bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Cryo-EM structure of the volume-regulated anion channel
LRRC8
Go Kasuya1*, Takanori Nakane1†*, Takeshi Yokoyama2*, Yanyan Jia3, Masato
Inoue4, Kengo Watanabe4, Ryoki Nakamura1, Tomohiro Nishizawa1, Tsukasa
Kusakizako1, Akihisa Tsutsumi5, Haruaki Yanagisawa5, Naoshi Dohmae6,
Motoyuki Hattori7, Hidenori Ichijo4, Zhiqiang Yan3, Masahide Kikkawa5, Mikako
Shirouzu2, Ryuichiro Ishitani1, Osamu Nureki1‡
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; 2Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa 230-0045, Japan; 3State Key Laboratory of Medical Neurobiology, Ministry of Education Key Laboratory of Contemporary Anthropology, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai 200438, China; 4Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; 5Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; 6RIKEN, Global Research Cluster, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; 7State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai 200438, China. * These authors contributed equally to this work. † Present address: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, United Kingdom ‡Correspondence and requests for materials should be addressed to O.N. ([email protected])
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Abstract
Maintenance of cell volume against osmotic change is crucial for proper cell functions,
such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8)
proteins are anion selective channels, and were recently identified as pore components
of the volume-regulated anion channels (VRACs), which extrude anions to decrease the
cell volume upon cell-swelling. Here, we present the human LRRC8A structure,
determined by a single-particle cryo-electron microscopy analysis. The sea
anemone-like structure represents a trimer of dimers assembly, rather than a
symmetrical hexameric assembly. The four-spanning transmembrane region has a gap
junction channel-like membrane topology, while the LRR region containing 15
leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis
and constricted on the extracellular side, where the highly conserved polar and charged
residues at the tip of the extracellular helix contribute to the anion and other osmolyte
permeability. Comparing the two structural populations facilitated the identification of
both compact and relaxed conformations, suggesting that the LRR region is flexible and
mobile with rigid-body motions, which might be implicated in structural transitions
upon pore opening. Overall, our structure provides a framework for understanding the
molecular mechanisms of this unique class of ion channels.
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Introduction
Cell volume regulation in response to changes in the extracellular osmotic pressure is a
fundamental homeostatic mechanism for all organisms1–3. Alteration of the cell volume
leads to numerous physiological processes, such as cell proliferation and migration. A
continuous decrease in the cell volume under isotonic conditions is related to the
activation of the programmed cell death pathway, and is known as apoptotic volume
decrease (AVD)4. Various proteins including membrane ion channels and transporters,
as well as cytoplasmic kinases, are essential components for cell volume regulation.
Among these proteins, the volume regulated anion channel (VRAC) is a particularly
interesting type of ion channel5. VRAC is activated by cell-swelling and releases Cl‒
ions or other osmolytes on the extracellular side and concurrently ejects water
molecules, leading to a decrease in the cell volume. This process, called regulatory
volume decrease (RVD), is observed in almost all types of eukaryotic cells.
The molecular entity of VRAC was unknown until quite recently, although extensive
experiments have been performed over the past decades, and several characteristics of
VRAC, including substrate selectivity, activation upon cell-swelling, outward
rectification, and inactivation kinetics, were functionally characterized6,7. In 2014, by
combining high-throughput RNAi screening with electrophysiological recordings, two
independent groups successfully identified the leucine-rich repeat-containing 8A
(LRRC8A) protein, which when mutated causes an innate immune disease,
agammaglobulinemia8, as the essential component of VRAC9,10. The LRRC8A protein
belongs to the LRRC8 gene family, which includes five members (LRRC8A-E) with
high amino-acid sequence similarity. All of the LRRC8 isoforms consist of about
800-850 amino acid residues11,12. Based on a sequence analysis, the N-terminal half of
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the LRRC8 isoforms is predicted to form four transmembrane (TM) helices, while the
C-terminal half of the LRRC8 isoforms is predicted to contain up to 17 leucine-rich
repeats (LRR). A previous bioinformatics analysis suggested that the N-terminal halves
of the LRRC8 isoforms share weak homology to pannexin, connexin, and innexin13.
