Main

In the simplest model for the conformational dynamics of GPCRs10 there is an equilibrium between two states, R and R*. The inactive state R preferentially binds inverse agonists and the activated state R* preferentially binds agonists11. Only R* can couple and activate G proteins. Although there are far more complex schemes12 describing intermediates between R and R*, studies on rhodopsin have indicated that there is only one major conformational change that significantly alters the structure of the receptor3. Thus the structures of dark-state rhodopsin13,14 and of opsin15,16 are considered to be representative structures for the R and R* state, respectively. Structures of six different GPCRs8,13,17,18,19,20,21 in conformations closely approximating the R state have now been determined and it is clear that they are similar to each other, with root mean squared deviation (r.m.s.d.) between any pair of structures in the transmembrane domains being less than 3 Å. As observed in light activation of rhodopsin, the major structural difference between R and R* is the movement of the cytoplasmic ends of helices 5 and 6 away from the receptor core by 5–6 Å, opening up a cleft in the centre of the helix bundle where the carboxy terminus of a G protein can bind16. Recently, the structure of an agonist-bound β-adrenoceptor (β2-AR) was determined in complex with an antibody fragment (nanobody Nb80)5. This structure of β2-AR is very similar to the structure of opsin, which indicates that the nanobody mimicked the action of a G protein by maintaining the receptor structure in an activated state. Given the structural similarities between opsin and the β2-AR–Nb80 complex, it is likely that the structures of the R* states of other GPCRs are also highly similar. This is consistent with the same heterotrimeric G proteins being able to couple to multiple different receptors22. However, it is unclear whether conserved structures of R and R* indicate that all agonists activate the receptors in an identical fashion. The recent structures of a thermostabilized β1-AR bound to four different agonists indicated that a defining feature of agonist binding to this receptor is the formation of a hydrogen bond with Ser5.46 on transmembrane helix 5 that accompanies the contraction of the ligand-binding pocket7. Here we describe two structures of the adenosine A2A receptor (A2AR) bound to two different agonists, which indicates that the initial action of agonist binding to A2AR has both similarities and differences compared to agonist binding in β-ARs.

The native human A2AR when bound to its endogenous agonist adenosine or to the high-affinity synthetic agonist NECA is unstable in detergent, so crystallization and structure determination relied on using a thermostabilized construct (A2AR-GL31) that contained four point mutations, which markedly improved its thermostability. Pharmacological analysis showed that the mutant receptor bound the five antagonists tested with greatly reduced affinity (1.8–4.3 log units), whereas four agonists bound with similar affinity to the wild-type receptor (Supplementary Fig. 1). However, A2AR-GL31 is only weakly activated by the agonist CGS21680 (Supplementary Fig. 2), which indicates that the thermostabilizing mutations might also decouple high-affinity agonist binding from the formation of R*. The conformation of A2AR-GL31 is not consistent with it being in the fully activated G-protein-coupled state, because we do not observe a 42-fold increase in affinity for NECA binding measured for Gαs-coupled A2AR (ref. 23). These data all indicate that A2AR-GL31 is in an intermediate conformation between R and R*, which is consistent with the structural analysis presented later.

The two structures we have determined are of A2AR-GL31 bound to adenosine and NECA with resolutions of 3.0 Å and 2.6 Å, respectively (Supplementary Table 1). Global alignments of the A2AR-GL31 structures with A2A-T4L (A2AR with T4 lysozyme inserted into inner loop 3) bound to the inverse agonist ZM241385 were performed based on those residues in the region of the ligand-binding pocket that show the closest structural homology (Fig. 1 and Supplementary Text). This gave an r.m.s.d. in Cα positions of 0.66 Å for the 96 atoms selected, which include all residues involved in binding either adenosine or NECA, with the exception of those in H3. Using this transformation, the adenine-like moiety of the two ligands superimposes almost exactly (r.m.s.d. 0.56 Å). The most significant differences between the two structures are seen in a distortion and a 2 Å shift primarily along the helical axis of H3, a bulge in H5 (resulting from non-helical backbone conformation angles of residues Cys 185 and Val 186) that shifts residues into the binding pocket by up to 2 Å and also a change in conformation of the cytoplasmic ends of H5, H6 and H7 (Fig. 1). Comparison of the A2AR-GL31 structure with the agonist-bound β2-AR–Nb80 complex indicates that these differences are similar to the conformational changes in the β2-AR that are proposed to be responsible for the formation of the R* state5. However, it is unlikely that the structure of A2AR-GL31 represents the fully activated state, because comparison with opsin bound to the C-terminal peptide of the G protein transducin shows that there is insufficient space in A2AR-GL31 for the C terminus of the G protein to bind (Supplementary Fig. 3). This is on the basis of the assumption that all G proteins bind and activate GPCRs in a similar fashion, but given the highly conserved structures of both G proteins and GPCRs this seems a reasonable hypothesis.

