CryoEM structure of mGlu6 captures receptor activation prior to G protein coupling

Domain: chemistry
Published: 2026-03-09
Editorial outcome: minor_revision

Abstract
The metabotropic glutamate receptor 6 (mGlu6) is essential for synaptic communication of rod
photoreceptors, and mutations in mGlu6 lead to a blinding disorder. However, its structural
organization remains unknown. Here, we present the structure of agonist-bound mGlu6, revealing
an asymmetric dimer arrangement in the absence of a G protein. This indicates that agonist binding
alone can induce the homodimeric receptor asymmetry in metabotropic glutamate receptors and
structurally prime mGlu6 for activation by pre-organizing the transmembrane domain dimer
interface for G protein binding. The structure also identifies noncanonical interactions between the
cysteine-rich domain and extracellular loop 2, forming a unique interface that likely stabilizes the
activation state. Mutational analyses of this interface reveal its role in maintaining rapid Gαo
activation and surface targeting. The structure also permits mechanistic investigation of congenital
stationary night blindness and reveals diverse effects of pathogenic mutations on surface
trafficking, Gαo coupling, and activation dynamics, including unexpected gain-of-function. These
results provide critical insight into the intermediate asymmetric structure of mGlu6 and offer a
molecular framework for understanding the pathogenesis of inherited retinal disorders.

Introduction
Vision is an essential sensory function that allows organisms to detect, interpret, and respond to
their environment, shaping both perception and behavior. In the mammalian retina, visual
processing begins through distinct vertical pathways established at the synapse between
photoreceptors and bipolar cells (BCs)1. In the dark, photoreceptors continuously release glutamate
from their synaptic terminals. However, exposure to light causes photoreceptors to hyperpolarize,
leading to a reduction in glutamate release2. This reduction is sensed by the postsynaptic partners
of photoreceptors, the two types of bipolar cells3,4. The ON-bipolar cells (ON-BCs) depolarize
when light intensity increases, while OFF-bipolar cells hyperpolarize3. While cone photoreceptors
signal to both ON- and OFF-BCs, rods transduce their signals exclusively to ON-BCs. Thus, the
synaptic dysfunction in ON-BCs leads to an inability to see in dim light, a genetically diverse
group of retinal disorders known as congenital stationary night blindness (CSNB).

A key component of synaptic transduction machinery in ON-BCs is the metabotropic
glutamate receptor (mGlu6), a principal neurotransmitter receptor which belongs to class C of G-
protein-coupled receptors (GPCRs). In darkness, glutamate released by photoreceptors binds to
mGlu6, leading it to activate the G protein Gαo. Activated Gαo then inhibits TRPM15-7, an effector
channel that mediates depolarizing response of ON-BCs. The rapid inactivation of Gαo requires a
GTPase-activating protein, known as a regulator of G protein signaling, which interacts with and
is regulated by another class C GPCR, GPR1798-10. However, the cascade is ultimately controlled
by changes in mGlu6 signaling upon light-driven fluctuation of synaptic glutamate concentration.
The molecular architecture of mGlu6 remains unknown, limiting our understanding of visual
signal transduction at the synapse between photoreceptors and ON-BCs.

mGlu6 belongs to the group III metabotropic glutamate receptor (mGluR) family, which also
includes mGlu4, mGlu7, and mGlu8, among the eight known mGluRs (mGlu1 to mGlu8).
Notably, mGlu6 is the only mGluR known to mediate direct synaptic transmission in the nervous
system11. Like other mGluRs, mGlu6 contains a large extracellular domain (ECD) that harbors the
orthosteric ligand-binding site12,13. The ECD is predicted to contain a ligand-binding Venus flytrap
(VFT) domain and a cysteine-rich domain (CRD). Canonically, in mGluRs, the VFT is linked to
the transmembrane domain (TMD) via the CRD, which acts as a hinge, transmitting
conformational rearrangements induced by glutamate binding to the VFT, ultimately leading to
TMD rearrangement. A key hallmark of class C GPCR activation is the reorientation of TM6 at
the dimer interface14-29. However, in the absence of structural information, the precise mechanism
by which glutamate binding to the VFT of m Glu6 induces conformational changes in the TMD to
facilitate subsequent Gαo activation remains unclear.

Agonist binding to GPCRs triggers structural rearrangements that promote subsequent G
protein coupling17,24,30-32. A hallmark of class C GPCR activation is the emergence of asymmetry
between the two protomers, typically allowing G protein binding to only one. However, whether
this asymmetry arises prior to or only upon G protein engagement remains unresolved. While
previous studies have shown that positive allosteric modulators (PAMs) can induce asymmetry in
mGluRs before G protein binding—thereby favoring coupling to the PAM-bound protomer33—it
is unclear whether such asymmetry can arise intrinsically in the absence of external modulators.
This uncertainty is primarily due to the lack of structural snapshots of intermediate states preceding
G protein association.

Here, we present the cryo-EM structure of human mGlu6 bound to L-serine O-phosphate (L-
SOP), a potent agonist of group III mGluRs. Unexpectedly, the structure reveals distinct features
not observed in other homodimeric mGluRs in the absence of G protein binding, including an
asymmetric domain arrangement that appears to structurally prime the receptor for G protein
coupling, and noncanonical interactions between the CRD and extracellular loop 2 (ECL2). The
mGlu6 structure enabled structure-function analysis of CSNB-associated mutations in the receptor
providing essential insights into the pathogenic mechanisms of CSNB.

Results
Structure determination of human mGlu6 homodimer bound to L-SOP
To better understand the activation mechanism of mGlu6, we purified the full-length human
receptor in the presence of L-SOP, a selective group III mGluR agonist with similar efficacy to
glutamate but with higher affinity34,35. Using cryo-EM, we resolved the structure of the human
mGlu6 homodimer bound to L-SOP at an overall resolution of 3.2 Å in C1 symmetry (Fig. 1,
Supplementary Figs. 1, 2, and Supplementary Table 1). The mGlu6 protomer comprises three key
structural domains: the VFT, the CRD, and the TMD (Fig. 1a). Reconstruction of the L-SOP-
bound mGlu6 homodimer was performed without imposing symmetry (C1), which revealed
substantial protomer bending that gives rise to a clear asymmetry between the two subunits. This
observation prompted us to perform further particle classification without alignment using the C1
reconstruction (Supplementary Fig. 1f), which yielded three distinct classes that primarily differed
in the degree of protomer bending and were refined to resolutions of 3.39 Å, 3.27 Å, and 3.51 Å,
respectively (Supplementary Figs. 1f and 2c–e). Unless otherwise noted, the consensus structure
prior to classification is referred to throughout the text.

