM-GUI and embedded in a bilayer that consisted of 200 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids by replacement methods. The membrane systems were then solvated into a periodic TIP3P water box supplemented with 0.15 M NaCl. The CHARMM36m Force Filed was used to model protein molecules, CHARMM36 Force Filed for lipids and salt with CHARMM General Force Field (CGenFF) for the agonist molecules BCM and cortisol.
Then, the system was subjected to minimization for 10,000 steps using the conjugated gradient algorithm and then heated and equilibrated at 310.13 K and 1 atm for 200 ps with 10.0 kcal mol−1 Å−2 harmonic restraints in the NAMD 2.13 software. Next followed five cycles of equilibration for 2 ns each at 310.13 K and 1 atm, for which the harmonic restraints were 5.0, 2.5, 1.0, 0.5 and 0.1 kcal mol−1 Å−2 in sequence.
Production simulations were run at 310.13 K and 1 atm in the NPT ensemble using the Langevin thermostat and Nose–Hoover method for 200 ns. Electrostatic interactions were calculated using the particle mesh Ewald (PME) method with a cut-off of 12 Å. Throughout the final stages of equilibration and production, 5.0 kcal mol−1 Å−2 harmonic restraints were placed on the residues of GPR97 that were within 5 Å of Go in the BCM (or cortisol)–GPR97–Go complex to ensure that the receptor remained in the active state in the absence of the G protein. Trajectories were visualized and analysed using Visual Molecular Dynamics (VMD, version 1.9.3)
cAMP ELISA detection in Y-1 cells
Y-1 cells were transfected with Gpr97 siRNA (si-97, GUGCAGGGAAUGUCUUUAA) or control siRNA (si-Con) for 48 h. After starvation for 12 h in serum-free medium, the cells were further stimulated with cortisone (8 nM), forskolin (5 μM) (Sigma-Aldrich) or control vehicle for 10 min. Then, cells were washed three times with pre-cooled PBS and resuspended in pre-cooled 0.1 N HCl containing 500 μM IBMX at a 1:5 ratio (w/v). The samples were neutralized with 1 N NaOH at a 1:10 ratio (v/v) after 10 min. The supernatants were collected after centrifugation of the samples at 600g for 10 min. The supernatants were then prepared for cAMP determination using the cAMP Parameter Assay Kit (R&D Systems) according to the manufacturer’s instruction. The Gpr97 expression level under various conditions were further confirmed using quantitative real-time PCR.
Mouse adrenocorticotoma cell line Y-1 cells were transfected with Gpr97 siRNA (si-97) or control siRNA (si-Con) for 48 h. Then, the cells were treated with serum-free medium for 12 h. After that, cortisone (16 nM) or ACTH (0.5 μM) were added to cells for 30 min. The supernatants of the cell culture medium were collected for measurements of corticosterone by ELISA according to the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA of cells was extracted using a standard TRIzol RNA isolation method. The reverse transcription and PCR experiments were performed with the Revertra Ace qPCR RT Kit (TOYOBO FSQ-101) using 1.0 μg of each sample, according to the manufacturer’s protocols. The quantitative real-time PCR was conducted in the Light Cycler apparatus (Bio-Rad) using the FastStart Universal SYBR Green Master (Roche). The mRNA level was normalized to GAPDH in the same sample and then compared with the control. The forward and reverse primers for GPR97 used in the experiments were CAGTTTGGGACTGAGGGACC and GCCCACACTTGGTGAAACAC. The mRNA level of GAPDH was used as an internal control. The forward and reverse primers for GAPDH were GCCTTCCGTGTTCCTACC and GCCTGCTTCACCACCTTC.