Figure 1: Structure of the adenosine A 2A receptor bound to NECA compared to other GPCR structures.
figure 1

a, The structure of NECA-bound A2AR is shown as a cartoon (yellow) aligned with the structure of A2A-T4L bound to the inverse agonist ZM241385 (blue; PDB code 3EML8). NECA is shown as a space-filling model (C, green; N, blue; O, red). b, c, Sections through the aligned receptors in a that highlight the differences in the intracellular face of the receptors (b) and in the ligand-binding pocket (c), with the bulge in H5 shown as an inset. d, e, Alignment of NECA-bound A2AR (yellow) with agonist-bound β2-AR–Nb80 (red; PDB code 3P0G5) showing the intracellular face of the receptors (d) and the ligand-binding pocket (e). NECA is shown as a space-filling model in c and e. The figures were generated using CCP4mg31. Analogous alignments to opsin are depicted in Supplementary Fig. 7.

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The fact that the structure of A2AR-GL31 represents an agonist-binding state is consistent with how A2AR-GL31 was engineered. Thermostabilizing mutations were selected by heating the NECA-bound detergent-solubilised receptor, so the mutations are anticipated to stabilize the agonist-bound state either by stabilizing helix–helix interactions and/or biasing the conformational equilibrium between the agonist-bound R* state and the agonist-bound R state24,25,26. The two most thermostabilizing mutations, L48A and Q89A, are in regions of the receptor that are involved in transitions between R and R*, providing a possible explanation for their thermostabilizing effect (Supplementary Fig. 4). The other two mutations, A54L and T65A, are at the receptor–lipid interface and the reason for their thermostabilizing effect is unclear. Although the overall shape of the ligand-binding pockets of A2AR and β2AR are different, the structural similarities with the β2-AR–Nb80 (ref. 5) and the structural differences to ZM241385-bound A2A-T4L8 indicate that the structure of the binding pocket in A2AR-GL31 is a good representation of the agonist-bound binding pocket of the wild-type receptor (Fig. 1).

Adenosine and NECA bind to A2AR-GL31 in a virtually identical fashion; in addition, the adenine ring in the agonists interacts with A2AR in a similar way to the chemically related triazolotriazine ring of the inverse agonist ZM241385 (Fig. 2). Thus the hydrogen bonds between exocyclic adenosine N6 (Supplementary Fig. 5) with both Glu 169 in extracellular loop 2 (EL2) and Asn 2536.55 in H6 are similar, with the significant π-stacking interaction with Phe 168 in EL2 also conserved. One of the major structural differences between ZM241385 and the agonists is the presence of a furan substituent on C20 of triazolotriazine in the inverse agonist, whereas agonists contain a ribose substituent linked to N9 of adenine (Fig. 2 and Supplementary Fig. 5). In ZM241385, the furan group forms a hydrogen bond with Asn 2536.55 in H6 and van der Waals contacts with other residues in H3, H5 and H6 (ref. 8). In contrast, the ribose moiety in agonists forms hydrogen bonds with Ser 2777.42 and His 2787.43 in H7, in addition to van der Waals interactions with other residues in H3 and H6 (Fig. 2). In particular, Val 843.32 has to shift its position upon agonist binding owing to a steric clash with the ribose ring, which may contribute to the 2 Å shift observed in H3 (Fig. 3). These differences in binding between ZM241385 and either adenosine or NECA indicate that the residues that bind uniquely to agonists (Ser 2777.42 and His 2787.43) have a key role in the activation of the receptor, as previously shown by mutagenesis studies27,28. This is analogous to the situation in the activation of β1-AR, where only full agonists cause the rotamer conformation changes of Ser5.46 in H5, whereas the inverse agonist ICI118551 prevents receptor activation by sterically blocking the rotamer change7,29. However, the details of the activation differ in that the critical residues that bind agonists and not antagonists are in H5 in the β1-AR, but in H7 in the A2AR (Fig. 4).