To improve the local resolution of the TMD, we performed local reconstruction, which
yielded an improved TMD map at a resolution of 3.82 Å (Supplementary Figs. 1f and 2b). As a
result, the overall densities of the VFT domain, CRD, and TMD, as well as L-SOP, were well-
resolved. These domains assemble into a homodimer, with the entire complex measuring
approximately 95 Å × 70 Å × 175 Å (Fig. 1a). Notably, we identified potential glycosylated
asparagine residues (N296, N451, and N567) with extra densities extending from their side chains
(Supplementary Fig. 1g, h).

Cryo-EM structure of L-SOP-bound mGlu6
The mGlu6 dimer adopts a dimerization mode characterized by an active conformation at the TM6
interface (Fig. 1a) and represents key structural features of active mGluRs. First, the VFT domain
of mGlu6 adopts an ‘Acc’ (active closed–closed) conformation, a typical conformation of mGluRs
upon agonist binding (Fig. 1b)14-16,18,20,32,33,36-39. The inter-bottom lobe 2 (LB2) distances are 11.7
Å when measured from the Cα position of E227 and 21.7 Å from Q235, which is consistent with
measurements reported for active mGlu2 and mGlu533 as agonists typically reduce the inter-lobe
distance at LB2 of the ligand-binding domain (Supplementary Fig. 3a)14. Additionally, the Acc
conformation is accompanied by a reorientation of the VFT dimer interface, resulting in a reduced
angle of B-C helix14,36,38. In the L-SOP-bound mGlu6 structure, this angle is approximately 60°,
reduced from the 125° observed in the inactive mGlu7 of group III and consistently decreased
relative to other inactive mGluRs (Fig. 1b and Supplementary Fig. 3b)16. Moreover, an electrostatic
interaction is observed between D102 (helix B) and R186 (D/E loop) from the opposing chain in
the L-SOP-bound mGlu6 structure (Fig. 1b). This interaction is absent in the inactive mGlu7
structure (Fig. 1b), suggesting its potential role in stabilizing the active conformation. A previous
single-molecule FRET study of mGlu2 demonstrated that the corresponding interaction between
D95 and R177 (structurally equivalent to D102 and R186 in mGlu6) contributes to stabilizing the
Acc conformation of the VFT domain40. This finding indicates a conserved role of this electrostatic
interaction in supporting receptor activation. To assess the functional significance of the R186–
D102 interaction, we introduced an R186A mutation, disrupting the salt bridge, and examined its
effect on L-glutamate and L-AP4 potency. The mutation significantly increased the half-maximal
effective concentration (EC50) for L-glutamate- or L-AP4-induced G protein activation, indicating
reduced potency (Fig. 1c, d and Supplementary Fig. 4c, d). Additionally, a small but significant
increase in maximal efficacy (Emax) was observed, while cell surface expression levels remained
unchanged (Fig. 1d and Supplementary Fig. 4a, b). These data suggest that the R186–D102
interaction is crucial for both agonist potency and efficacy.

Second, the mGlu6 structure adopts a compact CRD conformation upon activation. The
distance measured from the Cα of C555 is 24 Å, aligning with the 20–25 Å range for active mGlu2,
mGlu3, and mGlu533. Additionally, the distance for V534 (corresponding to E527 in mGlu5), a
known activation marker in mGlu5, is reduced to approximately 10 Å, consistent with features of
the active conformation (Fig. 1e)14. Third, in the TMD, the dimer interface is formed by the TM6
helices of the two protomers, with a distance of 5 Å at I802 (Fig. 1e). This distance is a well-
established marker of activation in mGluRs14-20,32,38,39 (Supplementary Fig. 3c, d). The TMD dimer
interface is predominantly stabilized by hydrophobic interactions (Fig. 1e). Fourth, the ionic
locks—a conserved electrostatic interaction between residues K6713.50 and E7816.35 of TM3 and
TM6, along with the less well-conserved residues R6743.53 and E777 of TM3 and ICL3—are
disrupted in the L-SOP-bound mGlu6 structure due to TMD rearrangements, as observed in other
active mGluRs (Supplementary Fig. 3e)14,16,24,41-43.

Asymmetric domain architecture of L-SOP-bound mGlu6
To investigate the structural characteristics arising from mGlu6 homodimer asymmetry, chain A
and chain B were superimposed based on their TMD alignment (Fig. 2a). In the LB1 region of the
V the
VFT, the distance between helix C and helix C’ (where the prime denotes the corresponding
segment from the adjacent protomer) at residue L166 was approximately 12 Å. In the LB2 region,
the angle between helix F and helix F’ differed by 9.8°, further highlighting the asymmetry
between chain A and chain B (Fig. 2b). A comparison of helix F-F’ angles in other mGluR
structures with C1 symmetry shows similar values: mGlu2 (PDB: 7MTR) exhibits a twist of 10.9°,
while mGlu5 (PDB: 8TAO) has a twist of 11.6°, aligning closely with the asymmetry observed in
mGlu6 (Supplementary Fig. 5a).

For a more detailed comparative analysis of asymmetry, the hinge region between the VFT
and CRD was designated as hinge 1 (S516), while the hinge region between the TMD and CRD
was designated as hinge 2 (L576) (Fig. 2a). When chains A and B of L-SOP-bound mGlu6 were
compared by superimposing either LB2 or the TMD, the relative orientations of the CRD varied
significantly (Fig. 2c, d). Based on the LB2 alignment, the CRD orientation around hinge 1
revealed that chain A exhibits a slightly larger angle than chain B (Fig. 2c, e). When aligned by
the TMD, the CRD rotation around hinge 2 showed that chain A undergoes a slightly greater
rotation than chain B (Fig. 2d, f). These results suggest that mGlu6 displayed asymmetry at these
hinges, which may underlie the overall asymmetric organization of its domains.