cAMP inhibition assay
To measure the inhibitory effects on forskolin-induced cAMP accumulation of different GPR97 constructs or mutants in response to different ligands or constitutive activity, the GloSensor cAMP assay (Promega) was performed according to previous publications12,13. HEK293 cells were transiently co-transfected with the GloSensor and various versions of GPR97 or vehicle (pcDNA3.1) plasmids using PEI in six-well plates. After incubation at 37 °C for 24 h, transfected cells were seeded into 96-well plates with serum-free DMEM medium (Gibco) and incubated for another 24 h at 37 °C in a 5% CO2 atmosphere. Different ligands were dissolved in DMSO (Sigma) to a stock concentration of 10 mM and followed by serial dilution using PBS solution immediately before the ligand stimulation. The transfected cells were pre-incubated with 50 μl of serum-free DMEM medium containing GloSensor cAMP reagent (Promega). After incubation at 37 °C for 2 h, varying concentrations of ligands were added into each well and followed by the addition of forskolin to 1 μM. The luminescence intensity was examined on an EnVision multi-label microplate detector (Perkin Elmer).
The Gqo protein activation BRET assay
According to previous publications, the BCM dipropionate-induced GPR97 activity could be measured by chimeric Gqo protein assays25. The Gqo BRET probes were generated by replacing the six amino acids of the C-terminal of Gq-RlucII with those from GoA1, creating a chimeric Gqo-RlucII subunit47. GFP10 was connected to Gγ. The Gqo protein activation BRET assay was performed as previously described54. In brief, HEK293 cells were transiently co-transfected with control D2R and various GPR97 constructs, plasmids encoding the Gqo BRET probes, incubated at 37 °C in a 5% CO2 atmosphere for 48 h. Cells were washed twice with PBS, collected and resuspended in buffer containing 25 mM HEPES, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mM NaHCO3, 5.6 mM d-glucose, 0.5 mM MgCl2 and 0.37 mM NaH2PO4. Cells that were dispensed into a 96-well microplate at a density of 5–8 × 104 cells per well were stimulated with different concentrations of ligands. BRET2 between RLucII and GFP10 was measured after the addition of the substrate coelenterazine 400a (5 μM, Interchim) (Cayman) using a Mithras LB940 multimode reader (Berthold Technologies). The BRET2 signal was calculated as the ratio of emission of GFP10 (510 nm) to RLucII (400 nm).
Measurement of receptor cell-surface expression by ELISA
To evaluate the expression level of wild-type GPR97 and its mutants, HEK293 cells were transiently transfected with wild-type and mutant GPR97 or vehicle (pcDNA3.1) using PEI regent at in six-well plates. After incubation at 37 °C for 18 h, transfected cells were plated into 24-well plates at a density of 105 cells per well and further incubated at 37 °C in a 5% CO2 atmosphere for 18 h. Cells were then fixed in 4% (w/v) paraformaldehyde and blocked with 5% (w/v) BSA at room temperature. Each well was incubated with 200 μl of monoclonal anti-FLAG (F1804, Sigma-Aldrich) primary antibody overnight at 4 °C and followed by incubation of a secondary goat anti-mouse antibody (A-21235, Thermo Fisher) conjugated to horseradish peroxide for 1 h at room temperature. After washing, 200 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added. Reactions were quenched by adding an equal volume of 0.25 M HCl solution and the optical density at 450 nm was measured using the TECAN (Infinite M200 Pro NanoQuant) luminescence counter. For determination of the constitutive activities of different GPR97 constructs or mutants, varying concentrations of desired plasmids were transiently transfected into HEK293 cells and the absorbance at 450 nm was measured.
The FlAsH-BRET assay
HEK293 cells were seeded in six-well plates after transfection with GPR97-FlAsH with Nluc inserted in a specific N-terminal site. Before the BRET assay, HEK293 cells were starved with serum for 1 h. Then cells were digested, centrifuged and resuspended in 500 μl BRET buffer (25 mM HEPES, 1 mM CaCl2, 140 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 0.37 mM NaH2PO4, 5.5 mM d-glucose and 12 mM NaHCO3). The FlAsH-EDT2 was added at a final concentration of 2.5 μM and incubated at 37 °C for 60 min. Subsequently, HEK293 cells were washed with BRET buffer and then distributed into black-wall clear-bottom 96-well plates, with approximately 100,000 cells per well. The cells were treated with a final concentration of BCM and cortisol at 10−5 to 10−11 and then coelenterazinc H was added at a final concentration of 5 μM, followed by checking the luciferase (440–480 nm) and FlAsH (525–585 nm) emissions immediately. The BRET ratio (emission enhanced yellow fluorescent protein/emission Nluc) was calculated using a Berthold Technologies Tristar 3 LB 941 spectrofluorimeter. The procedure was modified from those described previously34,55,56.