Since the previous structural analyses of the gap junction channels, connexin and
innexin, revealed that their hemichannels form a hexamer and an octamer,
respectively14–16, the LRRC8 proteins are also predicted to form hexameric or higher
TM region oligomers to create the channel pore13. Further electrophysiological analyses,
using cells expressing LRRC8 isoforms and LRRC8 isoforms embedded in lipid
bilayers, demonstrated that the heteromeric LRRC8 complex, consisting of LRRC8A
and at least one of the four other LRRC8 isoforms, is indispensable for the channel
activity in cells, while the homomeric LRRC8A protein still retains the hypotonically
induced channel activity under lipid embedded conditions10,17. This heteromeric
assembly of LRRC8A and the other LRRC8 isoforms contributes to the diversity of the
channel properties, including the gating kinetics and modulation, as well as the substrate
specificity. For example, cells expressing the LRRC8A/C heteromer are inactivated
more slowly than those expressing the LRRC8A/E heteromer10. In addition, the activity
of the LRRC8A/E heteromer is potentiated by intracellular oxidation, while the
activities of the LRRC8A/C and LRRC8A/D heteromers are inhibited by intracellular
oxidation18. Moreover, cells expressing the LRRC8A/D heteromer release uncharged
osmolytes such as taurine, and uptake anti-cancer drugs, including cisplatin, while those
expressing the LRRC8A/C/E heteromer release charged organic osmolytes19–21.
As the overall sequence of LRRC8 has little similarity to any other known classes of
ion channels, the detailed structural information of the LRRC8 protein has long been
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awaited to elucidate the molecular architecture of LRRC8 and to clarify the molecular
mechanisms, such as substrate permeability. Here, we report the cryo-electron
microscopy (cryo-EM) structure of the homomeric human LRRC8A, at an average
resolution of 4.25 Å (local resolution of the TM region is ~3.8 Å). The present structure
provides a framework for understanding the mechanisms of this unique class of ion
channels.
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Results
Structure determination of homomeric HsLRRC8A.
We first screened the LRRC8 proteins from various vertebrates by
Fluorescence-detection Size-Exclusion Chromatography (FSEC) and an FSEC-based
thermostability assay (FSEC-TS)22,23, and found that the human LRRC8A protein
(HsLRRC8A; the “Hs” refers to Homo sapiens), the best characterized protein among
the known LRRC8 proteins, produced symmetric and monodisperse FSEC peaks when
solubilized with digitonin (Extended Data Fig. 1a). We also confirmed the
hypotonicity-induced channel activity of the purified HsLRRC8A protein in
reconstituted liposomes (Extended Data Fig. 1b,c). Although the LRRC8 proteins
reportedly function as heteromers in cells10, the structure of the homomeric HsLRRC8A
protein may provide important insights into the basic architecture of the LRRC8
proteins, given the sequence similarity between the isoforms (Extended Data Fig. 2).
Therefore, we set out to elucidate the structure of the homomeric HsLRRC8A. The
purified HsLRRC8A protein was vitrified on grids, and images of the grids were
recorded by electron microscopy (Extended Data Fig. 3a). In the 3D classification step,
classes #1 and #2 showed clearer densities and similar assemblies, respectively, as
compared to the other classes. Therefore, we chose these two classes for further
refinement (Extended Data Fig. 3b,c). Since these classes exhibited 3-fold rotational
symmetry, C3 symmetry was imposed in the subsequent reconstructions. In the final 3D
map reconstructed to an overall 4.25 Å resolution (local resolution of the TM region is
~3.8 Å) (Extended Data Fig. 3d,e), the densities of the six protomers of HsLRRC8A
(namely, the α to ζ subunits) were clearly observed, including the long arc-like densities
corresponding to the leucine-rich repeat (LRR) region. This density map enabled us to
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assign the topologies of the LRR and other regions, including the extracellular,
transmembrane (TM), and intracellular regions (Fig. 1a-c, Extended Data Fig. 3b, c).
