Giuseppe Cannone

Ludovic Berto

Fanny Malhaire

Gavin Ferguson

Aurelien Foullien

Stéphanie Balor

Joan Font-Ingles

Amadeu Llebaria

Cyril Goudet

Abhay Kotecha

Kutti R Vinothkumar

Guillaume Lebon
email: guillaume.lebon@igf.cnrs.fr

Conformational diversity in class C GPCR positive allosteric modulation

 . Here, we report the cryoEM structures of metabotropic glutamate receptor 5 (mGlu 5 ) purified with three chemically and pharmacologically distinct PAMs. We find that PAMs modulate the receptor equilibrium through their different binding modes, revealing how their interactions in the 7TMs impact the mGlu 5 receptor conformational landscape and function. In addition, we identified a PAMfree but agonist-bound intermediate state that is stabilised by interactions mediated by intracellular loop 2. The activation of mGlu 5 receptor is a multi-step sequential process in which the binding of the PAMs in the 7TM modulates the equilibrium towards the active state.

Main

The major excitatory neurotransmitter in the mammalian nervous system, L-glutamate binds to various receptors in the membrane to modulate the activity at the synapse [START_REF] Gregory | CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors[END_REF] . Among them, the metabotropic glutamate receptors (mGlu) are class C G-protein receptors. This family is composed of eight subtypes, divided into three groups based on their pharmacology, sequence similarity, and primary cellular signalling pathways. The structure of mGlu receptor is modular with an extracellular venus-fly trap domain (VFT) that binds the agonist Lglutamate and the cysteine-rich domain (CRD), which connects the VFT to the transmembrane domain (7TM) [START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF] . Typical of all class C GPCRs, mGlu receptors also form obligate homo-and heterodimers and oligomerization is essential for their biological function. Binding of the orthosteric agonist, L-glutamate induces the closure of the VFTs that mediates a large conformational change resulting in the signal being transmitted to the 7TM and binding of the G-protein [START_REF] Wang | Structural insights into dimerization and activation of the mGlu2-mGlu3 and mGlu2-mGlu4 heterodimers[END_REF][START_REF] Seven | G-protein activation by a metabotropic glutamate receptor[END_REF][START_REF] Lin | Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4[END_REF][START_REF] Koehl | Structural insights into the activation of metabotropic glutamate receptors[END_REF][START_REF] Du | Structures of human mGlu2 and mGlu7 homo-and heterodimers[END_REF][START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] . Surprisingly, no natural ligands that bind to the 7TM of mGlu receptors have been identified yet but several small synthetic molecules can bind to 7TM, acting as positive or negative allosteric modulators, PAM and NAM, which enhance or inhibit receptor signalling, respectively.

The different mGlu receptors are widely expressed in the human brain where they modulate glutamatergic transmission, thus contributing to many important physiological processes [START_REF] Gregory | CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors[END_REF] . Aberrations in glutamatergic transmission result in many neurological disorders, making mGlu receptor targets of therapeutic interest [START_REF] Gregory | CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors[END_REF][START_REF] Stansley | Neuropharmacological Insight from Allosteric Modulation of mGlu Receptors[END_REF][START_REF] Nicoletti | Metabotropic glutamate receptors: from the workbench to the bedside[END_REF] . The mGlu 5 subtype is an important modulator of synaptic plasticity involved in learning and memory. The mGlu 5 positive allosteric modulation has strong potential for alleviating different symptoms of schizophrenia [START_REF] Dogra | Metabotropic Glutamate Receptors As Emerging Targets for the Treatment of Schizophrenia[END_REF] . However, developing safe mGlu 5 PAMs is a difficult task since some of them have been associated with adverse side-effects [START_REF] Parmentier-Batteur | Mechanism based neurotoxicity of mGlu5 positive allosteric modulators--development challenges for a promising novel antipsychotic target[END_REF][START_REF] Rook | Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity[END_REF] . Understanding the effects of PAMs is a real challenge, and how they bind and impact on the dynamics of the mGlu activation mechanism is of great physiological and pharmacological interest.

Pharmacologically, PAMs trigger diverse signalling outcomes, resulting in reported biased signalling [START_REF] Rook | Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents[END_REF][START_REF] Sengmany | Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: Implications for optimizing preclinical neuroscience drug discovery[END_REF][START_REF] Hellyer | Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site[END_REF] likely associated with multiple receptor conformations. Amongst the large number of PAMs that have been developed till date, some are defined as pure PAM that only potentiate the glutamate action (VU0409551 [START_REF] Rook | Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents[END_REF] and VU29 [START_REF] Chen | Interaction of novel positive allosteric modulators of metabotropic glutamate receptor 5 with the negative allosteric antagonist site is required for potentiation of receptor responses[END_REF] ), but they can display a low agonist-like activity (intrinsic agonist activity) depending of the signalling pathway and cellular context [START_REF] Sengmany | Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: Implications for optimizing preclinical neuroscience drug discovery[END_REF][START_REF] Sengmany | Differential contribution of metabotropic glutamate receptor 5 common allosteric binding site residues to biased allosteric agonism[END_REF][START_REF] Gregory | Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships[END_REF][START_REF] Noetzel | Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function[END_REF] . Others PAMs, such as VU0424465 [START_REF] Rook | Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity[END_REF] , are called ago-PAM that display a strong intrinsic agonist activity and can induce receptor signal transduction without the need for glutamate binding to the VFTs, either by activating the 7TM domain alone or by displacing the equilibrium between the inactive and active state of the receptor.

Several studies have reported the PAM-bound conformation of class C GPCRs [START_REF] Koehl | Structural insights into the activation of metabotropic glutamate receptors[END_REF][START_REF] Du | Structures of human mGlu2 and mGlu7 homo-and heterodimers[END_REF][START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF][START_REF] Park | Symmetric activation and modulation of the human calcium-sensing receptor[END_REF] and how the PAM and the G protein modulate the receptor conformation [START_REF] Wang | Structural insights into dimerization and activation of the mGlu2-mGlu3 and mGlu2-mGlu4 heterodimers[END_REF][START_REF] Seven | G-protein activation by a metabotropic glutamate receptor[END_REF][START_REF] Lin | Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4[END_REF][START_REF] Shen | Structural basis of GABAB receptor-Gi protein coupling[END_REF] . The class C GPCR activation mechanism is asymmetric i.e., with only one G protein being activated at a time by the receptor dimer. The 7TMs form asymmetric interface in the activated state both in the presence and the absence of G protein [START_REF] Seven | G-protein activation by a metabotropic glutamate receptor[END_REF][START_REF] Lin | Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4[END_REF][START_REF] Gao | Asymmetric activation of the calcium-sensing receptor homodimer[END_REF] . In mGlu 2 receptor, only one ago-PAM was found bound to the G protein-coupled 7TM [START_REF] Seven | G-protein activation by a metabotropic glutamate receptor[END_REF] but in other class C receptors, such as the calcium receptor it has been observed that PAMs are bound to both protomers in the absence of G protein, although they adopt different binding pose or mode [START_REF] Gao | Asymmetric activation of the calcium-sensing receptor homodimer[END_REF] . Recent single molecule-FRET analysis proposed a multi-state process including an agonist bound intermediate-active conformation between the inactive and active state [START_REF] Lecat-Guillet | Concerted conformational changes control metabotropic glutamate receptor activity[END_REF] . mGlu activation mechanism is a dynamic, multi-step process and PAMs are likely to modulate this receptor state equilibrium, promoting the stabilisation of the active state. Despite the conserved domain architecture in mGlu, the differences in the 7TM sequence provides an opportunity to selectively modulate the mGlu's activity in an isoform dependent manner and may lead to the discovery and design of new molecules with therapeutic potential and high level of selectivity. Thus, understanding the mechanism of PAM binding and the activation of the receptor is highly relevant. While structures of mGlu 5 7TM with NAM bound have been reported [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF][START_REF] Doré | Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain[END_REF][START_REF] Christopher | Fragment and Structure-Based Drug Discovery for a Class C GPCR: Discovery of the mGlu5 Negative Allosteric Modulator HTL14242 (3-Chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile)[END_REF][START_REF] Christopher | Structure-Based Optimization Strategies for G Protein-Coupled Receptor (GPCR) Allosteric Modulators: A Case Study from Analyses of New Metabotropic Glutamate Receptor 5 (mGlu5) X-ray Structures[END_REF] , the binding mode of PAM in the mGlu 5 7TM and their impact on mGlu 5 conformations has remained elusive.

Here, using cryogenic-sample electron microscopy (cryoEM), we report structures of full-length mGlu 5 in detergent micelles bound to high affinity orthosteric agonist quisqualate and to chemically distinct PAMs: the ago-PAM VU0424465 and the PAMs VU0409551 and VU29. Combined with molecular pharmacology, we decipher the diversity of PAM binding mode and how they impact mGlu 5 receptor conformation and function, providing insights into the effect of PAMs on the conformations and activity of mGlu 5 receptor.

Structures of PAM-bound mGlu 5 receptor. The structures of PAM-bound mGlu 5 receptor was solved by taking advantage of thermostabilised mGlu 5 receptor that contains five thermostabilising mutations (T742A 5.42 , S753A 5.53 , T777A 6.42 , I799A 7.29 , A813L 7.43 ) and truncation at its C-terminus after residue A856 (mGlu 5 -Δ856) [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF][START_REF] Nasrallah | Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs[END_REF] . We have previously shown that the thermostabilised receptor is fully functional, retaining its capability of activating the G protein when expressed in HEK293 cells [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF][START_REF] Nasrallah | Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs[END_REF] . The first structure described here is the ago-PAM VU0424465-bound mGlu 5 receptor at a global resolution of 3.1 Å (we define ago-PAM VU0424465 as PAM1, data set 1 as PAM1_d1) with no symmetry imposed. Density is well defined in the ECD but is of lower resolution in the 7TM, a common feature in all the data sets described in this study (Supplementary Fig. 1,2). The density for inner TM helices is better resolved and there is unambiguous density for the PAM in both protomers and for the surrounding residues (Fig. 1A andB; Extended Data Fig. 1 and7; Supplementary Fig. 1).