A one-way ANOVA test was performed to evaluate the statistical significance between various versions of GPR97 and their mutant in terms of expression level, potency or efficacy using GraphPad Prism. For all experiments, the standard error of the mean of the values calculated based on the data sets from three independent experiments is shown in respective figure legends.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
A ‘Build and Retrieve’ methodology to simultaneously solve cryo-EM structures of membrane proteins
Vinothkumar, K. R. & Henderson, R. Single particle electron cryomicroscopy: trends, issues and future perspective. Q. Rev. Biophys. 49, 1–25 (2016).
Herzik, M. A.Jr, Wu, M. & Lander, G. C. High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Nat. Commun. 10, 1032 (2019).
Ho, C. M. et al. Malaria parasite translocon structure and mechanism of effector export. Nature 561, 70–75 (2018).
Morgan, C. E. et al. Cryo-electron microscopy structure of the Acinetobacter baumannii 70S ribosome and implications for new antibiotic development. mBio 11, e03117–e03119 (2020).
Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Daligault, H. E. et al. Whole-genome assemblies of 56 Burkholderia species. Genome Announc. 2, e01106–e01114 (2014).
Doughty, D. M. et al. The RND-family transporter, HpnN, is required for hopanoid localization to the outer membrane of Rhodopseudomonas palustris TIE-1. Proc. Natl Acad. Sci. U S A 108, E1045–E1051 (2011).
Sousa, F. L. et al. The superfamily of heme-copper oxygen reductases: types and evolutionary considerations. Biochim. Biophys. Acta 1817, 629–637 (2012).
Abramson, J. et al. The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site. Nat. Struct. Biol. 7, 910–917 (2000).
Yap, L. L. et al. The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli. Biochim. Biophys. Acta 1797, 1924–1932 (2010).
Choi, S. K. et al. Location of the substrate binding site of the cytochrome bo3 ubiquinol oxidase from Escherichia coli. J. Am. Chem. Soc. 139, 8346–8354 (2017).
Kumar, N. et al. Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN. Proc. Natl Acad. Sci. U S A 114, 6557–6562 (2017).
Centers for Disease Control and Prevention. Bioterrorism agents/diseases (U.S. Department of Health and Human Services, 2018); https://emergency.cdc.gov/agent/agentlist-category.asp
Wagar, E. Bioterrorism and the role of the clinical microbiology laboratory. Clin. Microbiol. Rev. 29, 175–189 (2016).
Christopher, G. W., Cieslak, T. J., Pavlin, J. A. & Eitzen, E. M.Jr Biological warfare. A historical perspective. JAMA 278, 412–417 (1997).
Nierman, W. C. et al. Structural flexibility in the Burkholderia mallei genome. Proc. Natl Acad. Sci. U S A 101, 14246–14251 (2004).
Schweizer, H. P. Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis. Future Microbiol. 7, 1389–1399 (2012).
Malott, R. J., Steen-Kinnaird, B. R., Lee, T. D. & Speert, D. P. Identification of hopanoid biosynthesis genes involved in polymyxin resistance in Burkholderia multivorans. Antimicrob. Agents Chemother. 56, 464–471 (2012).
Malott, R. J. et al. Fosmidomycin decreases membrane hopanoids and potentiates the effects of colistin on Burkholderia multivorans clinical isolates. Antimicrob. Agents Chemother. 58, 5211–5219 (2014).
Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).
Cowan, S. W. et al. Crystal structures explain functional properties of two E. coli porins. Nature 358, 727–733 (1992).