The local resolution of the core of the TM region is estimated to be ~3.8 Å, and its
density is sufficiently resolved to enable the tracing of the α-helices and the bulky
amino-acid side chains (Fig. 1a, b). The densities corresponding to most of the
extracellular and intracellular regions are also resolved well enough to trace the
secondary structures, using the structure of the previously determined nematode Innexin
6 (CeInnexin-6; the “Ce” refers to Caenorhabditis elegans) (PDB ID: 5H1Q) as a guide.
Two loops connecting the TM helices in these regions are disordered (Fig. 1d, e). In
contrast to the clear densities of the extracellular, TM and intracellular regions, the local
resolution of the LRR regions is around 5.5 Å, and only the main chain of the LRR
region is resolved in the density map (Fig. 1a, b). Therefore, we generated the
homology model of the LRR region of HsLRRC8A based on the previously determined
LRR protein structure (PDB ID: 4U08) using the Phyre2 server24, and used it to guide
the model building. The final model of HsLRRC8A, in which about 90% of the residues
were assigned, contains the residues R18-D806 and is missing parts of the extracellular
loop (T61-D91) and the intracellular loop (T176-R226 in the α, γ, and ε subunits;
T176-L232 in the β, δ, and ζ subunits).
Overall structure and subunit fold of HsLRRC8A.
The hexameric structure of HsLRRC8A adopts an upside-down sea anemone-like
shape, when viewed parallel to the membrane (Fig. 1c). The subunit structure consists
of four regions; i.e., the extracellular region (Q49-H119 and H287-L314), TM region
(W23-T48, W120-F144, I259-V286, and A315-R345), intracellular region (K145-D258
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and R346-T412), and LRR region (L413-D806) (Fig. 1c-e), with both the N- and
C-termini residing on the cytoplasmic side. The overall structures of the six subunits are
similar, except for several points as described later, and thus we mainly discuss the
structure of the α subunit of the hexamer. The extracellular region protrudes by ~35Å
above the cell membrane, and forms the channel pore with the TM region. The LRR
region is protruded toward the intracellular side, and is twisted in a clockwise manner as
viewed from the cytoplasmic side (Fig. 1c). The TM region consists of the four TM
helices (TM1-4), which are connected by the two extracellular loops (EL1 and EL2) of
the extracellular region, and the intracellular loop (IL1) of the intracellular region (Fig.
1d, e). IL2 in the intracellular region connects the TM and LRR regions (Fig. 1d, e).
EL1 possesses one α-helix (EL1H) and one β-strand (EL1β), while EL2 possesses two
β-strands (EL2β1 and EL2β2) (Fig. 1d, e). IL1 possesses three α-helices (IL1H1–3),
while IL2 possesses four α-helices (IL2H1–4) (Fig. 1d, e). EL1 and IL1 contain
disordered regions (T61–D91 and T176–R226), suggesting that these loops have
conformational flexibility (Fig. 1d, e, Extended Data Fig. 2). While a previous
bioinformatics analysis suggested that LRRC8 contains a 17 LRR repeat12, the present
structure revealed that the LRR region consists of the leucine-rich repeat N-terminal
helix (LRRNT), fifteen leucine-rich repeats (LRR1–15), and the following C-terminal
helices (LRRCT) (Fig. 1d, e, Extended Data Fig. 2).
Differences in structures and interactions among HsLRRC8A subunits.
With its global C3 symmetry, the hexameric HsLRRC8A structure exhibits the “trimer
of dimers” architecture; in other words, the structures of the α, γ, and ε subunits, and
those of the β, δ, and ζ subunits, are identical (Fig. 1c, 2a). In contrast, a distinctive
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structural difference is observed between the α and β subunits. The LRR region is
rotated by ~22° relative to the extracellular, TM, and intracellular regions (Fig. 2b). No
large structural difference is observed in each region, as their structures superimpose
well with RMSD values less than 1 Å (0.39 Å, 0.40 Å, 0.98 Å, and 0.47 Å for the
extracellular, TM, intracellular, and LRR regions, respectively), suggesting their
structural rigidity (Fig. 2b, Extended Data Fig. 4). Therefore, this structural difference
between the subunits can be ascribed to the rotation of the IL2H4 helix in the IL2 loop,
which connects the intracellular and LRR regions as a hinge (Fig. 2c).