We were also able to build the loop ICL2 in this data set (using the deepEMhancer 30 maps), which has been implicated in G-protein binding. A second independent data set of PAM1 bound receptor was collected with cold FEG and Falcon 4i detector housed at the bottom of Selectris energy filter and this led to obtaining of two different conformations of the receptor, at overall resolutions of 3.2 Å (PAM1, conformer 1 in dataset 2 as PAM1_d2_c1) and 3.5 Å (PAM1, conformer 2 in dataset 2 as PAM1_d2_c2) respectively (Extended Data Fig. 2). The difference between these two conformations includes some variability in PAM occupancy with global rmsd of 1.5 Å and 1.6 Å for the monomers. In these two conformations, the VFT (0.3 Å rmsd for Cα) overlay well but the CRD and the TMD show greater deviations (0.6 Å and 0.8 Å rmsd respectively). The PAM density is observed in both protomers but the conformer 1 has slightly weaker density when compared to PAM1_d2_c2, and PAM1_d1 (Extended Data Fig. 7). In addition, model superposition shows that these two conformations are the mirror of each other where PAM1_d2_c1 superimpose very well with PAM1_d1 (rmsd 0.7 Å and 0.8 Å for chain A and B respectively), while PAM1_d2_c2 (1.7 Å rmsd for chain A and 1.6 Å rmsd for chain B) differs from PAM1_d1 (Fig. 2D andE).

These independent data sets provide confidence in the mode of PAM binding. We observe an asymmetrical interface previously described for other class C receptor that includes mGlu 2 [START_REF] Seven | G-protein activation by a metabotropic glutamate receptor[END_REF] ,

CaSR [START_REF] Gao | Asymmetric activation of the calcium-sensing receptor homodimer[END_REF] and several mGlu heteromers [START_REF] Wang | Structural insights into dimerization and activation of the mGlu2-mGlu3 and mGlu2-mGlu4 heterodimers[END_REF] . All the structures with PAM bound in both protomers (PAM1_d1, PAM1_d2_c1, PAM1_d2_c2), has the top of TM6 shifted towards the extracellular side of the receptor (Supplementary Fig. 3). Altogether, this suggest that the mGlu 5 receptor can adopt a minimum of two discrete conformations with both agonist and PAM bound, highlighting the dynamic nature of the mGlu 5 dimer active state, prior to G protein binding.

We then performed cryoEM reconstructions of the PAMs VU29 and VU0409551 at global resolutions of ~3.2 Å (PAM2) and 3.0 Å (PAM3), respectively (Extended Data Fig. 3 and 4). The VU29 map had reasonably well-defined PAM density in only one of the monomers that allowed model building of the PAM (Fig. 2A and Extended Data Fig. 7) and of few bulky side-chains around the PAM binding sites. The second protomer displayed poor density for the ligand suggesting a lower occupancy and thus no PAM was built into this monomer in the map, but the residual density indicates a similar binding pose (Extended Data Fig. 7). In the case of VU0409551, only weak density for the PAM was observed in both protomers, preventing the model building (PAM3_c1) of this PAM. However, this data set has well resolved VFT allowing us to build few water molecules in both the protomers and the region around the agonist binding site and the 7TM is also better resolved than for VU29 structure (Supplementary Fig. 2).

Subsequently, we also determined the structure of the thermostabilised receptor with an additional mutation, W785A (W785 6.50 ), a key residue for NAM and PAM binding and signal transduction [START_REF] Gregory | Identification of Specific Ligand-Receptor Interactions That Govern Binding and Cooperativity of Diverse Modulators to a Common Metabotropic Glutamate Receptor 5 Allosteric Site[END_REF] . We co-purified the full-length mGlu 5 -5M W785A mutant bound to the ago-PAM VU0424465 and the 3D reconstruction has a global resolution of ~3.4 Å (Extended Data Fig. 5). Removing the bulky W785 side chain strongly impairs the PAM occupancy but reveals some density found in the same region as other data sets (PAM_d1, PAM_d2_c1 and _PAM_d2_c2). The ligand is not modelled but the map implicates the role of W785 in VU0424465 ago-PAM binding.

When processing multiple PAM-bound data sets (3 different PAMs and one mutant),

we also identified population of particles visually identical to mGlu 5 inactive state receptor (i.e., with the TM domain separated apart) but only in the PAM3 data set, the number of particles was sufficient for medium-resolution EM reconstruction to ~4.1 Å (Fig. 3;

PAM3_c2; Extended Data Fig. 6). This structure reveals a closed conformation (c) of the VFTs, with quisqualate bound, but stabilised in an inactive (R) state, which we propose to be in an intermediate-active conformation (Fig. 3). The statistics of data processing and model building of all data sets are provided in Extended Data Table 1.

mGlu 5 PAM binding mode

Previously, the class C GPCR structures of mGlu 2 revealed that only one ago-PAM is bound in the 7TM whereas two PAMs are bound in case of CaSR but in different binding modes [START_REF] Seven | G-protein activation by a metabotropic glutamate receptor[END_REF][START_REF] Gao | Asymmetric activation of the calcium-sensing receptor homodimer[END_REF] . Nevertheless, despite the differences, an asymmetrical dimer interface was reported in both cases. In contrast, we observe ago-PAM bound in similar binding pose in both protomers in mGlu 5 (Fig. 1B).

VU0424465 (PAM1) shares a common binding site with the NAMs such as alloswitch-1 [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] or M-MPEP 28 (Supplementary_Fig. 4). The fluorophenyl group is found between G624 2.45 , I625 2.46 , G628 2.49 , S654 3.39 , P655 3.40 , V806 7.36 , S809 7.39 with one specific additional interaction with A810 7.40 in mol A (Fig. 1D andE). Although the PAM is positioned in the same binding pocket as NAM, the main differences are localised at the top of TM6, where the ago-PAM induces the shift of TM6 towards the extracellular side with a kink at P790 6.55 for only one protomer (mol B). As a consequence, the PAM slightly tilted upwards (toward the vertical axis of the 7TM) and makes an interaction with the S658 3.43 and L813 7.43 , one of the thermostabilising mutation introduced in the receptor 11 (Fig. 1D).

Y659 3.44 is displaced towards T781 6.46 (Fig. 1F). In the second monomer (mol A), the W785 6.50 is stabilised by a polar interaction with S809 7.39 as found in the inactive state of the receptor suggesting that only one protomer is activated.

The pyridinecarboxamide of VU0424465 occupies the same pose in both protomer making van-der-walls interaction with M802 7.32 , L744 5.44 , and W785 6.50 in TM6 (Fig. 1D andE). For both VU0424465 molecules, the N-pyridine atom makes polar contact to the S805 7.35 and to the W785 carbonyl. The dimethylpropyl group of VU0424465 molecule contacts L744 5.44 , F788 6.53 , V789 6.54 , Y792 6.57 , F793 6.58 in both protomers, whereas in mol B it is shifted towards TM7 and makes an additional interaction with T801 7.31 . Overall the PAM is slightly straightened in mol B, pushing TM6 up, towards the extracellular side.

The binding modes in PAM1_d2_c1 and PAM1_d2_c2 are very similar. Notably PAM1_d2_c1 model display a hydrogen bond between S809 7.39 -W785 6.50 in mol A only, similar to PAM1_d1. The PAM1_d2_c2 model displays the same polar contact in mol A, whereas the second protomer has an additional interaction between Y659 and T781 6.46 . The small differences observed could be because of the resolution and the quality of the maps in particular at the TMD (Extended Data Fig. 1,2). With all these structures we can now define the signature of mGlu 5 PAM bound state.

VU29 occupies a binding site similar to VU0424465 but with some significant differences. The main difference comes from W785 6.50 being pushed away from the helical bundle by the phenyl ring of VU29 whereas F788 moves in to stabilise the PAM (Fig 2A).

VU29's nitrobenzamide group is localised in a similar position compared to the fluoro phenyl moiety of VU042465, but is likely to push away Y659 3.44 . VU29 is further stabilised by interactions with S658 3.43 and a polar contact with S809 7.39 . In addition, VU29 engages with several residues from TM5 including V740 5.40 and I751 5.51 . The PAM VU29 is the most affected by mutation of residues in the allosteric binding pocket (Fig. 4C andD). The 7TM has poor resolution and only few side-chains could be built with confidence, suggesting a dynamic nature of VU29 binding and, at the moment, we cannot exclude other binding modes.

An asymmetric but flexible dimer interface

We have determined six 3D reconstructions for the PAM-bound mGlu 5 receptor. It includes two conformations of the receptor bound to VU0424465, one bound to VU29, one to VU0409551, and one for the mGlu 5 -5M-W785A receptor bound to VU0424465.

Surprisingly, only two of the VU0424465 structures (PAM1_d1 and PAM1_d2_c1) display an almost identical backbone superposition, from the VFTs to the 7TM when superposing the dimer (Fig. 2). When comparing monomers of each structure (i.e., PAM1_d1; Fig1 F), we observed that all VU0424465 structure (PAM1 models) display an asymmetrical interface, with a shift and a kink at P790 of TM6 for only one monomer, both much less pronounced in the W785 mutant and two other PAM structures (Fig. 2 B, C and F; Supplementary_Fig.

3). These are also the ones with the best occupancy for the PAM in each protomer. The 7TM interface is weaker for the two other PAMs suggesting that the lower occupancy in the binding site is likely associated to the weaker TM6-TM6 interface. This is also clearly reflected for the VU0409551 for which we were not able the model the PAM.

Even more surprising is the disruptive effect of the mutation W785A that affects not only the binding as reflected by the poor occupancy of VU0424465 but also disruption of the asymmetric interface between the 7TM as compared to PAM1 structure (PAM1_d1, d2_c1, d2_c2; Fig. 2D,E,F). Residue W785 is key for stabilising the VU0424465 ago-PAM in the allosteric binding site that mediates the active state asymmetrical dimer interface (Fig. 1B,C D and F; Supplementary Fig. 3). Indeed, in the VU29, VU0409551 and in the VU0424465+W785A mutant models, the distance between the Cα of P790 that is localised at the top of TM6 from each monomer significantly increase from ~7.5 Å in the VU0424465 models (PAM1-d1, d2-c1 and d2-c2) up to 11 Å for the VU29 structure. Interestingly, VU0424465 has the highest affinity and is likely to better stabilise the active conformation, at least in the absence of the G protein and thus we conclude that the occupancy of the PAM in 7TM strongly affects the stability of 7TM dimer interface and propensity of the mGlu 5 receptor active state.

Intermediate active-state, a quisqualate-bound Rcc conformation

The class C GPCRs undergo a number of conformational changes both in their ECD as well in the 7TM. In absence of glutamate or presence of orthosteric antagonist, the VFTs adopt an open (o) state and a resting (R) conformation, with respect to lobe-I orientation [START_REF] Pin | Organization and functions of mGlu and GABAB receptor complexes[END_REF][START_REF] Lecat-Guillet | Concerted conformational changes control metabotropic glutamate receptor activity[END_REF] .

Binding of L-glutamate or analogue such as quisqualate induces the closure of the VFTs and the reorientation of lobe-I that brings the lobe-II in proximity. This movement is translated to the 7TM that are far apart in the open and resting state of mGlu 5 , but form a dimeric interface in the active (A) state [START_REF] Koehl | Structural insights into the activation of metabotropic glutamate receptors[END_REF][START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] . The multiple states that the receptors can adopt have been defined

based on the conformation of VFT as open or close and the receptor as resting or active [START_REF] Vafabakhsh | Conformational dynamics of a class C G protein-coupled receptor[END_REF] .