Yankovskaya, V. et al. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science 299, 700–704 (2003).
Baslé, A., Rummel, G., Storici, P., Rosenbusch, J. P. & Schirmer, T. Crystal structure of osmoporin OmpC from E. coli at 2.0 Å. J. Mol. Biol. 362, 933–942 (2006).
Carpena, X., Melik-Adamyan, W., Loewen, P. C. & Fita, I. Structure of the C-terminal domain of the catalase–peroxidase KatG from Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 60, 1824–1832 (2004).
Capitani, G. et al. Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase. EMBO J. 22, 4027–4037 (2003).
Chorev, D. S. et al. Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry. Science 362, 829–834 (2018).
Ho, C. M. et al. Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu. Nat. Methods 17, 79–85 (2020).
Kastritis, P. L. et al. Capturing protein communities by structural proteomics in a thermophilic eukaryote. Mol. Syst. Biol. 13, 936 (2017).
Yi, X., Verbeke, E. J., Chang, Y., Dickinson, D. J. & Taylor, D. W. Electron microscopy snapshots of single particles from single cells. J. Biol. Chem. 294, 1602–1608 (2019).
Schmidli, C. et al. Microfluidic protein isolation and sample preparation for high-resolution cryo-EM. Proc. Natl Acad. Sci. U S A 116, 15007–15012 (2019).
Long, F. et al. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Nature 467, 484–488 (2010).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Terwilliger, T. C., Ludtke, S. J., Read, R. J., Adams, P. D. & Afonine, P. V. Improvement of cryo-EM maps by density modification. Nat. Methods 17, 923–927 (2020).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).
Glycoproteomics is coming of age, thanks to advances in instrumentation, experimental methodologies and computational search algorithms.
Glycosylation is one of the most common post-translational modifications, and glycoproteins play crucial roles in important biological processes like cell signaling, host–pathogen interaction, immune response and disease, including cancer and even the ongoing COVID-19 pandemic (Science 369, 330–333, 2020). Glycoproteomics aims to determine the positions and identities of the complete repertoire of glycans and glycosylated proteins in a given cell or tissue.
Glycans are everywhere. High-throughput glycoproteomics approaches offer insights. Credit: Katherine Vicari, Springer Nature
Mass spectrometry (MS)-based approaches allow large-scale global analysis; however, the structural diversity of glycans and the heterogeneous nature of glycosylation sites make comprehensive analysis particularly challenging. Glycans obstruct complete fragmentation of the protein backbone, and they were traditionally removed for simplicity at the cost of losing glycan information. The MS spectra tend to be complicated due to the presence of isomers, often requiring manual interpretation. Furthermore, database searching for spectral matches can quickly become a combinatorial problem and requires innovative bioinformatics solutions.
Recent developments in MS instrumentation, fragmentation strategies (J. Proteome Res. 19, 3286–3301, 2020) and high-throughput workflows have made analyzing intact glycoproteins a possibility. Several specific enrichment strategies have made even low-abundance glycans and glycopeptides detectable (Mol. Cell. Proteomics https://doi.org/10.1074/mcp.R120.002277, 2020). A variety of experimental workflows tailored for either N-linked glycans, which are found at consensus sites on the proteins, or O-linked glycans, which have no recognizable consensus sequence, have been developed (Nature 549, 538–542, 2017; Nat. Commun. 11, 5268, 2020; Nat. Methods 16, 902–910, 2019). New software packages based on fragment-ion indexing strategies offer substantial increases in speed for glycopeptide and site assignments (Nat. Methods 17, 1125–1132, 2020; Nat. Methods 17, 1133–1138, 2020).
With other -omics fields taking the lion’s share of attention in recent years, it is now time for glycoproteomics to shine. Comprehensive understanding of glycosylation at different levels of granularity is bound to serve both basic and translational research.
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Cite this article
Singh, A. Glycoproteomics. Nat Methods 18, 28 (2021). https://doi.org/10.1038/s41592-020-01028-9
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