We speculated that the bending observed in mGlu6 reflects dynamic flexibility rather than a
fixed conformation. To further investigate the structural heterogeneity of mGlu6, we performed
3D variability analysis44, which revealed a global bending motion of the mGlu6 structure
(Supplementary Movie 1). Consistently, particle classification without alignment using the C1
reconstruction (Supplementary Fig. 1f) resulted in three classes (referred to as classes 1–3), each
characterized by differing bending angles—smaller, comparable, or larger relative to the original
consensus map prior to classification (Fig. 2g). To quantify this variation, we aligned structures
by their TMDs and measured the distance between the Cα atoms of L166 in helix C from each
protomer, capturing the extent of inter-subunit bending (Fig. 2h). Class 1 displayed minimal
bending and a nearly symmetric conformation, including at the TMD interface (Supplementary
Fig. 5b–d). Class 2 showed a 9.5 Å increase in bending compared to class 1, closely matching the
configuration of the consensus structure. Class 3 exhibited the most substantial bending, with an
additional 5.9 Å increase in L166 separation compared to class 2, although the degree of
asymmetry at the TM6–TM6 interface remained similar (Fig. 2h). Together, these results suggest
that agonist-bound mGlu6 adopts a range of conformational states, accompanied by varying
degrees of inter-protomer asymmetry.

Interestingly, the C1 symmetry structure of L-SOP-bound mGlu6 exhibits an asymmetric
TM6–TM6 interface even in the absence of a PAM or G proteins. Chain B adopts a more overall
bent conformation compared to chain A, with TM6 shifting higher toward the VFT (Fig. 2i). In
the upward-shifted chain B protomer, Y790 points toward the opposing protomer, similar to the
conformation observed in the PAM-bound state of mGlu5 (Fig. 2i)38. This structural shift in TM6
and the associated domain orientation resemble conformations seen in other mGluR protomers
bound to G proteins (Supplementary Fig. 5e, f). These findings suggest that, despite the
symmetrical binding of L-SOP to both subunits, mGlu6 adopts an asymmetric conformation,
potentially priming chain B for G-protein binding in subsequent steps (Fig. 2j). Because full
agonists are known to engage nearly identical activation mechanisms and functional responses on
mGluR35,45, we speculate that this observed asymmetry is a general feature of mGlu6 activation
rather than a conformation specific to L-SOP, an idea that remains to be determined.

L-SOP binding mode at the glutamate binding site
We next examined the L-SOP binding site, where activation of mGlu6 is initiated upon agonist
binding (Fig. 3). L-SOP acts as a potent agonist for group III mGluRs, including mGlu635,46,47, and
is known to reduce cyclic AMP levels through mGluR-mediated inhibition of adenylyl cyclase
activity48. In our structure, L-SOP was bound to the canonical orthosteric site within the VFT
domain (Fig. 3a), consistent with the L-AP4 binding site in mGlu8 (PDB: 6BT5)49 and the L-SOP
binding site in mGlu4 (PDB: 8JD6)32,49 (Fig. 3c). However, the specific interactions with
surrounding residues were distinct in mGlu6.

In mGlu6, the phosphate group of L-SOP interacts with Q64 and R68 in the helix A region,
along with nearby residues K312 and K400 (Fig. 3a). Among these, Q64—previously suggested
to play a role in the selectivity of L-SOP for group III mGluRs35—forms a hydrogen bond with
the phosphate group of L-SOP (Fig. 3a, b). T177, which is also known to be involved in glutamate
binding, interacts with the serine group of L-SOP (Fig. 3a). The T177M mutation has been
associated with CSNB50. Structurally, this mutation likely disrupts ligand binding due to
alterations in side chain length and charge. Additionally, R192, another CSNB-associated mutation
site, does not directly interact with L-SOP but stabilizes the helix C/D loop involved in L-SOP
binding through interactions with the loop’s backbone Cα and van der Waals forces (Fig. 3a).
Mutations such as R192Q may disrupt these stabilizing interactions, compromising the loop’s
structural integrity and hindering ligand binding. The residues corresponding to R68, T177, D307,
and K400 are strictly conserved across all mGluR subtypes. Mutational analyses in other subtypes
have shown that mutations at these positions markedly reduce or abolish L-AP4- or L-glutamate-
induced binding and activation, indicating that they form part of the conserved orthosteric pocket.
Overall, our cryo-EM structure of L-SOP-bound mGlu6 identifies key residues involved in L-SOP
binding and provides structural insights into how CSNB-associated mutations near the glutamate
binding site destabilize the receptor, leading to functional impairment.

Allosteric coupling mediated by noncanonical interactions between the CRD and ECL2
It is well established that structural coupling from the VFT to the CRD is largely influenced by
CRD–ECL2 interactions14,16, ultimately driving rearrangements within the TMD. In this context,
to better understand how ligand-induced conformational changes in the VFT are communicated to
the 7TM domain via the CRD, we examined the lower portion of mGlu6 extending from the L-
SOP binding site (Fig. 4). Upon L-SOP binding, significant conformational changes in the VFT
bring the two CRDs closer together, forming an interface crucial for mediating structural
transitions in mGluRs14,16,18,51. In the active mGlu2 structure (PDB: 7EPB), this CRD interface is
stabilized by hydrophobic interactions16.

Notably, in the L-SOP-bound mGlu6 structure, the inter-CRD distance is even shorter than
in active mGlu4 (PDB: 8JD6), another group III mGluR. Based on the distance between the Cα
atoms of H539 residues located in β-sheet XVIII, the inter-CRD distance measures 9.2 Å in
mGlu6, while it is 12.2 Å in mGlu4 (Supplementary Fig. 6a, b). This unique configuration is
maintained by hydrophobic interactions involving V531 and V534, polar interactions between
H539 and the backbone of M530 in the opposing protomer, and histidine-histidine contact pairs
between H539 residues (Fig. 4a). These distinctive H539-H539’ and H539-M530’ interactions
define a CRD interface in mGlu6 that differs from those observed in mGlu2 and mGlu5 (Fig. 4a
and Supplementary Fig. 6c, d).

Notably, in the L-SOP-bound mGlu6 structure, we observed noncanonical CRD–ECL2
interactions. Compared to other mGluR groups, group III mGluRs feature a relatively longer
ECL2, which establishes an additional interface along the CRD (Fig. 4c, d)16,17. Specifically, E736
in ECL2 forms salt bridges with R563 and R572 in the CRD, contributing to the unique structural
configuration of mGlu6 (Fig. 4e). Additionally, R731 in ECL2 forms hydrogen bonds and a
cation–π interaction with the backbone of G560 and the side chain of Y546 in the CRD (Fig. 4f).
The lower CRD and ECL2 regions are further stabilized by hydrophobic interactions, which are
highly conserved in group III mGluRs (Fig. 4f).