Thus, the antagonist bound receptors typically adopt Roo conformation [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] (for the resting and open conformation of both VFTs) and the fully active receptor are called Acc (for active and closed conformation of both VFTs) [START_REF] Koehl | Structural insights into the activation of metabotropic glutamate receptors[END_REF][START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] . The receptor can also adopt other conformations including the Rcc conformation as proposed for the mGlu 2 receptor [START_REF] Lecat-Guillet | Concerted conformational changes control metabotropic glutamate receptor activity[END_REF] .

Both the agonist and the PAM have been included throughout the purification of the receptor used in the study, and we still identified a sub-set of particles that showed distinguishable 2D classes compared to the PAM-bound active state in the data sets presented in this study. We could calculate only low-resolution maps from particles of the VU0424465 and VU29 data sets, but a reasonable medium resolution map at ~4.1 Å was reconstructed from the PAM3 data set (Extended Data Fig. 6) and has slight anisotropy when compared to other data sets (Supplementary Fig. 6). The overall map of the receptor dimer is well resolved and the 7TMs are separated apart, as previously reported in the antagonist LY341495-bound mGlu 5 receptor (Fig. 3A, B, C andD), although this structure represents an additional and different conformation of the receptor (Fig. 3D, H andI).

In the map, the VFT of one of the monomers is better resolved than the other and shows quisqualate bound in the classical pose as in other structures determined. The ligand is stabilised by several polar interactions (Y64, S152, S173, T175, G280, D305, R310, K396)

and van-der-walls contact (W100, G150, S151, A174, Y223, E279, G306,) (Fig. 3E,F,G).

In the second VFT, the quality of the map is lower indicating flexibility and the quisqualate adopts a different pose, the carboxylic group orients towards Lobe-II, making fewer van-derwalls contacts with residues Y233, E279, F277, S304, G306, and two polar interactions with D305 and S173, and suggesting an intermediate and transient binding mode (Fig. 3G; Supplementary Fig. 5). The weak density for one of the VFT is reflective of the receptor dynamics. Indeed, the 3D variability analysis (3DVA) of this subset of particles and subsequent analysis of the models by phenix.varref [START_REF] Afonine | Conformational space exploration of cryo-EM structures by variability refinement[END_REF] showed that one VFT is closed and in a stable conformation whereas the second VFT samples multiple states, from a closed to a more open conformation, compatible with quisqualate being weakly bound (Supplementary Movie 1 and 2). Although, we could not build the Lobe-1 in this monomer confidently due to poor density, we however can see that the helices B and C are starting to move and to undergo the transition from the R towards the A state when compared with the orthosteric antagonist LY341495-bound VFT conformation (Supplementary Fig. 5). This transition suggests that whilst one VFT is stabilised in the closed state, the second VFT is oscillating in multiple states between open (o) and close (c) conformations displacing the receptor equilibrium towards a more active-like A state initiating the motion to be translated to the CRD and to the 7TM. The results presented here illustrate the sequential but dynamic events occurring initially at the VFTs.

An interesting feature of this intermediate state is the ICL2, which extends from TM3, similar to the VU0424465-bound conformation (PAM1_d1), (Fig. 3A,B,C; Extended Data Fig. 6). In the Rcc conformation, the ICL2 from each protomer face each other, most likely stabilising the intermediate (Fig. 3C andI). From the intracellular side of the receptor the motion of TM3 clearly shows that ICL2 represents an intermediate state between the fully inactive state bound LY341485 and the active state presented here (Fig. 3I). The ICL2 loops of mGlu 5 has a unique motif KKICTKK and mutating any of these residues affects the efficacy of the PAM signalling with a dramatic effect in particular for the residue I680

(Extended Data Fig. 8; Supplementary Table 1). The ICL2 loop has been proposed to be important for G protein interaction of class C GPCRs [START_REF] Gomeza | The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins[END_REF] and it is tempting to speculate that this intermediate conformation prevents the binding of the G-protein.

Despite a modest global resolution for this state of the receptor, several key residues involved in PAM binding have defined density but no clear density could be detected for the PAM. The side-chains of W785 [START_REF] Goudet | Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors[END_REF] i.e., even in the Rcc conformation [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] .

mGlu 5 PAM activation mechanism

The selection of the PAMs for this study was initially based on their difference in chemical scaffold, affinity, as well as their different intrinsic agonist activity. In the literature, VU0424465 is described as an high affinity ago-PAM [START_REF] Rook | Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity[END_REF] contrary to VU29 and VU0409551, which are less able to activate mGlu 5 by themselves [START_REF] Sengmany | Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: Implications for optimizing preclinical neuroscience drug discovery[END_REF][START_REF] Gregory | Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships[END_REF] . Here, we confirm that VU0424465 possesses a high intrinsic agonist activity in absence of the agonist quisqualate, being able to almost fully activate mGlu 5 at 1 µM (83 ± 6 % of the maximal activation induced by quisqualate in our assay) (Extended Data Fig. 9; Supplementary Table 2). In contrast, VU29 and VU0409551 solely display a low intrinsic agonist activity, being able to partially activate the receptor at 1µM (20 ± 9% and 15 ± 1% of maximal activation induced by the agonist quisqualate for VU29 and VU0409551, respectively) (Extended Data Fig. 9; Supplementary Table 2). W785 6.50 , a key residue for GPCR activation mechanism [START_REF] Gregory | Identification of Specific Ligand-Receptor Interactions That Govern Binding and Cooperativity of Diverse Modulators to a Common Metabotropic Glutamate Receptor 5 Allosteric Site[END_REF] , differentially affects the activity of the PAMs (Fig. 4; Supplementary Table 3). The mutation of this residue to alanine has no significant effect on the signalling activity induced by VU0424465 (Fig. 4A and B), likely due to its strong affinity. In contrast, the potency of the other two PAMs VU29 (Fig. 4C andD) and VU0409551 (Fig. 4E andF) is significantly affected in the W785A mutant (Supplementary Table 3). The allosteric effect of VU29 is improved in this mutant but the effect by VU0409551 is reduced. In the model proposed here, VU29 binding pushes the W785 6.50 side chain out and thus removing the side-chain is likely to facilitate its efficient binding to the receptor (Fig. 2A). In the other words, when mutating W785 6.50 in alanine, more space is created for the PAM VU29 than for ago-PAM VU0424465 and VU0409551.

Another key residue for all three PAM is Y659 3.44 . Replacing this tyrosine abolishes the VU29 and VU0409551 modulation and strongly impairs the effect of VU0424465, although some activation was measured (Fig. 4; Supplementary Table 3). Y659 is localised at the bottom of the binding site and was found in a network of polar contact in the inactive conformation of the 7TM structure solved to high resolution by X-ray crystallography 11 (Fig. 4G). Here, the Y659 3.44 is displaced towards T781 6.46 in PAM1_d1 and moves away from S809 disrupting the Y659 3.44 -S809 7.39 interaction. The Y659 3.44 also interacts with T781 6.46 in one monomer only for the model PAM1_d2_c2. Pharmacology presented here highlight the role Y659 3.44 but also of T781 6.46 , that display reduced potency for VU0424465 as well as for VU0409551. In the protomer where the VU29 is modelled, the nitrobenzamide group pushes the Y659 3.44 towards TM5 and the interaction with S809 is also broken.

A third important residue is the F788 that undergoes the most dramatic movement when all structures are compared (Fig. 4G-J). In the Rcc conformation, the side chain of F788 6.53 is facing the core of 7TM (Fig. 4H) as previously reported for the M-MPEP bound conformation, and is likely to prevent the PAM binding (Supplementary Fig. 4). The position of the F788 6.53 may adopt multiple conformations without affecting the pharmacology of PAM or NAM. Indeed, in the structure of alloswitch-1, a photoswitchable NAM has forced the F788 6.53 out (Fig. 4G) but maintains strong hydrogen bond network with S809 7.39 , Y659 3.44 and W785 6.50 . However, in presence of PAM VU0424465, F788 6.53 is facing out (Fig. 4I), whereas it moves and interact with VU29, stabilising its binding as suggested by its reduced potency in F788A mutation (Fig. 4C, D andJ).

We also observe interactions between N796 (ECL3) and D722, P726 and Y730 (ECL2) of the active-like protomer in the PAM1_d1 model that may help to stabilise the active conformation (Extended Data Fig. 10; Supplementary Table 4). Such interaction is also observed in the two other VU0424465 structures (PAM1_d2_c1 and PAM1_d2_c2).

However, mutating these residues has only weak effect on the efficacy of PAM.

The structural and functional analysis of ago-PAM and PAM-bound receptor presented here reveals a dynamic/flexible network of contacts between residues in the ligand binding site stabilising the mGlu 5 receptor active state and to ultimately promote G protein binding. The global differences of the 7TM between the inactive state and the active state are not extensive and revolve around the residues Y659 3.44 , W785 6.50 , S809 7.39 , F788 6.53 and T781 6.46 . These residues have been shown to be important for receptor inactive state and for transmitting receptor signalling [START_REF] Gregory | Probing the metabotropic glutamate receptor 5 (mGlu₅) positive allosteric modulator (PAM) binding pocket: discovery of point mutations that engender a 'molecular switch' in PAM pharmacology[END_REF] . The analysis of the pharmacological characterisation clearly illustrates that residues Y659 3.44 , S809 7.39 , and T781 6.46 affect PAM's signal transduction, as an activation module. Associated with the structural characterisation presented here, the W785 6.50 appears to provide account for the complexity of mGlu 5 allosteric modulation and for the diversity of PAM binding mode.

Discussion

Allosteric modulation of class C GPCRs is the subject of intense research with the grail of discovering high affinity and selective molecules for specific receptor subtype. The main therapeutic potential for mGlu 5 PAMs is in the treatment of different symptoms of schizophrenia [START_REF] Dogra | Metabotropic Glutamate Receptors As Emerging Targets for the Treatment of Schizophrenia[END_REF] . However, the development of a safe mGlu 5 PAM with antipsychotic properties has remained a real challenge [START_REF] Rook | Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity[END_REF][START_REF] Rook | Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents[END_REF] . One of the main reasons is the complexity of the mGlu 5 signal transduction and the adverse side effects associated with some of these PAMs in preclinical studies. The complex signalling of mGlu 5 receptor has been associated to biased signalling [START_REF] Hellyer | Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site[END_REF] . Conformational diversity of the class C GPCRs is also directly linked to signalling profiles of the receptor but remains elusive.

Here, we selected three different PAMs with different scaffold and signalling profile.