Figure 2: Comparison of receptor–ligand interactions for A 2A R bound to the inverse agonist ZM241385 and the agonists NECA and adenosine.
figure 2

a–c, Structures of the human A2AR in cartoon representation are shown bound to the following ligands: a, ZM241385 (PDB code 3EML8); b, NECA; and c, adenosine. d, e, Polar and non-polar interactions involved in agonist binding to A2AR are shown for NECA (d) and adenosine (e). Amino acid residues within 3.9 Å of the ligands are depicted, with residues highlighted in blue making van der Waals contacts (blue rays) and residues highlighted in red making potential hydrogen bonds with favourable geometry (red dashed lines, as identified by HBPLUS, see Methods) or hydrogen bonds with unfavourable geometry (blue dashed lines, donor acceptor distance more than 3.6 Å). Where the amino acid residue differs between the human A2AR and the human A1R, A2BR and A3R, the equivalent residue is shown highlighted in orange, purple or green, respectively. Panels a–c were generated using PyMOL (http://www.pymol.org/). Omit densities for the ligands are shown in Supplementary Fig. 6 and densities for water molecules in Supplementary Fig. 8.

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Figure 3: Positions of adenosine and ZM241385 in the A 2A R ligand-binding pocket.
figure 3

The structures of adenosine-bound A2AR-GL31 and ZM241385-bound A2A-T4L were aligned using only atoms from the protein to allow the ligand positions to be compared, with adenosine in yellow and ZM241385 in pink (N, blue; O, red). The ligands are shown in the context of the binding pocket of A2AR-GL31, with transmembrane helices of A2AR-GL31 shown in yellow and the surfaces of the receptor, including the cavity of the ligand binding pocket, shown in grey. The side chains of Val 84 and Leu 85 that interact with the ribose moiety of the agonist are shown in green.

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Figure 4: Comparison of the positions of agonists in the binding pockets of the A 2A R and β 1 -AR.
figure 4

a, The structures of the A2AR bound to adenosine and the β1-AR bound to isoprenaline (PDB code 2Y03)7 were aligned by superimposing equivalent atoms in the protein structure and the positions of both ligands are shown as stick models with the carbon atoms in blue/green (isoprenaline) or yellow (adenosine); N, blue; O, red. The A2AR structure is shown, with H5 and H7 as space-filling models (C, grey; N, blue; O, red) and the remainder of the structure as a cartoon (pale green). Some water molecules are shown as red spheres, hydrogen bonds as red dashed lines and the polar contacts as blue dashed lines. The orientation of the figure is identical to that shown in Fig. 2. b, Structure of the A2AR bound to adenosine viewed from the extracellular surface. c, Structure of β1-AR bound to isoprenaline (PDB code 2Y03)7 viewed from the extracellular surface. In panels b and c, equivalent side chains in the respective structures that make contacts to both isoprenaline and adenosine in their respective receptors are shown as space-filling models and they have the following Ballesteros–Weinstein numbers (amino acid side chains are shown in parentheses for the A2AR and β1-AR, respectively): 3.32 (V84, D121); 3.36 (T88, V125); 5.42 (N181*, S211); 6.51 (L249, F306); 6.55 (N253, N310); 6.52 (H250*, F307); 7.39 (I274, N329); 7.43 (H278, Y333). An asterisk indicates residues that only make indirect contacts to the agonists via a water molecule.