To investigate the structural significance of the CRD–ECL2 interaction, we examined the
roles of R731 and E736 through functional analyses of mGlu6 mutants (R731A, E736A, and the
double mutant R731A/E736A), assessing surface expression, Gαo coupling efficacy, and
activation kinetics (Fig. 4g–j). Surface biotinylation studies revealed that the R731A and
R731A/E736A mutants exhibited significantly reduced surface expression, while E736A showed
levels comparable to wild-type (WT) (Fig. 4g and Supplementary Fig. 4a, b), indicating a key role
for R731 in maintaining proper surface expression of mGlu6. We then assessed Gαo activation
using a BRET-based kinetic assay, which detects L-glutamate-induced Gβγ dissociation from Gα
in real time (Fig. 4h and Supplementary Fig. 4e, f). Despite their reduced surface expression, the
R731A and R731A/E736A mutants generated maximal response amplitudes and pEC50
comparable to those of the WT receptor (Fig. 4i and Supplementary Fig. 4e, f). This indicates that
the receptor concentrations in our BRET assay were saturating, supporting the conclusion that the
observed slowness of activation (Fig. 4h, j) is unlikely to be a consequence of reduced receptor
abundance.

Having excluded receptor abundance as a confounding factor, we next compared activation
kinetics across mutants. Activation kinetics were notably slower in the R731A and R731A/E736A
mutants, as indicated by a reduced activation rate (1/τ) (Fig. 4h, j), whereas the E736A mutant was
unchanged. To confirm that this kinetic deficit was not simply a consequence of their reduced
surface expression (Fig. 4g), we titrated the expression of WT mGlu6 by transfecting varying
amounts of DNA (Supplementary Fig. 4g). This experiment revealed that the activation rate of
WT receptors remained rapid and was independent of the surface expression level, even when its
expression was reduced to levels comparable to the R731A mutants. This result validates that the
R731 residue is essential for rapid Gαo activation. Together, these results demonstrate that R731,
part of the unique ECL2 extension in group III mGluRs, is essential for efficient surface expression
and rapid Gαo activation, reflecting the functional importance of the CRD–ECL2 interaction in
mGlu6 signaling.

Functional characterization of CSNB-associated mGlu6 mutations
mGlu6 is essential for relaying signals from photoreceptors to ON-BCs, and mutations in its gene
cause CSNB52-54. To assess the functional impact of CSNB-associated mutations, we analyzed all
clinically reported mGlu6 variants identified in CNSNB patients. For each mutant we measured total
and surface expression, Gαo coupling efficacy, and Gαo activation kinetics (Fig. 5a–c). The
majority of these mutants exhibited reduced surface expression—less than 50% of that observed
in WT mGlu6. Among them, the W20_A24del mutant, located in the signal peptide region, showed
nearly undetectable localization of the surface. In contrast, G150S and D183N mutants displayed
only mildly reduced expression, while the V513M mutant maintained expression levels
comparable to WT mGlu6 (Supplementary Fig. 7a, b).

We next evaluated Gαo activation using a BRET-based kinetic assay, which measures L-
glutamate-induced Gβγ dissociation from Gα in real time (Fig. 5a and Supplementary Fig. 7c). To
account for variations in surface expression that could influence response generation, we
constructed a correlation curve by titrating WT mGlu6 cDNA. The relative surface expression
levels of the CSNB mutants, quantified from surface biotinylation data, were then compared with
their corresponding maximum response amplitudes. Mutants G51V, V513M, and E781K fell
within or near the 95% confidence interval, suggesting normal G protein coupling efficacy,
whereas most other mutants fell below the threshold, indicating significantly impaired signaling
(Fig. 5c).

To further evaluate activation kinetics, we calculated the activation rate (1/τ). Interestingly,
even among mutants with preserved coupling efficacy, a reduced activation rate was observed (Fig.
5b and Supplementary Fig. 7c, g). Although many of the CSNB mutants exhibited functional
impairments, the V513M mutant retained normal surface expression, G protein coupling efficacy,
and activation kinetics, suggesting that this mutation does not directly compromise mGlu6
signaling. The pathogenicity of this variant is therefore dubious, as evident from the literature55,56.
Notably, the R352C and I405T mutants displayed complex phenotypes. They exhibited clear
gain-of-function behavior in maximal efficacy (Emax), with responses significantly above the WT
confidence interval (Fig. 5c). However, this was not reflected in their potency values (pEC50) or
activation kinetics which remained comparable to WT (for R352C) or decreased (for I405T)
(Supplementary Fig. 7d–g). As R352 and I405 are located on the surface of the VFT domain (Fig.
5d, e), the structural basis for this gain-of-function remains unclear. Overall, these results show
that most CSNB-associated mGlu6 mutants exhibit defects in surface expression, G protein
coupling efficacy, or activation kinetics. While many act as loss-of-function mutations, as expected
for disease-associated variants, the unexpected gain-of-function observed in R352C and I405T
provides intriguing insights that warrant further investigation. Despite their enhanced G protein
coupling capacity, the reduced surface expression of R352C and I405T likely limits their access
to extracellular glutamate (Supplementary Fig. 7a, b). Together with their previously reported loss
of ELFN1 binding and synaptic mislocalization at ON-BCs57, these combined defects likely
ultimately lead to an overall loss-of-function phenotype from the physiological perspective.

Our L-SOP-bound mGlu6 structure provides the direct physical rationale for many of these
functional defects. The CSNB-associated mutation sites are distributed across the VFT domain,
CRD, and TMD of mGlu6 (Fig. 5d and Supplementary Figs. 7h, i and 8). For instance, G150S
resides in the VFT domain within a tightly packed hydrophobic environment composed of L44,
V148, A151, V156, and V160. These residues, located in the upper lobe, contribute to maintaining
its structural integrity. Substituting glycine with serine at position 150 likely disrupts these
hydrophobic interactions, destabilizing the upper lobe and impairing proper VFT activation (Fig.
5f). G464R, also in the VFT domain, is expected to compromise structural stability due to steric
clashes with nearby residues, including R446, resulting from the larger arginine side chain (Fig.
5f).