The VU0424465 is defined as an ago-PAM whereas as VU29 and VU0409551 are considered as pure PAM for biased signalling as observed in specific cellular context [START_REF] Sengmany | Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: Implications for optimizing preclinical neuroscience drug discovery[END_REF][START_REF] Arsova | Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 as Tool Compounds to Study Signaling Bias[END_REF] .

They also display different affinity that translates into different potency [START_REF] Chen | Interaction of novel positive allosteric modulators of metabotropic glutamate receptor 5 with the negative allosteric antagonist site is required for potentiation of receptor responses[END_REF][START_REF] Rook | Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity[END_REF][START_REF] Rook | Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents[END_REF] . In fact, VU0424465 displays the highest affinity and potency, while VU0409551 has the weakest potency of the PAM, as analysed by the Gq protein activation and inositol phosphate-1 production. (Fig. 4; Supplementary Table 3).

The high occupancy and better density of the ago-PAM VU0424465 reported in this study correlates with higher affinity than PAM VU29 18 and VU0409551 [START_REF] Rook | Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity[END_REF] . The receptor occupancy affects the conformation and stability of the mGlu 5 active state (Fig. 2B andC), correlating with the difference in receptor signal transduction in cells i.e., a higher proportion of mGlu 5 receptor in the active state will bind and activate the G protein. An important observation is that the ago-PAM VU0424465 is bound to both protomers and stabilises the asymmetric interface characteristic of class C receptor dimer and better stabilises the active state (Fig 1 and2). We observe density in both protomer for the PAMs VU29 and VU0409551 albeit at lower occupancy, which probably allows the receptor to explore more conformations.

Perhaps due to the lower affinity of PAM3, a larger population of the receptor is able to exist in the closed VFT and in the inactive resting (R) state allowing us to determine the structure of an active intermediate state (Rcc) conformation. The L-glutamate analogue, quisqualate, is bound to both VFTs but adopts different poses: a standard pose, as previously reported [START_REF] Koehl | Structural insights into the activation of metabotropic glutamate receptors[END_REF][START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] and seen in all active state solved here, and an alternate pose that is likely to be energetically unfavourable, suggesting significant degree of VFT dynamics (Fig. 3E,F We have unveiled the binding site for the ago-PAM VU0424465 and identified a binding pose for VU29 illustrating the differential contribution of TM6 residues mainly W785 6.50 and F788 6.53 Fig ( 4I andJ). Functional analysis confirms the importance of the mGlu 5 receptor residues W785 6.50 , F788 6.53 , Y659 3.44 , S809 7.39 and T781 6.46 in PAM allosteric modulation, which is highly valuable in the design of new PAMs.

Comparing the PAM-free and PAM-bound conformations allowed us to illustrate the motion of key residues that control the binding of PAM and subsequently stabilise the active state, by sampling the 7TM conformation landscape. PAMs differentially displace the Y659 3.44 and F788 6.53 . The W785 6.50 is central to differential stabilisation of the PAM in the binding site, either positively for VU0424465 and VU0409551 or negatively for VU29.

Mutating this residue to alanine has a direct effect on both the PAM occupancy and the active state conformation that may be comparable to low affinity PAM such as VU0409551. The differences between the inactive and active state at the 7TM is subtle and in agreement with the absence of TM6 motion as defined as the molecular signature for the class A and B GPCR active conformations [START_REF] Venkatakrishnan | Molecular signatures of G-protein-coupled receptors[END_REF] . Indeed, comparison of the structures of NAM-bound mGlu 5

show some similarity between the NAM and PAM binding site (Supplementary Fig. 4) [START_REF] Doré | Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain[END_REF][START_REF] Christopher | Structure-Based Optimization Strategies for G Protein-Coupled Receptor (GPCR) Allosteric Modulators: A Case Study from Analyses of New Metabotropic Glutamate Receptor 5 (mGlu5) X-ray Structures[END_REF] .

The residues Y659 3.44 , S809 7.39 , W785 6.50 are making tight polar network that also involves T781 6.46 through a water molecule, in which S809 was shown important for mediating NAM effect [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] . Here we show that this tight network of polar interaction is reorganised, and key interaction such as Y659 3.44 -S809 7.39 polar interaction is destabilised. Y659 on the receptor active state is likely to trigger a much higher level of signal transduction than orthosteric L-glutamate, and may induce possible undesired side-effect.

The multi-step activation process of the mGlu 5 receptor perhaps includes internal check-points such as the ICL2 and the conformations of the residues in the PAM binding sites that can be modulated by small molecules and biologics. The strong stabilising potency of an ago-PAM such as VU0424465 is likely to trigger a much higher level of signal transduction by potentiating orthosteric L-glutamate as would a pure PAM perform, but also due to intrinsic agonist activity that triggers signal transduction in absence of L-glutamate, likely giving account for undesired side-effect. Thus, the structures reported here provide a template for exploring positive allosteric modulation and to be integrated in structure-based drug discovery program for identifying new PAM for mGlu 5 receptor.

Methods

Expression and purification of the human full-length thermostabilized mGlu 5 receptor bound to activators

The thermostabilised mGlu 5 -5M receptor and mGlu 5 -5M_W785A mutants were expressed and purified as previously reported [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] . mGlu 5 -5M receptor and mGlu 5 -5M_W785A constructs were subcloned into the BacMam vector. The constructs contain peptide signal from baculovirus GP64 envelope surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV baculovirus), the flag (DYKDDDDK), 10xHis tag, and the precision protease recognition site (LEVLFQGP) at the N terminus, followed by the human mGlu 5 -5M or mGlu 5 -5M_W785A. The construct also contains a H350L mutation, initially designed for binding of a nanobody and a N445A mutation to remove a glycosylation site.

The mGlu 5 -5M_W785A includes the additional mutation W785A and the N445A glycosylation mutation was reversed.

High-titre recombinant baculovirus was obtained using the Bac-to-Bac Baculovirus expression system (Invitrogen) as described by the manufacturer. Briefly, Sf9 cells grown at 28°C in EX-CELL 420 medium (Sigma Aldrich) were infected at a density of 3x10 6 cells using human mGlu 5 -5M and mGlu 5 -5M_W785A virus and P2 virus were harvested by centrifugation 48 hours post-infection. HEK293S cell line lacking Nacetylglucosaminyltransferase I 39 (HEK293S GnTI -) cells were grown at 37°C in humidified 5% CO 2 in shaking incubator (110 rpm/minute) in Freestyle medium complemented by 10%

of Fetal Bovine Serum (Sigma Aldrich). HEK293S GnTI (-) cells were infected at a density of 3x10 6 cells per mL with 6% of freshly produced P2 virus (60 mL per litre) and 10 mM sodium butyrate was added 24 hours post-infection. Cells expressing mGlu 5 -5M and mGlu 5 -5M_W785A were harvested 96 hours post-infection and stored at -80°C for further purification.

For membrane preparation, frozen cell were resupended in a lysis buffer containing bound mGlu 5 FL receptor in detergent micelles, at 5 mg/mL was applied to a Quantifoil ® Au 0.6/1.0 µm 300 mesh grids. Grids were rendered hydrophilic by glow-discharging for 10 seconds at 25 mA using a PELCO EasiGlow instrument. Grids were plunge-frozen using EM GP2 Plunge Freezer (Leica) with the chamber equilibrated at 95% RH and 4°C. Grids were blotted with Whatman no. 1 filter paper for 4.5-6 seconds and plunge frozen in liquid ethane.

The frozen grids were stored in liquid nitrogen until further use. Data were collected on Titan Krios G3i (ThermoFisher) at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) with a K3 detector in counting mode with a quantum energy filter and 20 eV slit width. Data was collected from one grid with a dose of ~15.2 e -/p/s, and 2.4 second exposure at nominal magnification of 105000x, corresponding to pixel size of 0.84 Å. A total of 19830 movies were used for image processing. Movie files were imported into cryosparc 3.2 [START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] .

Motioncorr was carried out with cryosparc in-built patch motion correction (multi) and CTF estimation was performed in-built patch CTF estimation (multi). Initial particles picking was performed using Blob picker in cryosparc and particles were initially extracted with large box size in order to have the particles centered and for subsequent analysis they were down sampled. From initial large set of particles obtained after 2D classification, ab-initio model generation was performed followed by several round of 2D and 3D classifications to select good particles and obtain final map using refined and reconstructed using non-uniform refinement [START_REF] Punjani | Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction[END_REF] (NU) with C1 symmetry. The complete workflow is described in Extended Data

VU0424465 (PAM1) with Falcon4i detector :

Another independent data set of the mGlu 5 bound with quisqualate and PAM VU0424465

was collected with a Falcon4i detector (PAM1_d2). Grids were made by application of the receptor (6mg/ml) to a Quantifoil ® Au 0.6/1 µm 300 mesh grids. Grids were treated with 9:1 Ar:O 2 plasma (in a Fischione 1070 chamber) for 60 s to render them hydrophilic. Grids were plunge-frozen using Vitrobot Mk IV (ThermoFisher) with the chamber equilibrated at 100% RH and 10°C. Grids were blotted with Whatman no. 1 filter paper for 5 seconds with a blot force of 10 without any waiting or draining time and plunge frozen in liquid ethane.

The data acquisition was carried out using the cold field emission gun (CFEG) Titan Krios G4 microscope (ThermoFisher), at the ThermoFisher NanoPort facility in Eindhoven.

The CFEG microscope was equipped with a Falcon 4i detector operating in electron counting mode and was integrated at the base of a Selectris Energy Filter. The microscope featured fringe-free imaging (FFI) and aberration-free image shift (AFIS) [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF] , enabling the acquisition of multiple images before stage movement.

Data was collected with a sampling rate of 0.727 Šper pixel, employing an electron flux of ~5 e -/Ų/s and a total fluence of 50 e -/Ų. Movies were processed using Relion 4.0 [START_REF] Scheres | Implementation of a Bayesian approach to cryo-EM structure determination[END_REF] .

A total of 19,115 movie frames underwent motion correction using Relion's built-in program, followed by contrast transfer function (CTF) estimation carried out with CTFFIND4 [START_REF] Rohou | CTFFIND4: Fast and accurate defocus estimation from electron micrographs[END_REF] . Initial particle picking was executed using Topaz with a specified threshold of -2 [START_REF] Bepler | Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs[END_REF] . Reference-free two-dimensional (2D) classification was employed to identify high-quality particles. A subset of these particles was then employed to train a new Topaz model, which was subsequently utilized for repicking particles. These particles were extracted with a box size of 360 pixels and down sampled to speed up processing. From the 2D classification, a total of 396,207 particles were selected and subsequent 3D classification resulted in 221,875 particles. These selected particles were refined in 3D with C1 symmetry, resulting in a resolution of 3.