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Adenosine and NECA activate the A2AR through interactions with H3 and H7 that are absent in the interactions between the receptor and the inverse agonist ZM241385 (Fig. 2). The inward shift of H7, the movement of H3 and the consequent formation of a bulge in H5 are all observed in the structures of agonist-bound A2AR-GL31 and β2-AR–Nb80 (Fig. 1). The formation of the bulge in H5 of the β2-AR–Nb80 structure was linked to a series of conformational changes that generate the 60° rotation of H6 about Phe 2826.44, resulting in the cytoplasmic end of H6 moving out from the receptor centre and opening the cleft where the C terminus of a G protein is predicted to bind as observed in opsin5,6. There are analogous side-chain movements in A2AR-GL31 that result in a 40° rotation of H6, but the cytoplasmic end of H6 remains partially occluding the G-protein-binding cleft (Supplementary Fig. 3), perhaps because the fully active conformation requires the binding of G proteins to stabilize it. Interestingly, the structure of β2-AR6 with a covalently bound agonist is also not in the fully activated R* conformation, which is only seen after the nanobody Nb80 is bound5. The importance of the bulge in H5 in the activation of A2AR is highlighted by how inverse agonists bind. Formation of the H5 bulge results in the inward movement of Cys 1855.46 (Cβ moves by 4 Å), which in turn causes the movement of Val 186 and ultimately a shift of His 2506.52 by 2 Å into the ligand-binding pocket, thereby sterically blocking the binding of ZM241385 (Supplementary Fig. 4). Hence, when the inverse agonist binds, it is anticipated that the H5 bulge is unlikely to form owing to the opposite series of events and hence the formation of the R* state is inhibited.

Thus, in both β-ARs and A2AR, the formation of the Η5 bulge seems to be a common action of agonists, whereas inverse agonists seem to prevent its formation. However, the energetic contributions to its formation may be different between the two receptors. In β-ARs there is a major contribution from direct interaction between the agonist and Ser5.46, whereas in A2ARs, the major interaction seems to come from interactions between the agonist and H3, combined with polar interactions involving residues in H7. Despite these differences, agonist binding to both receptors involves strong attractive non-covalent interactions that pull the extracellular ends of H3, H5 and H7 together, which is the necessary prerequisite to receptor activation.

While this manuscript was in review, a related manuscript appeared30, describing the structure of the A2A-T4L chimaera bound to the agonist UK432097, which is identical to NECA except for two large substituents on the adenine ring. The structure of UK432097-bound A2A-T4L is very similar to the structures presented here in the transmembrane regions (r.m.s.d. 0.6 Å), although there are differences in the extracellular surface due to the bulky extensions of UK432097 interacting with the extracellular loops and the absence of density for residues 149–157. Xu et al.30 conclude that the structure of UK432097-bound A2A-T4L is in an “active state configuration”, whereas we conclude that the NECA- and adenosine-bound structures are best defined as representing an intermediate state between R and R*.

Methods Summary

Expression, purification and crystallization

The thermostabilized A2AR-GL31 construct contains amino acid residues 1–316 of the human A2AR, four thermostabilizing point mutations (L48A2.46, A54L2.52, T65A2.63 and Q89A3.37) and the mutation N154A to remove a potential N-glycosylation site. A2AR-GL31 was expressed in insect cells using the baculovirus expression system and purified in the detergent octylthioglucoside using Ni2+-NTA affinity chromatography and size exclusion chromatography (see Methods). The purified receptor was crystallized in the presence of cholesteryl hemisuccinate by vapour diffusion, under conditions described in Methods.

Data collection, structure solution and refinement

Diffraction data were collected in multiple wedges (20° per wedge) from a single cryo-cooled crystal (100 K) for the GL31–NECA complex at beamline ID23-2 at the European Synchrotron Radiation Facility and from four crystals for the GL31–adenosine complex, at beamline I24 at the Diamond Light Source. The structures were solved by molecular replacement using the ZM241385-bound A2A-T4L structure (PDB code 3EML)8 as a model (see Methods). Data collection and refinement statistics are presented in Supplementary Table 1 and omit densities for the ligands are shown in Supplementary Fig. 6.

Online Methods

Expression, purification and crystallization

The human A2A construct, GL31, contains four thermostabilizing point mutations (L48A2.46, A54L2.52, T65A2.63 and Q89A3.37), the mutation N154A to remove the potential N-glycosylation site and a truncation at the C terminus after Ala 316 (ref. 32). A polyhistidine tag (His10) was engineered at the C terminus, separated from the receptor by a TEV protease cleavage site. Baculovirus expression and membrane preparation were performed as described previously for the β1-AR33.