In the CRD and VFT, C423, located in the upper VFT domain, forms a disulfide bond with
C420, which stabilizes the interaction between helix L and the L/M loop (Fig. 5f). The C423R
mutation likely disrupts this disulfide bridge, weakening the domain’s structural cohesion.
Similarly, the C522Y mutation in the CRD disrupts a critical disulfide bond with C540, and the
bulkier tyrosine side chain introduces steric clashes with adjacent residues such as W538, further
compromising domain stability. Both C423R and C522Y have been reported to impair ER-to-
surface trafficking of mGlu6, possibly due to misfolding and reduced structural stability57,61.

In the TMD, R653, located in TM3, forms stabilizing interactions with M641 and A643. The
R653C mutation likely disrupts these electrostatic interactions, destabilizing the TMD. E781, a
residue involved in forming an ionic lock in the inactive conformation, becomes disengaged upon
L-SOP binding (Fig. 5f). The E781K mutation likely prevents proper ionic lock formation,
hindering normal activation of the receptor. Together, our structural mapping provides crucial
insights into how CSNB-associated mutations destabilize key domains, ultimately leading to the
observed functional impairments.

Methods
Expression and purification of mGlu6
The human mGlu6 WT (residues 1–877) sequence was cloned into a pFastBac vector with EGFP
and Twin-StrepII tags at the C-terminus, both of which were removable by 3C protease cleavage
(mGlu6 forward 5′-CGG ATC TCG GTC CGA AAC CGC CAC CAT GGC TCG GCC TCG
GAG-3′, mGlu6 reverse 5′-GTA CCG TGG ACG CTC TAG ATT CGA ATT TGT GGG CCT
CGG CAT CTT-3′). mGlu6 was expressed in Spodoptera frugiperda (Sf9) cells (ATCC) using the
Bac-to-Bac baculovirus expression system. Sf9 cells were authenticated by the vendor. For
expression, Sf9 cells at a density of 1.2 × 106 cells/ml were infected with baculovirus and cultured
at 27°C. Cells were harvested by centrifugation 48 h post-infection and stored at −80°C.

For purification, thawed cells were resuspended in a buffer containing 20 mM HEPES-NaOH
(pH 7.5), 200 mM NaCl, 20% glycerol, and 5 mM L-serine O-phosphate (L-SOP) with protease
inhibitors (1 mM PMSF, 4.7 μM leupeptin, 2 μM pepstatin A, and 2 μM aprotinin). To solubilize
the membrane, 1% (w/v) lauryl maltose neopentyl glycol (LMNG) and 0.1% (w/v) cholesteryl
hemisuccinate (CHS) were added, and the lysate was incubated at 4°C for 2 hours. Cell debris was
removed by ultracentrifugation at 100,000×g for 1 h.

The supernatant was incubated with Strep-Tactin resin (IBA Lifesciences) at 4°C for 3 hours.
The resin was washed with buffer containing 20 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 10%
glycerol, 5 mM L-SOP, 0.01 mg/ml brain lipid, 0.01% (w/v) LMNG, and 0.001% (w/v) CHS. The
protein was eluted using buffer containing 100 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 10%
glycerol, 5 mM L-SOP, 0.01 mg/ml brain lipid, 10 mM D-desthiobiotin, 0.01% (w/v) LMNG, and
0.001% (w/v) CHS.

The eluted protein was concentrated and incubated with 3C protease for 1 hour. The sample
was then loaded onto a Superose 6 Increase 10/300 column equilibrated with buffer containing 20
mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 1% glycerol, 5 mM L-SOP, 0.01 mg/ml brain lipid,
0.005% (w/v) LMNG, and 0.0005% (w/v) CHS. Peak fractions containing mGlu6 were pooled,
concentrated to 1.0–1.5 mg/ml, flash-frozen, and stored at −80°C for grid preparation.

Cryo-EM grid preparation and data acquisition
For grid preparation, 4 μl of protein sample was applied to glow-discharged Quantifoil 1.2/1.3
300-mesh Cu holey carbon grids (Electron Microscopy Sciences). The grids were blotted for 4.0 s
at 4°C under 100% humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI).
The prepared grids were imaged at the Institute for Basic Science (IBS) on a 300 kV Krios G4
cryo-EM (Thermo Fisher Scientific, TFS, USA) equipped with a Gatan K3 BioQuantum detector
using automated data acquisition. A total of 11,002 movies were collected at a pixel size of 0.848
Å per pixel, with a defocus range of −0.7 to −1.7 μm.

Data processing and 3D reconstruction
All data processing was performed using CryoSPARC v4.4.062. First, motion correction and CTF
estimation were carried out using Patch Motion Correction and Patch CTF Estimation, followed
by curate exposure to exclude micrographs with a CTF resolution worse than 5 Å.
Particles were initially selected using blob-based particle picking to generate templates for the
subsequent template-based particle picking. This resulted in a total of 5,718,287 particles picked
from 10,821 movies.

Particles were extracted with a box size of 432 pixels and then binned by 2 for more efficient
initial processing. This binned particle stack was subjected to several rounds of reference-free 2D
classification, and 433,734 high-quality particles were retained for further processing. An ab initio
reconstruction was performed to generate an initial map, followed by heterogeneous refinement.
The best class (244,570 particles) was re-extracted at full scale and subjected to homogeneous
refinement. Non-uniform (NU) refinement without symmetry constraints resulted in a 3.20 Å
resolution map, based on gold-standard Fourier shell correlation (GSFSC).

To assess conformational heterogeneity, 244,484 particles were re-classified in 3D
classification without alignment. The initial five classes were regrouped into three classes
depending on the degree of protomer bending. The regrouped particles were subjected to NU
refinement, yielding three structures with resolutions of 3.39 Å, 3.27 Å, and 3.51 Å, respectively.
To improve the TM domain map quality, 433,734 particles from ab initio reconstruction underwent
homogeneous refinement, NU refinement, and local refinement without symmetry, producing a
map with a resolution of 3.82 Å.

Model building and refinement
The initial model was generated using AlphaFold 3 on the AlphaFold Server
(https://alphafoldserver.com/about)63 and fitted into the global density map in UCSF ChimeraX64.
The model was manually adjusted using COOT65 and further refined with real-space refinement
in Phenix66. To improve the model of the transmembrane domain (TMD), the TMD portion of the
model was first refined into the 3.82 Å locally refined map. This improved TMD model was then
combined with the extracellular domain model (built into the 3.20 Å consensus map). This final
composite model was then subjected to global real-space refinement against the 3.20 Å consensus
map to optimize the overall geometry and resolve clashes. All structural figures were generated
using ChimeraX.

cDNA Plasmids
The mGlu6 construct (Cat# GRM6000000) was obtained from the Missouri S&T cDNA Resource
Center. All mGlu6 mutants were generated using conventional site-directed mutagenesis methods.
All constructs were confirmed by Eurofins Nanopore sequencing.