VU29-bound mGlu 5 receptor :

The agonist quisqualate-bound mGlu 5 WT in detergent micelles, at 5 mg/mL and PAM VU29 was applied to a Quantifoil ® Au 0.6/1.0 µm 300 mesh grids. Grids were rendered hydrophilic by glow-discharging for 10 seconds at 25 mA using a PELCO easiGlow instrument. Grids were plunge-frozen using EM GP2 Plunge Freezer (Leica) with the chamber equilibrated at 95% RH and 4°C. Grids were blotted with Whatman no. 1 filter paper for 4.5-6 seconds with no waiting/drying time and plunge frozen in liquid ethane.

The data acquisition was carried out using the cold field emission gun (CFEG) Titan Krios G4 microscope (ThermoFisher), at the ThermoFisher NanoPort facility in Eindhoven equipped with a Falcon 4i detector integrated at the base of a Selectris Energy Filter. Data was collected using fringe-free imaging (FFI) and aberration-free image shift (AFIS) with a sampling rate of 0.727 Šper pixel, employing an electron flux of ~6.1 e -/Ų/s and a total fluence of 49.62 e -/Ų. The processing of this data set was done with Cryosparc [START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] . Movie files (9469) were imported into cryosparc 3.2. Motioncorr was carried out with cryosparc in-built patch motion correction (multi) and CTF estimation was performed in-built patch CTF estimation (multi). Initial particles picking was performed using Blob picker in cryosparc and particles were initially extracted with large box size in order to have the particles centered and for subsequent analysis they were down sampled. From initial large set of particles obtained after 2D classification, ab-initio model generation was performed followed by several round of 2D and 3D classifications to select good particles and obtain final map using refined and reconstructed using non-uniform refinement [START_REF] Punjani | Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction[END_REF] (NU) with C1 symmetry. The complete workflow is described in Extended Data Fig. 3. Local resolutions were estimated with Relion 42 .

VU0409551-bound mGlu 5 receptor :

The agonist quisqualate-bound mGlu 5 WT in detergent micelles, at 5 mg/mL and PAM VU0409551 was applied to a Quantifoil ® Au 0.6/1.0 µm 300 mesh grids. Grids were rendered hydrophilic by glow-discharging for 10 seconds at 25 mA using a PELCO easiGlow instrument. Grids were plunge-frozen using EM GP2 Plunge Freezer (Leica) with the chamber equilibrated at 95% RH and 4°C. Grids were blotted with Whatman no. 1 filter paper for 4.5-6 seconds with no waiting/drying time and plunge frozen in liquid ethane. Data was collected on a Titan Krios G3i (ThermoFisher) at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) with a K3 detector in counting mode with a quantum energy filter and 20 eV slit width. Data collection was performed with a dose of ~18.4 e -/p/s, an exposure of 1.9 seconds at 105000x, corresponding to pixel size of 0.84 Å. A total of 19943 movies were used for image processing. Movie files were imported into cryosparc 3.2 [START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] . Motioncorr was carried out with cryosparc in-built patch motion correction (multi) and CTF estimation was performed in-built patch CTF estimation (multi). Initial particles picking was performed using Blob picker in cryosparc and particles were initially extracted with large box size in order to have the particles centered and for subsequent analysis they were down sampled for 2D classification. From initial large set of particles obtained by 2D classification, ab-initio model generation was performed, which identified two different conformations followed by several round of 2D and 3D classifications to select good particles and obtain final maps using using non-uniform refinement [START_REF] Punjani | Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction[END_REF] (NU) with C1 symmetry for both the active and the RCC conformation. For the Rcc conformation, 3D variability analysis in cryosparc was performed and the resulting maps was used for analysis with Phenix.varref [START_REF] Afonine | Conformational space exploration of cryo-EM structures by variability refinement[END_REF] (described below). The complete workflow are described in Extended Data Fig. 4 and6. Local resolutions were estimated with Relion [START_REF] Scheres | Implementation of a Bayesian approach to cryo-EM structure determination[END_REF] .

mGlu 5 W785A : The agonist bound mGlu 5 W785A in detergent micelles, at 6 mg/mL with quisqualate and PAM VU0424465 was applied to a Quantifoil ® Au 1.2/1.3 µm 300 mesh grids. Grids were rendered hydrophilic by glow-discharging for 1 min at 25 mA using a PELCO EasiGlow instrument. Grids were plunge-frozen using Vitrobot Mk IV (ThermoFisher) with the chamber equilibrated at 100% RH and 16°C. Grids were blotted with Whatman no. 1 filter paper for 3-3.5 seconds with a blot force of 0 and plunge frozen in liquid ethane. The frozen grids were stored in liquid nitrogen until further use.

Data were collected on Titan Krios G3i (ThermoFisher) at the National CryoEM facility in Bangalore with a Falcon 3 detector in counting mode and faster data acquisition with AFIS [START_REF] Weis | Combining high throughput and high quality for cryoelectron microscopy data collection[END_REF] . Data were collected from two independent grids made on different days with a dose of ~0.54 e -/p/s and an exposure of 60 seconds at 75000x, corresponding to pixel size of 1.07 Å. Images from both data sets were manually curated to remove crystalline and thicker ice, followed by import of movie files into Relion 4.0 [START_REF] Scheres | Implementation of a Bayesian approach to cryo-EM structure determination[END_REF][START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF] . Motioncorr was carried out with relion's in-built program and CTF estimation was performed with CTFFIND4 [START_REF] Rohou | CTFFIND4: Fast and accurate defocus estimation from electron micrographs[END_REF] . Based on CTF resolution and upper limit of defocus to 3.5 µ, a total of 2751 and 3001 images from each batch were selected further processing (the complete workflow is described in Extended Data Fig. 5). Low-pass filtered templates from prior data sets were used for automated particle picking with Gautomatch (https://www2.mrc-lmb.cam.ac.uk/download/gautomatch-053/) and particles were extracted with 4.28 Å sampling and 2 rounds of 2D classification was performed to select good particles. These selected particles were extracted with a box size of 384 and 1.07 Å sampling and further enriched for good particles with 2D classification. The particles from batch 1 and batch 2 were individually refined in 3D with C1 symmetry resulting in 4.8 and 6.1 Å respectively after postprocessing (all resolutions described here are of FSC at 0.143). Subsequently, the particles were subjected to Bayesian

Polishing [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF] and both B-factor weighted particles (shiny.star) were joined and refined. The For all the data sets, the anisotropy or the sphericity was estimated using the 3DFSC server 47 (Supplementary Fig. 6).

Model building and refinement :

Previous agonist bound active state model (7fd8) was used for rigid body fitting into PAM1_d1 map using Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF] and manually inspected and rebuilt where required using Coot [START_REF] Emsley | Features and development of Coot[END_REF] . Multiple maps were used during model building in all data sets including the maps with different B-factor sharpening and the deepEMhancer maps [START_REF] Sanchez-Garcia | DeepEMhancer: a deep learning solution for cryo-EM volume post-processing[END_REF] . For instance, the ICL2 loop in PAM1_d1 was built using the deepEMhancer map generated with mask option and for modelling water molecules, sharpened maps with automatic B-factor estimation were used. Ligand restraint files were generated using JLigand [START_REF] Lebedev | JLigand: a graphical tool for the CCP4 template-restraint library[END_REF] . Omit maps were generated using servalcat [START_REF] Yamashita | Cryo-EM singleparticle structure refinement and map calculation using Servalcat[END_REF] and used to verify the ligands and solvent.

For the subsequent data sets, the refined model from PAM1_d1 was used as template and monomers were individually fit using Chimera. This global rigid body gave good fit for the VFT and the CRD and 7TM regions were manually fit to the density with Coot first using rigid body and then with real space refinement (in the case of W785A mutant, the residue was changed to alanine). Unlike the other data sets, in W785A mutant data set, there was also extra density at N445 for glycosylation and N445 was restored from alanine, but sugar has not been modelled. In all the data sets, the density for ICL2 loop is ordered to varying degree but only in PAM1_d1 and PAM3_c2, they have been modelled. For the PAM3_c2 data set, the VFT and CRD from PAM1_d1 was used and the 7TM was rigid body with 7fd9 as template along with 7p2L.

Water molecules in the VFT region of PAM1_d1 and PAM3_c1 were conservatively picked using Coot and they were manually checked using the hydrogen bonding distance and density as the criteria. Models were refined using Phenix [START_REF] Liebschner | Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix[END_REF][START_REF] Afonine | Real-space refinement in PHENIX for cryo-EM and crystallography[END_REF] in particular the real space refinement (ref) and Servalcat/Refmac in ccpEM [START_REF] Yamashita | Cryo-EM singleparticle structure refinement and map calculation using Servalcat[END_REF][START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF]55 . For the real space refinement of the model with Phenix, the map sharpened map with half the value of B-factor estimated by the relion postprocess was used and when refmac was used for refinement, the half-maps were provided. The statistics of the final models are provided in Extended Data Table 1.

For the RCC conformation, the maps from 3D variability analysis and the model without any ligands was used as input to Phenix.varref [START_REF] Afonine | Conformational space exploration of cryo-EM structures by variability refinement[END_REF] to obtain the dynamic nature of the VFT (as shown in Supplementary Movies).

Cell culture, transfections, expression and inositol phosphate one (IP 1 ) accumulation assay

Cell culture and transfection HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and were maintained at 37°C in a humidified atmosphere with 5% CO 2 . Absence of mycoplasma was checked regularly to guarantee the use of mycoplasma-free cells. An alanine mutant library of the SNAP-tagged human truncated mGlu 5 receptor (mGlu 5 -Δ856, designated as WT in the following experiments) was generated using Quick-change strategy (Agilent technologies) and verified by sequencing (Eurofins Genomics) [START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] . Cells were transiently transfected using electroporation with the indicated mGlu 5 construct. The DNA mixture for transient transfection included 0.6 µg of the indicated mGlu 5 mutants, 2 µg of the glutamate transporter EAAC1 cDNA (to reduce the influence of glutamate that may remain in the assay medium as released by the cells) per 10 million of cells. Cells are seeded at the density of 100,000 cells per well into white bottom 96-well culture plates (Greiner Bio-one). Following 24 hours of transfection, expression and IP 1 production are measured.

Cell-based pharmacological assays

Wild-type and mutant mGlu 5 receptor expression was determined by labelling the SNAP-tag receptors with BG-lumi4-Tb. In brief, cells were incubated for 1 hour at 37°C with 100 nM of BG-Lumi4-Tb in 50 µL of glutamate-free DMEM GlutaMAX-I. Cells were then washed twice with Tag-Lite buffer, and then 100 µL of Tag-Lite buffer was added to each well (Revvity). Lumi4-Tb fluorescence was then measured with BMG Pherastar.