Membranes were thawed at room temperature (20–25 °C), diluted with 25 mM HEPES pH 7.4, in the presence of protease inhibitors (Complete; Boehringer). Membranes were pre-incubated with NECA at 100 μM for 45 min at room temperature. The receptor–ligand complexes were then solubilised by adding decylmaltoside (DM) and NaCl to give final concentrations of 1.5% and 0.3 M, respectively, stirred for 30 min (4 °C) and insoluble material removed by ultracentrifugation (120,000g, 45 min, 4 °C). All protein purification steps were performed at 4 °C. The solubilised receptor sample was filtered through a 0.22 μm filter (Millipore) and applied to a 5 ml Ni-NTA superflow cartridge (Qiagen) pre-equilibrated with buffer (25 mM HEPES, pH 7.4, 0.1 M NaCl, 100 μM NECA, 0.15% DM, 2.5 mM imidazole). The column was washed sequentially with the same buffer supplemented with either 10, 40 or 80 mM imidazole, and then eluted with 250 mM imidazole. The eluted receptor–ligand complex was mixed with His6-tagged TEV protease to cleave the tag for 4–6 h, 4 °C, concentrated to 2 ml using an Amicon-ultra spin concentrator (Ultracel-50K, Millipore) and then imidazole was removed using a PD-10 column (GE Healthcare). Eluted fractions were further purified by binding the TEV and other contaminants to Ni-NTA (QIAGEN) pre-equilibrated in 25 mM HEPES pH 7.4, 0.1 M NaCl, 100 μM NECA, 0.15% DM, 40 mM imidazole, incubating for 30 min and then collecting the flow-through. For detergent exchange into 0.35% octylthioglucoside (OTG), the sample was concentrated using an Amicon-ultra concentrator (Ultracel-50K, Millipore), diluted tenfold in 25 mM HEPES pH 7.4, 0.1 M NaCl, 100 μM NECA, 0.35% OTG, and concentrated again to 0.3 ml. The protein sample was applied to a Superdex 200 10/300 GL size-exclusion column pre-equilibrated in 25 mM HEPES pH 7.4, 0.1 M NaCl, 100 μM NECA, 0.35% OTG and run at 0.5 ml min−1. Eluted receptor fractions (2–2.5 ml) were concentrated to 50–60 μl. Protein determination was performed using the amido black34 assay.

Before crystallization, cholesteryl hemisuccinate (CHS) and OTG were added to 1 mg ml−1 and 0.5% respectively and the protein concentration adjusted to 10–12.5 mg ml−1. NECA and adenosine A2A-GL31 crystal hits were obtained using a new PEG-based crystallization screen developed in house35. Crystals were grown at 4 °C in 100 nl sitting drops using 0.05 M ADA NaOH, pH 6.4, 23.6% PEG 400, 4% v/v 2-propanol for the NECA complex. Crystals were cryoprotected by soaking in 0.05 M ADA NaOH, pH 6.4, 45% PEG 400. For the adenosine complex, crystals were initially grown in 0.05 M TrisHCl, pH 7.6, 9.6% PEG 200, 22.9%. PEG 300. Crystals were cryoprotected by soaking in 0.05 M TrisHCl, pH 7.5, 15% PEG 200, 30% PEG 300. The crystals were mounted on Hampton CrystalCap HT loops and cryo-cooled in liquid nitrogen.

Data collection, structure solution and refinement

Diffraction data for the NECA complex were collected at the European Synchrotron Radiation Facility with a Mar 225 CCD detector on the microfocus beamline ID23-2 (wavelength, 0.8726 Å) using a 10 μm focused beam and for the adenosine complex on beamline I24 at the Diamond Light Source with a Pilatus 6M detector and a 10 μm microfocus beam (wavelength 0.9778 Å). The microfocus beam was essential for the location of the best diffracting parts of single crystals, as well as allowing several wedges to be collected from different positions. Images were processed with MOSFLM36 and SCALA37. The NECA complex was solved by molecular replacement with PHASER38 using the A2A-T4L structure (PDB code 3EML)8 as a model after removal of the coordinates for T4L, all solvent molecules and the inverse agonist ZM241384. This structure was then used as a starting model for the structure solution of the adenosine complex. Refinement and rebuilding were carried out with REFMAC539 and COOT40, respectively. In the final models, 98.1% of residues were in the favoured region of the Ramachandran plot with one outlier for the NECA complex, and 97.7% with no outliers for the adenosine complex. Smile strings for NECA and adenosine were created using Sketcher and dictionary entries using Libcheck. Hydrogen bond assignments for the ligands were determined using HBPLUS41.