Cell culture and transfection
HEK293T/17 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), minimum essential medium non-essential amino acids (Life
Technologies), 1 mM sodium pyruvate, penicillin (100 U/mL), and streptomycin (100 µg/mL) at
37°C in a humidified incubator with 5% CO2. For transfection, cells were seeded in 3.5 cm dishes
at a density of 1 × 106 cells/dish. After 4 hours, cells were transfected with the appropriate
expression constructs using Metafectene Pro (Biontex Laboratories GmbH) according to the
manufacturer’s instructions. Transfected cells were harvested and used for experiments
approximately 36 hours post-transfection.

Cell surface biotinylation and Western blotting
mGlu6 surface proteins were labeled using the PierceTM Cell Surface Protein Biotinylation and
Isolation Kit (Thermo Fisher Scientific, Cat# A44390) according to the manufacturer’s
instructions. Approximately 2 × 107 cells were used per condition. Total protein lysates and
biotinylated eluates were mixed with sample buffer containing β-mercaptoethanol and incubated
at 42°C for 10 minutes. SDS-PAGE and Western blotting were performed following blocking with
5% non-fat milk. Blots were incubated with anti-mGlu6 antibody (Invitrogen, Cat# PA5-28770)
and anti-GAPDH (Millipore Sigma, Cat# MAB374) at 1:1000 dilution in 5% milk. HRP-
conjugated secondary antibodies (Kindle Biosciences) were used at 1:2000 dilution in 5% milk.
Images were acquired using a KwikQuant Imager and KwikQuant ECL Solutions (Kindle
Biosciences). Band intensities were quantified using Fiji software.

BRET assays for G protein activation
Cells were transfected in 3.5 cm dishes with 0.1 µg of mGlu6, 0.25 µg each of GαoA, Venus 156–
239-Gβ1, Venus 1–155-Gγ2, and masGRK3ct-Nluc-HA, using Metafectene Pro (3 µL/dish). At
the time of transfection, 0.1% Matrigel (Corning, Cat# CB-40230A) was added to the culture
medium. The total amount of cDNA was adjusted to 2 µg/dish with the empty vector
pcDNA3.1(+). At 2 days post-transfection, cells were harvested, washed three times with PBS,
and seeded into 96-well white flat-bottom microplates (Greiner Bio-One) in Tyrode’s solution
(Thermo Fisher, Cat# AAJ67593AP) at a density of 5–10 × 104 cells/well. The NanoLuc substrate
(Nano-Glo® Luciferase Assay Reagent; Promega) was diluted 1:250 in Tyrode’s solution and
added to the wells. Glutamate (300 µM in Tyrode’s solution) was injected after obtaining a
baseline value over 10 seconds, with BRET signal acquisition every 100 ms using a PHERAstar
FSX microplate reader (BMG Labtech). All measurements were performed at room temperature.
BRET was calculated as the ratio of light emitted at 535 nm (Venus-Gβ1γ2) to that at 475 nm
(masGRK3ct-Nluc-HA). G protein activation was determined as the change in BRET ratio upon
L-glutamate addition. The rate of activation (1/τ) was calculated using GraphPad Prism.
For dose-response measurements of L-glutamate and L-AP4, the baseline value was recorded
for 5 seconds after NanoLuc addition. Ligand was then applied, and signals were collected for an
additional 5 seconds. The response amplitude was determined as the mean signal during the 5-
second period after ligand addition minus the baseline value.