Before the commencement of the IP 1 accumulation assay, HEK293 cells were incubated for 1.5 hours with glutamate-free DMEM GlutaMAX-I (Life Technologies), to reduce the extracellular concentration of glutamate. The IP 1 accumulation assay kit (Revvity, France)

was used for the direct quantitative measurement of IP 1 in HEK293 cells transiently transfected with mGlu 5 constructs. Cells were stimulated with various concentrations of allosteric compounds with a fixed concentration of the orthosteric ligand Quisqualate (10 nM) for 30 minutes at 37°C, 5% CO 2 . For the Schild experiments, cells were stimulated with various concentrations of Quisqualate with a fixed concentration of the allosteric compounds.

Then, cells were lysed using the lysis buffer for 15min and half of the lysate was incubated with conjugate-lysis buffer containing the d2-labeled IP 1 analog and the terbium cryptatelabeled anti-IP 1 antibody in 384well plate, according to the manufacturer's instructions. After

1 hour incubation at RT, the HTRF measurement was performed after excitation at 337 nm with 50 µs delay, terbium cryptate fluorescence and tr-FRET signals were measured at 620 nm and 665 nm, respectively, using a PheraStar fluorimeter (BMG Labtech).

Data were fitted by a 3 parameters equation and statistical analyses were perfomed using oneway ANOVA followed by a Dunnett's post-hoc test, using GraphPad Prism version 10 (San Diego, CA). Fig. 4. Functional role of PAM binding site residues on mGlu 5 activity. We evaluated the influence of different residues of the PAM binding site in the 7TM of mGlu 5 on the ability of the different PAM's to potentiate agonist-induced activity. Single residues were mutated into alanine and functional consequences were determined by measuring the production of inositol monophosphate (IP 1 ) in HEK293 cells transiently expressing the WT and mutated mGlu 5 receptors. Experiments were performed in the presence of a fixed dose of the orthosteric agonist quisqualate (10 nM) and different doses of PAMs: VU0424465 (A and B), VU29 (C and D) and VU0409551 (E and F). Bar plots in A, C and E are pEC50 average values of at least 3 independent experiments +/-SEM. Dose response curve B, D and F are presented for mutants displaying statistically significant effect as compared to the WT type receptor. Each curve is an average of 3 independent experiments. Network of polar interaction between Y659, S809 and W785 is reframed by PAM binding (G-J). mGlu 5 inactive state relies on W785, Y659, S809 forming polar contact with the NAM alloswitch-1 ligand (G; PDB 7P2L). W785 and F788 are both inwards orientated preventing the binding of the PAM in the 7TM PAM-free (H; Supplementary Fig. 4), Y659, S809 and W785 making polar contact as in NAM bound conformation. Ago-PAM VU0424465 binding destibilises polar interactions between Y659, S809 and W785 (I). VU29 pushes away Y659 and W785 but is stabilised by F788 (J). VU0424465 and VU29 stabilise different 7TM conformations.

  25 mM HEPES (pH 7.4), 10 mM MgCl 2 , 20 mM KCl, 5 mM EDTA, 1 mM PMSF (Sigma Aldrich), and the complete protease inhibitor cocktail tablet (Roche). The pellets were washed two times with the lysis buffer and an additional wash performed using high salt concentration of sodium chloride (25 mM HEPES (pH 7.4), 10 mM MgCl 2 , 20 mM KCl, 1 M NaCl). Membrane fraction was harvested by centrifugation at 45,000 rpm (≈200.000g) for an hour and resuspended with a lysis buffer supplemented with 40% glycerol and then stored at -80°C. Membrane preparation from 2 litres of cell culture expression were resuspended into a final volume of 120 mL of 25 mM HEPES buffer (pH 7.4), 0.4 M NaCl, 10% Glycerol,10 µM quisqualate (Abcam), 10 mM iodoacetamide (Sigma), and the complete protease inhibitor cocktail tablets and complemented with 10 µM of either PAM such as, VU0424465[START_REF] Nasrallah | Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations[END_REF] , PAMs VU29 (med chem express) VU0409551 (R&D system), The mixture was then incubated at room temperature (RT) for 1 hour and the membranes were solubilised by adding 1% (w/v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace), and 0.2% (w/v) cholesteryl hemisuccinate (CHS, Anatrace) for 1.5 hours at 4°C, under shaking. Solubilised receptors were harvested by centrifugation at 45,000 rpm for 1 hour and supplemented with 10 mM imidazole (Sigma) before loading onto a Nickel-NTA (Ni 2+ ) resin (HisTrap HP, GE Healthcare) at 0.5 mL/min, overnight at 4°C. The resin was pre-equilibrated in 25 mM HEPES (pH 7.4), 400 mM NaCl, 10 µM quisqualate, 10 µM of PAM, 5% (v/v) glycerol, 0.2% (w/v) DDM, 0.04% (w/v) CHS, and 10 mM imidazole. The resin was washed using a step gradient of imidazole with concentrations of 10mM, 20mM, 40mM and 50 mM and the receptor was eluted with 25 mM HEPES (pH 7.4), 400 mM NaCl, 10 µM quisqualate, 10 µM of PAM, 5% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, and 250 mM imidazole.The eluted receptors were incubated onto a Flag affinity resin (Anti-Flag M1, Sigma) for 1 hour at 4°C, washed with 10 column volumes of a wash buffer (25 mM HEPES (pH 7.4), 150 mM NaCl, 10 µM quisqualate, 10 µM PAM, 0.03% (w/v) DDM, 0.006% (w/v) CHS, and 2 mM Ca 2+ ). The receptor was eluted with 0.2 mg/mL Flag peptide in a buffer containing 25 mM HEPES pH 7.4, 0.15 M NaCl, 10 µM quisqualate, 10 µM VU0424465, 0.03% DDM (w/v), 0.006% CHS (w/v), 2 mM EDTA. The eluted fractions were then concentrated using a Amicon Ultra-4 centrifugal concentrator 100 kDa (Millipore), centrifuged for 10 minutes at 80,000 rpm to eliminate aggregates, and then loaded on a size exclusion chromatography (SEC) column (Superdex 200 Increase 10/300, GE Healthcare) pre-equilibrated with 25 mM HEPES pH 7.4, 0.15 M NaCl, 10 µM quisqualate and 10 µM PAM, 0.03% DDM (w/v) and 0.006% CHS (w/v). Eluted and purified mGlu 5 -5M receptor and mGlu 5 -5M_W785A receptor mutant bound to quisqualate and PAM were concentrated to 5-6 mg/ml using Amicon Ultra-4 centrifugal concentrator 100 kDa (Millipore) for cryoEM grid preparation and analysis.CryoEM sample preparation, data collection and image processing:VU0424465 (PAM1) with K3 detector : The agonist quisqualate and PAM VU0424465

Fig. 1 .

 1 Fig. 1. Local resolutions were estimated with Relion 42 .

  5 Å after postprocessing, as determined by Fourier shell correlation (FSC) at 0.143. The map was further enhanced through one round of Bayesian Polishing and two rounds of CTF refinements following the method outlined by Zivanov et al. (2018)[START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF] , resulting in a final resolution of 2.7 Å. C2 symmetry expansion and 3D classification with focus on the TMD (using a T value of 128 and no image alignment) revealed the presence of two conformers.Particles corresponding to these two conformers were re-refined separately in Relion[START_REF] Scheres | Implementation of a Bayesian approach to cryo-EM structure determination[END_REF] .Particles associated with these two conformers were subsequently imported into Cryosparc v4.3.0[START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] . Duplicate particles arising from symmetry expansion were eliminated before the import. Non-uniform (NU) refinement, local CTF refinement, and local refinement were carried out independently for each conformer. These refinements yielded overall resolutions of 3.2 Å (2.9 Å) and 3.5 Å (3.0 Å) for conformer 1 (PAM1_d2_c1) and conformer 2 (PAM1_d2_c2), respectively (the numbers in brackets are the resolution with tight mask and corrected FSC in cryosparc). The complete workflow is described in Extended Data Fig.2. Local resolutions were estimated with Relion 42 .

  combined data with 260,251 particles after 3D refinement and postprocessing resulted in a 4.3 Å map.The combined data was subsequently imported into Cryosparc v4.3.0[START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] and ab-initio reconstruction was performed with 5 classes and maximum resolution of 12 Å followed by heterogenous refinement. Three of the best classes or volumes were combined (that had the same resolution) and both homogenous and Non-uniform (NU) refinement[START_REF] Punjani | Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction[END_REF] were performed resulting in 4.3 Å and 3.6 Å maps respectively. Local and global CTF refine was then carried out on the particles with the volume from NU refinement and subsequent refinement resulted in an overall resolution of 3.4 Å. Local resolution was estimated in relion and the local resolution of the maps revealed that the VFT of the receptor had higher resolution than the CRD and the TMD as observed for the other data sets described in this study.

Fig. 1 .

 1 Fig.1. CryoEM structure of orthosteric agonist quisqualate and VU0424465 ago-PAMbound conformation of the mGlu 5 receptor. (A) mGlu 5 structure display symmetrical binding of the ago-PAM VU0424465 to both 7TM as well as both VFTs display bound quisqualate. (B) CryoEM map (magenta) sharpened with B=-95 Å 2 at 8 σ showing the density for the ago-PAM VU0424465 in both monomers surrounded by the TM helices and key side chain residues are shown in stick representation (carbon atoms in blue and green) and ligand carbon atoms in yellow. (C) The active state is stabilised by a limited number of residues localised at the top of TM6 from both 7TM, with an asymmetrical distribution. Molecular contact between N796 (ECL3) and D722, P724, Y730 (ECL2) observed only in one protomer. (D, E) Ligand binding site of VU0424465 in each protomer. VU0424465 make polar interaction with S805 and with the W785 carbonyl. It is stabilised by hydrophobic contact involving M802, V806, S809 in TM7; W785, Y793, F788, L789 in TM6 pushing out F788. (F) VU0424465 induces a shift of TM6 along its helical axis and towards the extra cellular side of the receptor. We could resolve the density for ICL2 that displays an extended helix 3 that stretches out of the detergent micelles. Figures were generated usingpymol.

Fig. 2 .

 2 Fig.2. Conformational diversity of positive allosteric modulation of mGlu 5 receptor dimer. (A) CryoEM structure of the PAM VU29-bound mGlu 5 with only one molecule modelled and the VU29 binding mode. (B -F) Structure comparison of mGlu 5 receptor dimer bound to ago-PAM VU0424465, PAMs VU29 and VU0409551 and VU0424465-bound to mGlu 5 W785A mutant was performed. 3D alignment of residues 25-191 and 331-461 of both lobe-1 of the PAM1_d1 data set (one protomer in green and the other in blue) (VU0424465bound mGlu 5 receptor) was performed for VU29 (PAM2, yellow); VU0409551 (PAM3, light purple); the different conformations of VU0424465 including PAM1_d2_c1 (salmon) and PAM1_d2_c2 (cyan) and VU0424465-bound mGlu-5 W785A mutant (PAM3_c2, olive).