To facilitate a structural comparison between ZM241385-bound A2A-T4L and the thermostabilized A2A-GL31 with bound agonist, the structures were superimposed based on those residues in the region of the ligand-binding pocket that show the closest structural homology. This was achieved using the lsq_improve option of program O42 and an initial transformation based on residues at the C terminus of helix 6 and the N terminus of helix 7. The final superposition, based on residues 16–21 in H1, 51–70 in H2 and ECL1, 132–140 in H4 and ECL2, 142–146 in ECL2, 166–182 in ECL2 and H5 and 245–283 in H6, ECL3 and H7, gave an r.m.s.d. in Cα positions of 0.66 Å for the 96 atoms and includes almost all residues involved in binding either ligand with the exception of those in H3. Using this transformation, the adenine moiety of the agonist superimposes well with the equivalent atoms of the triazolotriazene bicyclic ring of ZM241385 (r.m.s.d. 0.56 Å). Validation of the final refined models was carried out using Molprobity43. Omit densities for the ligands are shown in Supplementary Fig. 6. All figures in the manuscript were generated using either Pymol (DeLano Scientific) or CCPmg31.

Binding of agonists and antagonist to A2AR-GL31 expressed in CHO cells

Chinese hamster ovary (CHO) cells were maintained in culture in DMEM HAMs F12 media containing 10% FBS. Cells were transfected with plasmids expressing either wild-type adenosine A2AR or A2AR-GL31 using GeneJuice according to manufacturer’s instructions (EMD Biosciences). Forty-eight hours after transfection, cells were harvested, centrifuged at 200g for 5 min at 4 °C and the pellet re-suspended in 20 mM HEPES, 10 mM EDTA buffer (pH 7.4). The membrane suspension was homogenized and centrifuged at 200g for 15 min at 4 °C. The supernatant was collected, the pellet re-suspended in 20 mM HEPES, 10 mM EDTA (pH 7.4) buffer and the solution homogenized and centrifuged as described previously44. The collected supernatant was centrifuged for 30 min at 40,000g at 4 °C. Pellets were re-suspended in 20 mM HEPES, 0.1 mM EDTA to a protein concentration of 1 mg ml−1 and stored at −80 °C .

Membranes from CHO cells transiently expressing wild-type or A2AR-GL31 (10–15 μg per well) were assessed using competition [3H]NECA binding in buffer containing 50 mM Tris-HCl (pH 7.4) as described previously44. Inhibition curves were fitted to a four-parameter logistic equation to determine IC50 values, which were converted to Ki values using Kd values determined by saturation binding and the [3H]NECA concentration of 10 nM.

G-protein-coupling activity of A2AR-GL31 measured in whole cells

A2AR–His6 and A2AR-GL31–His6 (amino acid residues 1–316 of human A2AR) were subcloned into plasmid pcDNA5/FRT/TO using KpnI and NotI restriction sites. Flp-in T-Rex HEK293 cells were maintained at 37 °C in a humidified atmosphere in Dulbecco’s modified Eagle’s medium without sodium pyruvate, supplemented with 4,500 mg l−1 glucose, l-glutamine, 10% (v/v) FBS, 1% penicillin/streptomycin mixture and 10 μg ml−1 blasticidin. To generate stable cell lines, the cells were transfected with a ratio of 1:9 receptor cDNA in pcDNA5/FRT/TO vector and pOG44 vector using Genejuice as per manufacturer’s instructions (EMD Biosciences). After 48 h, media were replaced with fresh medium supplemented with 200 μg ml−1 hygromycin B to select for stably expressing clones. Colonies were combined and tested for doxycycline-induced receptor expression. To induce receptor expression clones were treated with either 1 ng ml−1 or 3 ng ml−1 doxycyline for 16 h.

Cells were seeded at a density of 25,000 per well in a poly-l-lysine coated 96-well half area plate. Cells were induced with doxycyline (3 or 1 ng ml−1) for 16 h. After 16 h media were removed and replaced with fresh media containing 100 μM Ro-201724 and 2 U ml−1 adenosine deaminase. Cells were incubated at 37 °C for 30 min before addition of varying concentrations of agonist (25 °C, 30 min). As a control cells were also incubated for 30 min (25 °C) with 10 µM forskolin. Cells were then lysed and cAMP produced detected using the CisBio cAMP kit according to manufacturer’s instructions before plates were read on a PolarStar fluorescence plate reader.