Statistics
Data are presented as mean ± SEM. Comparisons were made using two-tailed, Student’s t-tests.
Dose-response curves were fitted to a non-linear regression curve giving pEC50. Each statistical
analysis included a minimum of three biological replicates. All statistical analyses were performed
using GraphPad Prism version 10.4. Significant difference was expressed as *P < 0.05, **P < 0.01,
***P < 0.001, and ****P < 0.0001. Not significant (ns) was indicated where appropriate.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports the 3D structure at 3.5A resolution of the agonist bound mGlu6 receptor. Although only three agonist-bound structures are reported, illustrating structural flexibility, it is of great importance for two main reasons. First, the receptors from the class C GPCRs, including mGlu6, are surprising allosteric machines that seems to convert a apparent symmetric conformational change at the level of the binding domains, to an asymmetric organization of the 7TM domains. This asymmetry has been reported long ago, well-illustrated by the observation that only one subunit can activate a G protein at a time. This was confirmed with the resolution of active class C GPCRs, including various mGlu, the CaS or the GABAB receptors in their active state bound to the nucleotide-free G protein, showing a single G protein per dimer. Asymmetric organization of the 7TM domain dimer was also observed with various receptors bound to both agonists and PAMs. However, the question remains whether the asymmetry is stabilized either by the G protein, or the PAMs, that may have similar conformational effects of the G protein, or if, the asymmetry can be solely the result of agonist binding in the binding domains. The present paper is the first to show that in an mGlu receptor homodimer, agonist binding leads to a slight asymmetric organization at the bottom of the dimer of binding domains, while the transmembrane domains show a clear asymmetric organization, demonstrating that the asymmetry is an intrinsic property of these receptors. The second major aspect of this work, is that when mutated, the GRM6 gene, that encodes mGlu6, is responsible for night-blindness in humans, as being the main receptor responsible for the ON response in the retina. With the structure in hand, and a full characterization of the functional consequences of many of these human mutations, the authors have been able to provide structural explanation for the effect of these mutations.\nDespite the importance of this study, we have raised a number of points that need to be clarified.\n1- The authors claim that “The structure reveals an unexpected asymmetric dimer arrangement in the absence of G protein binding”. The authors must be more precise, as asymmetry has already been reported with the mGlu2-4 receptor (Huang et al., Nat Commun 2024), as well as the GABAB receptor (Shaye et al. Nature 2020) bound to agonist only. In contrast, the mGlu3 homodimer bound to agonist (Fan et al., CellRes 2022) did not reveal much asymmetry at the 7TM domain dimer. The authors should mention that this is the first case observed in an homodimeric class C GPCR.\n2- If such a process has been selected during evolution, it has likely a role that the authors should discuss.\n3- The authors say that this \"intermediate conformation primes mGlu6 for G protein binding, it means it makes it easier for G protein to bind to this conformation\". There no direct demonstration for this conclusion, although it is likely, one could argue that a symmetric organization would even be better for G protein interaction with the active receptor, as both subunits could do it.\n4- Introduction: passage from l. 55 to l. 59 seems only dedicated to promoting the team’s past work as it is off topic with regards to mGlu6. It should be removed.\n5- L60: “The molecular architecture of mGlu6 remains largely unknown,” – there is no structure solved yet of mGlu6, the authors should be clearer on this point.\n6- L113-114: “From the map, we successfully identified a homodimer assembly (95 × 70 × 175 Å³) (Fig. 1a).” this not very clear, since the authors purified a homodimer was anything else to be expected from the reconstruction? And what are the numbers referring to? please rephrase.\n7- L120-123: The authors cannot make the claim that mGlu6 transitions from a ‘Roo’ conformation in the apo state to their active structure as no structure of apo mGlu6 exists, hence they cannot know in which conformation the apo structure would be. This must be made clear in the text. May be the only evidence that mGlu6 behaves like the other mGlu receptors, is the observation that the FRET change observed upon activation of SNAP-versions of the mGlu receptors is found similar in mGlu6 and the other receptors, consistent with a similar change in the binding domain relative orientation (Scholler et al., Nat Chem Biol 2017).\n8- L128-130: the authors use the inactive structure of mGlu7 as a proxy for the inactive structure of mGlu6. Why not use mGlu4? They need to justify that choice. Indeed, mGlu7 is very different from the other mGluRs, with such a low affinity for glutamate. In that respect, mGlu4 is more similar to mGlu6. The diversity of structures obtained from the mGluR family demonstrates that there can be some variability of conformations even within the subgroups. It would probably be more appropriate to make those comparisons to several mGluR structures unless they have a good argumentation not to.\n9- L141-143: \"a small but significant increase in efficacy was observed, suggesting that the conformation of the G protein binding site may also be affected\". The curves shown do not saturate, as the authors used a low affinity ligand (L-Glu). It is difficult (indeed not accurate) to compare efficacies, and EC50s from non-saturating curves. The authors may use another more potent agonist (L-SOP, L-AP4 or others), to reach saturation. Moreover, we disagree on the conclusion that the conformation of the G protein site may also be affected. The mutation may simply affect the R-A equilibrium of the VFT dimer, leading to a partial activity of the mutant. A careful quantification of both receptor forms at the cell surface must also be provided, before a comparison of the efficacies can be discussed.\n10- L162 : \" while in the LB2 region, and the inclination between helix F and helix F’ differed by 9.8°\": unclear, rephrase.\n11- L180-183: what was the rational for excluding 2 out of the five classes for further refinement? Could the authors try classification and refinement with more than 5 classes to see if more conformations can be extracted from their dataset?\n12- Fig2: the legend is not informative enough, eg for panel h, there is clearly an alignment and measure made to place the vertical line but not clear from what.\nPanel h: it’s not clear from this figure that conformation 1 is nearly symmetrical. Because this is a strong point of the paper – the conformational diversity of mGlu6, it would be nice to see more alignments of the different classes (could be in supplementary) to document this asymmetry or lack of.\nPanel j: it’s not clear what ‘intrinsic’ asymmetry refers to… seems like wording redundancy and simply ‘asymmetry’ is more appropriate.\n13- L-SOP binding site description: there is a need to validate the proposed model of L-SOP binding (based on a 3.5 A resolution) using point mutation of some residues (Q64, R68, K312? K400, T177), or refer more clearly on previous data reported by others if they exist.\n14- the expression ‘propagation of activation’ suggests a sequential mode of activation, but tis remains to be demonstrated. If the expression is kept, it must be justified.\n15- L259-260: We cannot conclude on the role of R731 in trafficking by simply measuring surface expression. Could be a misfolding issue etc.. The authors should remove this sentence, or mention other explanations.\n16- L263-265: wording issue, the sentence is not clear.\n17- L266-267: Can the change in kinetics be due to difference in receptor numbers? Could the authors try the same experiments but with varying levels of mGlu6 expression to compare similar level of surface expression between wt and mutants. They should already have the data from the plot in figure 5.\n18- L277: wording issue.\n19- L294-295: Could this be due to the altered surface expression? The authors should plot the t value as they have for max BRET in Fig.5c to confirm or infirm this hypothesis.\n20- L300: I would like to see the whole-dose response curves for all the disease mutants – could be in the supplementary, and their EC50 values. Because mutants R352C and I405T are claimed to be gain-of-function by the authors it would be important to see those curves, especially since the t value for both these mutants are very low – which could also confirm that low t is simply a reflection of low surface expression.\n21- L318-322: Why would G464R disrupt glycosylations? Glycines are not glycosylated. This paragraph needs to be clarified.\nMethod section\n22- Please mention the binning size for the initial processing steps.\n23- The authors should include in the methods the full EM workflow, meaning the 3D classification no refinement, associated particle numbers and rational for class selection.