Fig. 3

 3 Fig.3 CryoEM structure of intermediate-active state (Rcc) with quisqualate bound but PAM free. (A and B) Two views of cryoEM map of the Rcc conformation illustrating molecular interaction between the ICL2 loops. (C) 3D model of the Rcc quisqualate-bound state show closed conformation of the VFTs induced by quisqualate binding. (D) Comparsion of monomers of Rcc model with antagonist-bound Roo state (PDB 7FD9) and the agonist and PAM-bound Acc state (PAM1_d1; green) conformation. (E) Superimposition of VU0424465-bound mGlu 5 structure (PAM1_d1; green) and intermediate active state (Rcc) bound quisqualate but PAM free (PAM3_c2; purple). (F and G) Quisqualate standard binding pose as observed in VU0409551-bound mGlu 5 structure and its alternate binding mode in intermediate-active state (Rcc). (H and I) The 7TMs of Rcc quisqualate-bound state undergo a tilt along the perpendicular axis to the membrane as P790 at the top of TM6 moves aways from each other compared to fully inactive state, before they come into close proximity in the Acc active state of the receptor. In the intracellular side the ICL2s cross and meet in the Rcc intermediate active state. ICL2 movement is illustrated by the L673 and A690 since the loop is not modelled in the fully inactivated state with open VFTs (o) and resting conformation (R), Roo (PDB 7FD9).

Extended Data Figure 1 .Extended Data Figure 2 .Extended Data Figure 4 .Extended Data Figure 9 .

 1249 CryoEM analysis of VU0424465 and quisqualate bound to full-length mGlu 5 (mGlu 5 -Δ856). (A) A representative micrograph of the VU0424465 and quisqualate bound FL mGlu 5 in detergent micelles imaged on ice and collected on K3 detector. Scale bar is 500 Å. (B) Select reference-free 2D class averages of mGlu 5 with a box size of 476 pixels and sampled at 0.84 Å. (C) The image processing workflow of the mGlu 5 with all the steps performed in cryosparc. The final 3D maps with sharpened with a B-factor of -45 Å 2 and generated with deepEMhancer using the halfmaps and a mask are shown. The arrow marks the ICL2 loop in the deepEM map that was used for model building. D) CryoEM maps coloured according to the local resolution plot of FL mGlu 5 as determined with Relion 4.0 are shown in two different views. Much of the VFT is at a higher resolution and the CRD and the TMD are at lower resolution along with the detergent-lipid micelle. (E) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 3.1 Å and the one of map vs model at FSC 0.5 is 3.4 Å. CryoEM analysis of VU0424465 and quisqualate bound to full-length mGlu 5-5M (mGlu 5 -Δ856) with the data collected with Falcon 4i. (A) A representative micrograph of the VU0424465 and quisqualate bound FL mGlu 5 in detergent micelles imaged on ice and collected on cFEG, selectris and Falcon IV detector, with some particles marked with white circle. Scale bar is 100 Å. Selected reference-free 2D class averages of mGlu 5 with a box size of 256 pixels. The image processing workflow of the mGlu 5 with the steps performed in Relion and cryosparc. B) CryoEM maps coloured according to the local resolution plot of FL mGlu 5 conformer 1 as determined with Relion 4.0 are shown in two different views. Much of the VFT is at a higher resolution and the CRD and the TMD are at lower resolution along with the detergent-lipid micelle. (C) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown for conformer 1. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 3.2 Å and the one of map vs model at FSC 0.5 is 3.3 Å. D) CryoEM maps coloured according to the local resolution plot of full-length mGlu 5 conformer 2 as determined with Relion 4.0 are shown in two different views. Much of the VFT is at a higher resolution and the CRD and the TMD are at lower resolution along with the detergent-lipid micelle. (C) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown for conformer 2. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 3.5 Å and the one of map vs model at FSC 0.5 is 3.6 Å. Extended Data Figure 3. CryoEM analysis of VU29 and quisqualate bound to full-length mGlu 5 (mGlu 5 -Δ856). (A) A representative micrograph of the FL mGlu 5 with VU29 in detergent micelles imaged on ice and collected on Falcon 4i detector. Scale bar is 100 Å. (B) Selected reference-free 2D class averages of mGlu 5 with VU29 and quisqualate bound with a box size of 476 pixels and sampled at 0.84 Å. (C) The image processing workflow of the mGlu 5 with all the steps performed in cryosparc. The final 3D maps with the unsharpened and one generated with deepEMhancer using the half-maps and a mask are shown. D) CryoEM maps colored according to the local resolution plot of FL mGlu 5 as determined with Relion 4.0 are shown in two different views. Much of the VFT is at a higher resolution and the CRD and the TMD are at lower resolution along with the detergent-lipid micelle. The TMD of the VU29 data set is of lower quality than the other data sets with PAM bound. (D) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 3.3 Å and the one of map vs model at FSC 0.5 is 3.7 Å. CryoEM analysis of VU0409551 and quisqualate bound to full-length mGlu 5 (mGlu 5 -Δ856) in an active conformation. (A) A representative micrograph of the VU0409551 and quisqualate full-lenght mGlu 5 -5M in detergent micelles imaged on ice and collected on K3 detector. Scale bar is 500 Å. (B) Selected reference-free 2D class averages of VU0409551 mGlu 5 with a box size of 440 pixels and sampled at 0.84 Å. (C) The image processing workflow of the mGlu 5 with all the steps performed in cryosparc. The final 3D maps with either sharpened with a B-factor of -30 Å 2 and generated with deepEMhancer using the half-maps are shown. D) Cryo EM maps coloured according to the local resolution plot of FL mGlu 5 as determined with Relion 4.0 are shown in two different views. Much of the VFT is at a higher resolution and the CRD and the TMD are at lower resolution along with the detergent-lipid micelle. (D) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 3.0 Å (the tight mask in cryosparc estimates 2.8 Å) and the one of map vs model at FSC 0.5 is 3.4 Å. Extended Data Figure 5. CryoEM analysis of VU0424465 and quisqualate bound mGlu 5 W785A mutant. (A) A representative micrograph of the full-length mGlu 5 -5M_W785A in detergent micelles imaged on ice and collected on Falcon 3 detector. Scale bar is 500 Å. (B) Selected reference-free 2D class averages of mGlu 5 W785A mutant with a box size of 384 pixels and sampled at 1.07 Å. (C) The image processing workflow of the mGlu 5 W785A mutant. All the initial steps in image processing were performed with Relion 4.0 and the steps shown in red rectangle were performed with cryosparc 4.3.0. D) CryoEM maps coloured according to the local resolution plot of FL mGlu 5 W785A mutant as determined with Relion 4.0 are shown in two different views. Much of the VFT is at a higher resolution and the CRD and the TMD are at lower resolution along with the detergent-lipid micelle. (E) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 3.4 Å (the tight mask after correction in cryosparc estimates 3.3 Å) and the one of map vs model at FSC 0.5 is 3.7 Å. Extended Data Figure 6. CryoEM analysis of quisqualate bound to Full-lenght mGlu 5 (mGlu 5 -Δ856) in a Rcc conformation. (A) A representative micrograph of the quisqualate FL mGlu 5 in detergent micelles imaged on ice and collected on K3 detector. Scale bar is 500 Å. (B) Selected reference-free 2D class averages of Rcc conformation of mGlu 5 with a box size of 416 pixels and sampled at 0.84 Å. The separated TMD can be clearly seen in the class averages. (C) The image processing workflow of the mGlu 5 with all the steps performed in cryosparc. The final 3D maps with either sharpened with a B-factor of -129 Å 2 and generated with deepEMhancer using the half-maps and a mask are shown. The arrow marks the ICL2 loop in the deepEM maps that was used for model building. D) CryoEM maps coloured according to the local resolution plot of FL mGlu 5 as determined with Relion 4.0 are shown in two different views. Much of the VFT in one monomer are at higher resolution and due to dynamic nature, the other monomer is of lower resolution. The CRD and the TMD in both monomers are at lower resolution along with the detergent-lipid micelle. (D) The Fourier shell correlation (FSC) curves of two half-maps with a mask (blue) and the map and model (red) are shown. The estimated resolution by comparison of the two half-maps from refinement and postprocessing at FSC 0.143 is 4.1 Å (the tight mask after corrections in cryosparc estimates 3.9 Å) and the one of map vs model at FSC 0.5 is 4.3 Å.Extended Data Figure8. Functional consequences of mutations in the second intracellular loops (ICL2) of mGlu 5 on PAM activity. Six different residues from the ICL2 of mGlu 5 -Δ856 were mutated into alanine by site-directed mutagenesis. The influence of these mutations on the dosedependent potentiation of agonist-induced response was compared to the mGlu 5 -Δ856 receptor, using the IP-One HTRF assay in HEK293 cells transiently transfected with the indicated receptor. A, C, E. Comparison of the mean pEC50 ± SEM of VU0424465, VU29 and VU0409551, as determined from 3 independent dose-response experiments for all mutants except for K684A (n=4) and for mGlu 5 -Δ856 (n=7), in presence of a fixed concentration of the agonist quisqualate (10 nM) and different concentrations of the indicated PAM. B, D, F. Dose-dependent potentiation by VU0424465, VU29 and VU0409551 of the response induced by 10 nM of the agonist quisqualate on mGlu 5 -Δ856 and the mutants. Each data point corresponds to mean ± SEM of 3 independent experiments for all mutants except for K684A (n=4) and for mGlu 5 -Δ856 (n=7). Data are normalized to the response measured on the mGlu 5 -Δ856 receptor. Concentration-response curve parameters are listed in Supplementary Table3. Difference between mGlu 5 -Δ856 and mutants were analyzed by a One-way ANOVA followed by Dunnett's post hoc test. * p<0.05. Dose-dependent potentiation of agonist-induced response of mGlu 5 by VU0424465, VU29 and VU0409551. The ability of the PAMs to positively modulate the dosedependent response of mGlu 5 to the agonist quisqualate was determined by the measurement of IP 1 accumulation in HEK cells transiently transfected with mGlu 5 -Δ856. A. Effect of VU0424465. B. Effect of VU29. C. Effect of VU0409551. Data are normalized to the response measured for quisqualate alone and are mean ± SEM of at least three independent experiments performed in duplicate. Parameters from the dose-response curves are listed in Supplementary

10 .