As a result of this structural difference between the subunits, several variations are
observed in the interfaces between the subunits; e.g., the α subunit tightly interacts with
the β subunit, while it loosely interacts with the ζ subunit (Fig. 2a). Notably, in the
“tight” interaction, there are extensive contacts between the adjacent subunits from the
extracellular region to the LRR region, whereas in the “loose” interaction, there are
almost no contacts between the adjacent subunits in the lower part of the TM region, the
intracellular region, and the upper part of the LRR region (Fig. 2a). At the TM region,
the subunit interface is highly hydrophobic and mainly formed by the TM2 helix from
one subunit, and the TM1 and TM4 helices from the other adjacent subunit (Extended
Data Fig. 5a, b). There is a “lateral crevice” open to the lipid bilayer across the central
channel pore, and it is wider in the loose interface than in the tight interface (Extended
Data Fig. 5a, b). At the intracellular region, the subunit interface is highly hydrophilic
and formed by the IL1H1 and IL1H3 helices from one subunit, and the IL1H2, IL2H2
and IL2H3 helices from the other adjacent subunit (Extended Data Fig. 5c, d). In this
region, extensive interactions are formed in the tight interface, while few interactions
are observed in the loose interface (Extended Data Fig. 5c, d). At the LRR region,
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extensive interactions throughout all of the LRR repeats are observed in the tight
interface, while in the loose interface, fewer interactions are formed between the
C-terminal LRRCT helices from one subunit and the middle of the LRR repeats from
the other adjacent subunit (Extended Data Fig. 5e, f). Notably, a heterozygous LRRC8A
truncation mutant, in which the C-terminal 91 amino acids are replaced by 35 amino
acids encoded in the intron sequences, has been reported to suppress the LRRC8A
function, which causes an innate immune disease8,9. This disease-related truncation
might abrogate the interactions at this loose interface, thereby affecting the proper
formation of the hexameric channel structure. In contrast, the interactions in the tight
and loose interfaces of the extracellular region are almost the same (Extended Data Fig.
5g, h). The interactions are formed by the extensive contacts between the EL1H helix
and the three-stranded β-sheet (EL1β, EL2β1, and EL2β2) from the other adjacent
subunit (Extended Data Fig. 5g, h).
Overall, these structural comparisons between the subunits suggest that the flexibility
around the IL2H4 helix, which connects the intracellular and LRR regions, causes the
structural differences between the subunits, which may enable the formation of the
“trimer of dimers” architecture of the hexameric channel, with the wide crevice in the
loose interface.
Channel pore
HsLRRC8A has a channel pore along the central axis, perpendicular to the membrane.
The channel pore is mainly composed of the EL1H helix in the extracellular region and
the TM1 helix in the TM region, as well as the IL1H1 and IL1H3 helices in the
intracellular region (Fig. 3a). The diameter of the pore is about ~ 25 Å on the
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intracellular side, and it widens to about 50 Å at the lower part of the TM region, and
then narrows to less than 10 Å at the upper part of the extracellular region (Fig. 3b-e).
Therefore, the channel pore formed by the hexameric LRRC8A is accessible from both
the extracellular and intracellular sides, and thus the present structure of LRRC8A
probably represents the open conformation. The residues lining the channel pore are
mainly hydrophilic, suggesting that the pore is filled with water molecules in the present
structure. A previous experiment revealed that the T44C mutation alters the I– versus
Cl– anion selectivity of HsLRRC8A, and the MTSES (2-sulfonatoethyl
methanethiosulfonate) modification of the T44C mutant significantly reduced the
channel activity, suggesting that T44 is involved in the pore formation9. Consistently, in
our HsLRRC8A structure, T44 is located on TM1 and involved in the formation of the
solvent-accessible surface (Fig. 3b, d). The most-constricted site of the pore has a
diameter of about 7.6 Å, which is wide enough to permeate a hydrated Cl– ion. This
constriction site is mainly formed by the side chains of the positively charged R103
residues (Fig. 3b, c). In addition, a negatively charged residue, D102, is located next to
R103, and is positioned just above R103 like a “lid” for the constriction site. These
structural features suggest that the D102 and R103 residues play an important role in the
ion permeation (Fig. 3b, c). Furthermore, a recent electrophysiological analysis
demonstrated that three residues, corresponding to K98, Y99, and D100 in the LRRC8A
isoform, are involved in the voltage dependence and kinetics of the LRRC8 isoforms25.