\n24- The authors should add PDB and EMDB numbers for the 3 classes also deposited.\n25- L725-726: This sentence is not clear and I don’t understand what the authors mean. Have the authors used the TMD density to build/fit the TMD structure of the full length mGlu6? If yes, then they should have done a global real-space refinement in the consensus map post-fitting in the local map. From their method it is unclear how this was done.", "reviewer_2": "In the study, “CryoEM structure of mGlu6 captures receptor activation prior to G protein coupling,” The authors report the first structural determination of mGlu6 in an agonist (L-SOP)-activated state without bound G protein. Additionally, they employ surface expression and G protein activation assays to profile mutations implicated in congenital stationary night blindness (CSNB), as well as those predicted from the structure to be involved in receptor activation.\nWhile this work will undoubtedly be of significance to researchers studying congenital stationary night blindness, I believe it is unlikely to garner broad interest beyond this specific disorder. This limitation extends to the GPCR structural biology field, given the limited scope of the study. This in my view stems from the fact that full-length structures for all other mGlu receptors have already been determined. Several of these structures capture multiple states, including antagonist-bound, agonist-bound, and allosteric modulator-bound conformations, with some also bound to their G proteins. Consequently, this mGlu6 structure does not provide critical new insights into mGlu receptor activation mechanisms that are not already well established in the field. The mutation profiling is also interesting but is likely to be of interest only to the CSNB field. The asymmetric agonist-bound dimer arrangement that primes the receptor for G protein binding presents an interesting observation. However, it remains unclear whether this arrangement is unique to the specific agonist L-SOP employed in their work. Regardless of this uncertainty, I view this finding as a relatively minor detail in the broader context of mGlu receptor structural biology.\nThe addition of an mGlu6-G protein complex structure would likely significantly boost the impact of this work. Such a structure would likely enable more robust mechanistic examination of the mutants and their effects, and could provide insights on group III mGluR activation beyond what is currently known, potentially elevating the work's contribution to the field beyond its current scope.\nOverall, I believe the claims made are supported, but some are inferred from structures of other mGlu receptors. For instance, on Page 7, lines 120-123, “First, the VFT domain of mGlu6 transitions from a 'Roo' (resting open–open) conformation in the apo state to an 'Acc' (active closed–closed) conformation, a typical conformational change of mGluRs upon agonist binding (Fig. 1b).” This claim is inferred from the mGlu7 and other structures. Additional mGlu6 structures would provide more robust insight into the activation of this receptor.\nLastly, On Page 7, lines 139-141, “The mutation significantly increased the half- maximal effective concentration (EC50) for L-glutamate-induced G protein activation, indicating reduced potency (Fig. 1c and Supplementary Fig. 4c, d)”. Looking at the dose-response curve in Fig.1c, the potency of both WT and R186A look nearly identical. Were the fits of each dose-response curve replicate performed on the data plotted in log scale and as shown in Figure 1c to generate pEC50 values? If so, the distributions should be plotted on this scale as well and shown as pEC50 and not converted to uM. The statistical comparisons should also be performed on this scale. This should be the case for all such data reported in the manuscript. It would also help to indicate the Exact N values in the legends.", "reviewer_3": "The authors report the first active-state structure of the mGlu6 receptor bound to its potent agonist, L-serine O-phosphate (L-SOP), revealing how agonist binding primes the receptor for G-protein coupling through an asymmetric conformational rearrangement. This work highlights the activation mechanism of mGlu6 compared to other mGluR family members. In the structural study, authors find three distinct agonist-bound conformations of mGlu6. They perform functional experiments addressing cell expression and G-protein activation (maximum response and activation rate) to investigate the role of an extended loop in the ECL2 domain (unique to mGlu6 receptor) as well as to explore the pathogenic mechanisms of representative CSNB-associated mutations. The study combines cryo-EM structural analysis with cell-based functional assays, providing a comprehensive understanding of mGlu6 activation. The manuscript is clearly written and logically organized.\nHere are some point to address:\n1. Line 101-104: This statement is wrong, not all structures are processed in C2. Other studies on class C GPCRs identified asymmetry in receptor ligand bound state ( absence of G-protein).\n2. Line 106: “which yielded three distinct classes that primarily differed in the degree of protomer bending 106 and were refined to resolutions of 3.39 Å, 3.27 Å, and 3.51 Å, respectively” – I am curious about all three of these conformations. They seem to differ quite significantly. I think it would be interesting to further assess if they are indeed intermediates, is there any other differences than the bending of the subunit?. Also wondering if the density and pose of L-SOP is the same in all three.\n3. Line 134-136: Does the authors mean the close-close (cc) active state (A) of the VFTs (Acc). please specify.\n4. Line 139-143: Impact of the mutations on receptor potency seems to be statistically significant, even if the effect is rather small. However, regarding efficacy, the dose-response curves and bar graphs are not showing the same thing neither in the main figure 1 nor in the supplementary figure 4. For example, the efficacy of the WT Vs R186 mutant is not significant in Fig 1c (Dose-response curve), whereas Fig 1d show statistically relevant effect (bar plot), despite being very small as well. This is the message a get from this figure. It is very speculative to define a role of R168 for impacting on the receptor conformation based on such small functional effect.\n5. Line 152-155. Please display the density for the ionic- lock residues. Is it also broken in the 3 distinct conformations that were identified in this study or only in the consensus structure?\n6. Line 304: “While many act as loss-of-function mutations, as expected for disease-associated variants, the unexpected gain of-function observed in R352C and I405T provides intriguing insights that warrant further investigation.” Is any data showing if the phenotype of the disease is any different than in the loss of function mutants?\n7. Line 309: Paragraph entitled “Structural mapping of CSNB mutations in mGlu6”. This paragraph is highly speculative, indirect interpretation of the data are made, without data supporting the statements. This paragraph is a part of the discussion and not a result section.\n8. Line 687: “0.01 mg/ml brain lipid” – I wonder if it is common to add lipids to mGluR preps and if there are any ordered lipids visible in the map.\n9. Line 746: “~2X107 cells” needs formatting"}, "author_response": "We thank the reviewer for the insightful summary and constructive feedback, which have been invaluable in strengthening our manuscript. We are especially grateful for the acknowledgment of the two major contributions of our study: (1) the demonstration that agonist binding alone induces an intrinsic asymmetric organization of the 7TM domains, and (2) the structural basis our work provides for understanding the functional impact of human GRM6 mutations linked to night-blindness. We have addressed each comment point-by-point below and hope that our detailed responses and corresponding revisions are clear and satisfactory.\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors modified and clarified their manuscript as requested. We have no further comments.", "reviewer_2": "I thank the authors for their thoughtful and thorough responses to all of my comments. I also greatly appreciate their effort to try and determine the mGlu6–G protein complex structure by cryo-EM. Considerable work went into addressing the concerns raised by myself and the other reviewers, and I applaud the authors for these efforts. As I noted previously, the claims made are well supported, and the revisions have improved the interpretations throughout. I believe this work is suitable for publication, though I personally feel it is better suited for a structural biology–focused journal or one dedicated to the molecular mechanisms of CSNB/vision disorders.", "reviewer_3": "Authors addressed all my comments.\nOf note, there is a mistake in table presented in Supplementary Fig. 3. Activation features of VFT domain and TMD in L-SOP-bound mGlu6. b,: mGlu1 and mGlu5 are group 1 receptor and mGlu2 and mGlu3, group 2", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the reviewer for the meticulous evaluation and kind recognition of our revisions and additional data, which we believe have greatly strengthened our manuscript."}]

Decision letter