 10 Functional consequences of mutations in the second and third extracellular loop (ECL2, ECL3) of mGlu 5 on PAM activity. Three different residues from the ECL2 and one residue of the ECL3 of mGlu 5 were mutated into alanine by site-directed mutagenesis. The influence of these mutations on the dose-dependent potentiation of agonist-induced response was compared to the mGlu 5 -Δ856 receptor, using the IP-One HTRF assay. Comparison of the mean pEC50 ± SEM of VU0424465, VU29 and VU0409551, as determined from three independent doseresponse experiments in presence of a fixed concentration of the agonist Quisqualate (10 nM) and different concentrations of the indicated PAM (A, C and E). Dose-dependent potentiation by VU0424465, VU29 and VU0409551 of the response induced by 10 nM of the agonist quisqualate of mGlu 5 -Δ856 and mutants (B, D and F). Each data point corresponds to mean ± SEM of three independent experiments. Data are normalized to the response measured on the WT receptor. Concentration-response curve parameters are listed in Supplementary Table2. Difference between mGlu 5 -Δ856 and mutants were analyzed by a One-way ANOVA followed by Dunnett's post hoc test. * p<0.05, ** p<0.01.

a-

  The resolution mentioned in the parenthesis are with tight mask and after correction. b -The resolution range as estimated by local resolution c -The auto B-factor sharpened map was used for model refinement. For map interpretation/model building, maps sharpened with multiple B-factors and deepEMhancer maps were used. d -B-factor values for both chains are indicated e -The rotamer outliers calculated either with Molprobity within Phenix or with PDB validation server, which is shown in parenthesis.

  6.50 and F7886.53 are pointing towards the helical bundle (Fig

4H

). Although W785 stabilises the VU04244645 in PAM1 models (Fig.

1

), the presence of F788 is likely to cause steric hindrance and allows us to exclude the possibility that the PAM in the 7TM have lower occupancy in the intermediate active state Rcc. We defined this conformation as a PAM-free intermediate-active state. On hindsight, this observation supports the view that the PAMs stabilise the active Acc conformation. The weaker the PAM, the less stabilised is the Acc state with agonist quisqualate bound in the VFT, allowing particles to distribute between the intermediate-active Rcc and the Acc state. Ago-PAM that induces substantial mGlu 5 receptor signalling in absence of orthosteric agonist is likely due to the propensity of the PAM to stabilise the mGlu 5 receptor active state, although we cannot exclude that PAM can activate isolate 7TMs

  3.44 is a key residue for PAM signal transduction and for NAM inhibition of the receptor. Interestingly, Y659A mutant has no effect on signal transduction induced by the orthosteric agonist quisqualate, suggesting that the receptor dimer can still sample the 7TM asymmetric dimer interface, in absence of PAM. The strong stabilising potential of the ago-PAM VU0424465

Table 4 .

 4

	normalized [IP1] / no VU0424465	0 50 100 150
				-12	-10	-8	-6	-4	-2
						[Quisqualate]
	no VU0424465		+10nM VU0424465	+30nM VU0424465
	+100nM VU0424465	+300nM VU0424465	+1µM VU0424465
	normalized [IP1] / no VU0424465	0 50 100 150
				-12	-10	-8	-6	-4	-2
						[Quisqualate]
				no VU29		+100nM VU29	+300nM VU29
				+1µM VU29		+3µM VU29	+10µM VU29
	normalized [IP1] / no VU0409551	0 50 100 150
				-12	-10	-8	-6	-4	-2
						[Quisqualate]
	no VU0409551		+100nM VU0409551	+300nM VU0409551
	+1µM VU0409551		+3µM VU0409551	+10µM VU0409551

Cryo-EM data collection, refinement and validation statistics

	factor (Å 2 ) c
	Model composition
	Non-hydrogen atoms No. of polypeptide Protein residues Ligands Waters	mGlu 5 +quisqualate +VU0424465 (EMDB-37973) (PDB 8X0B) 12415 2 6140 51 33	mGlu 5 + quisqualate +VU0424465 (conf 1) (EMDB-37974) (PDB 8X0C) 12180 2 6039 51 -	mGlu 5 +quisqualate +VU0424465 12176 2 6037 (conf 2) (EMDB-37975) (PDB 8X0D) 51 -	mGlu 5 W785A +quisqualate +VU0424465 12086 2 6016 (EMDB-37976) (PDB 8X0E) 27 -	mGlu 5 +quisqualate +VU29 12073 2 5995 (EMDB-37977) (PDB 8X0F) 83 -	mGlu 5 +quisqualate +VU0409551 12129 2 6022 (EMDB-37978) (PDB 8X0G) 27 31	mGlu 5 12317 +quisqualate 2 Rcc state 6140 27 (EMDB-37979) (PDB 8X0H) -
	B factors (Å 2 ) d	PAM1_d1 155.4	PAM1_d2_c1 38.9	PAM1_d2_c2 98.1	183.8	PAM2 121.7	PAM2_c1 68.6	PAM3_c2 88.3
	Protein Data collection and NAG processing Magnification (x) QUS Voltage (kV) PAM Electron exposure (e-water	155.5/155.6 242.7/252.3 105000 85.2/76.6 300 150.2/148.2 51.7 73.5	39.0/38.9 39.7/39.4 165000 34.3/34.2 300 41.5/41.3 50 -	96.7/99.8 120.7/127.2 165000 38.5/36.3 300 72.9/88.7 50 -	185.7/182.0 258.5/232.7 75000 102.2/101.8 300 -28.25 -	10.8/122.5 117.8/118 165000 74.3/69.3 300 181.3 49.6 -	68.6/68.6 68.7/68.8 105000 67.3/67.2 300 -49.55 57.1	74.8/102.0 73.7/79.2 39.1/76.1 105000 -300 49.55 -
	/Å 2 ) Defocus range (µm) R.m.s. deviations Pixel size (Å) Bond lengths (Å) Symmetry imposed Bond angles (°)	-0.8 to -2.2 0.84 0.006 C1 1.0	-0.5 to -1.2 0.727 0.006 C1 0.95	-0.5 to -1.2 0.727 0.006 C1 1.05	-1.0 to -3.5 1.07 0.006 C1 1.0	-0.5 to -1.2 0.727 0.006 C1 0.7	-1 to -2.2 0.84 0.006 C1 0.95	-1 to -2.2 0.004 0.84 C1 0.7
	Initial particle images Validation (no.) Final particle images MolProbity score Clashscore (no.) Map resolution (Å) Rotamer outlier (%) e	2928252 551163 1.9 8.3 3.1 (3.0) 0.9 (1.2)	4481289 92933 1.9 9.4 3.2 (2.9) 0.5 (1.5)	4481289 41866 2.1 11 3.5 (3.0) 1.3 (1.5)	783666 188960 2.1 9.2 3.4 (3.3) 1.3 (1.4)	1418090 113563 2.1 13 3.5 (3.2) 0.23 (3)	3416003 290960 2.0 10.6 3.0 (2.8) 0.5 (1.7)	3416003 2.3 344232 17.1 4.1 (3.9) 0 (0.7)
	FSC threshold (0.143) a Map resolution range Ramachandran plot Favored (%) (Å) b Allowed (%)	2.8-12.8 91.9 7.6	2.8-12.9 94.7 5.2	3.1-15 93.2 6.7	3.2-13.6 91.8 8.0	3.1-10 91.5 8.5	2.8-14 91.9 7.8	3.6-14 89.1 10.8
	Refinement Disallowed (%)	0.5	0.1	0.1	0.2	0	0.3	0.1
	Initial model used	7FD8	PAM1_d1	PAM1_d1	PAM1_d1	PAM1_d1	PAM1_d1	7FD9+PAM1_d1
	(PDB code)
	Model resolution (Å)	3.4	3.3	3.7	3.7	3.7	3.4	4.3
	FSC threshold 0.5
	Model resolution range	191.5-3.1	184.1-3.2	184.1-3.5	177.6-3.4	183-3.2	186.5-3.2	186.5-4.1
	(Å)
	Map sharpening B	-95	-42	-36	-115	-78	-63	-130

Acknowledgement:

We acknowledge the MRC Laboratory Molecular Biology EM and computing core facilities (Cambridge, UK), the European Synchrotron Radiation Facility for provision of beam time on CM01 and we would like to thank Alessandro Grinzato and Gregory Effantin for assistance (Grenoble, France). We acknowledge the METi imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We acknowledge the platform of pharmacology Arpeges of the Institut de Génomique Fonctionnelle (Montpellier, France). We acknowledge the National cryoEM Facility at Bangalore for data collection. L.B. was supported by Ligue Contre le Cancer. GF was supported by SWITCH-ON (ANR-20-CE11-0019). J.F.-I., and A.L. were supported by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF -A way of making Europe (PID2020-120499RB-I00) and by the Catalan government (2021 SGR 00508). G.C. was supported by Medical Research Council grant MC-U105184322. K.R.V. acknowledges the DBT B-Life grant DBT/PR12422/MED/31/287/2014, and the support of the Department of Atomic Energy, Government of India, Government of India, under Project Identification No. RTI4006. KRV is part of EMBO Global Investigator Network. G.L. was supported by SWITCH-ON (ANR-20-CE11-0019) and Ligue Contre le Cancer.

Data availability. The cryoEM density map and model coordinate have been depositied in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) respectively, under the accession code : EMDB-37973 and PDB-8X0B (PAM1_d1, VU0424465-and quisqualate -bound mGlu 5 -5M, active state) ; EMDB-37974 and PDB-8X0C (PAM1_d2_c1, VU0424465-and quisqualate -bound mGlu 5 -5M, active state) ; EMDB-37975 and PDB-8X0D (PAM1_d2_c2, VU0424465-and quisqualate -bound mGlu 5 -5M, active state) ; EMDB-37976 and PDB-8X0E (PAM1, VU0424465-and quisqualate-bound mGlu 5 -5M-W785A, active state) ; EMDB-37977 and PDB-8X0F (PAM2, VU29-and quisqualate-bound mGlu 5 -5M, active state); EMDB-37978 and PDB-8X0G (PAM3_c1, VU0409551-and quisqualate-bound mGlu 5 -5M, active state); EMDB-37979 and PDB-8X0H (PAM3_c2, quisqualate-bound mGlu 5 -5M, intermediate-active state, Rcc).

Author contributions: GC and LB contributed equally to the work and share the co-first authorship. LB performed expression and purification of the full-length mGlu 5 -5M bound to quisqualate and three different PAM samples for cryoEM analysis, with the help of GF. LB, AF performed grid freezing. GC prepared cryoEM grids, screened, collected and processed cryoEM data. AK performed data collection at TFS cryoEM facility. FM performed and analysed pharmacological experiments on the mGlu 5 receptor and receptor mutants under the supervision CG. JFI synthesized the VU0424465 under the supevision of AL. KRV and GL performed image processing and building/refinement of full-length mGlu 5 -5M models. KRV and GL interpreted the data and wrote the manuscript with contributions from all the authors. GL designed, supervised and coordinated the project.

Competing interest. The authors declare no competing interestes

no VU -6,65 0,02 0,00 0,00 100,00 0,00