Single and combination mutants of these residues reportedly affected the rate and
voltage dependence of inactivation, the kinetics of inactivation, and the ion selectivity25.
Consistently, these three residues are located in the loop preceding the EL1H helix and
are exposed to the extracellular entrance of the channel pore in the present HsLRRC8A
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structure (Fig. 4a), suggesting their importance for the ion permeation.
Based on the present HsLRRC8A structure, as well as the previous functional analysis,
we hypothesized that the N-terminal tip of the EL1H helix (D102-Y106) plays a critical
role in the ion permeation, as it resides on the extracellular side of the channel pore and
constitutes its most-constricted site (Fig. 4a,b). To corroborate this notion, we generated
a series of single alanine mutants of residues D102 to Y106 in HsLRRC8A and
performed cell-based measurements of the VRAC activity, using assays originally
developed upon the discovery of the LRRC8A protein as a VRAC component9,10. First,
by a confocal microscopic analysis, we confirmed that the signals derived from these
mutants are observed at the cell membrane (Extended Data Fig. 6a). In addition, the
FSEC analysis of these mutants produced symmetric and monodisperse peaks, similar
to those of the wild-type channel, confirming the proper structural integrities of these
mutant channels (Extended Data Fig. 6b). We transfected the halide-sensitive YFP
mutant26 with or without HsLRRC8A wild-type (WT) into HEK293A cells, in which
the endogenous LRRC8A gene is knocked out, and verified the assay system:
HsLRRC8A WT could rescue the hypotonicity-induced iodide quenching response in
the LRRC8A KO cells (Fig. 4c, d). We then evaluated each of the HsLRRC8A mutants
within the range of WT expression levels (Extended Data Fig. 6c), and found that the
D102A, Q105A and Y106A mutants decreased the quenching speed as compared to the
wild-type channel (Fig. 4c, d, Extended Data Fig. 6d). Even though the reduction of the
quenching speed cannot exactly explain how the mutations affect the channel properties,
such as the rate of ion permeation and the channel open probability, the results support
our hypothesis that the N-terminal tip of the EL1H helix is important for the ion
permeation. Interestingly, the amino-acid sequences in this region of the other LRRC8
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isoforms deviate from that of LRRC8A (Fig. 4b). These differences in the amino acid
sequences in this region may alter the properties of this constriction site in the channel
pore, thereby modulating the substrate specificity of the heteromeric LRRC8 hexamers
containing different isoforms20,21.
Putative structural change of LRRC8
During the 3D classification, in addition to classes #1 and #2 used for the 4.25 Å
reconstruction, we found that class #7 also showed clearer LRR densities than the other
classes (Extended Data Fig. 3b). Interestingly, class #7 exhibited the distorted
arrangement of the LRR densities, as compared to those in classes #1 and #2, and thus it
may represent another structural population of LRRC8A. Therefore, we performed the
3D reconstruction of class #7 without imposing any symmetry. The overall resolution of
the resulting map was 9.17 Å, and we could assign the extracellular, TM, intracellular,
and LRR regions. We then constructed a model of class #7, based on the higher
resolution structure.
In this model constructed from class #7, the LRR region is substantially deviated and
more loosely packed as compared to that in the high-resolution structure from classes #1
and #2 (Fig. 5a, b). Accordingly, we refer to this class #7 structure as the “relaxed form”
(Fig. 5a), and the higher-resolution structure from classes #1 and #2 as the “compact
form” (Fig. 5b). In the relaxed form, the LRR regions are further tilted and expanded
away from the central axis, with their width elongated by ~20 Å and height shortened
by ~8 Å, as compared with the compact form (Fig. 5a, b). The C3 symmetry observed in
the compact form is absent and the arrangement of the LRR regions is completely
asymmetric. In contrast, the arrangement of the secondary structure elements in the
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extracellular, TM, and intracellular regions is quite similar to that observed in the
compact form. The structures of the LRR regions in both the relaxed and compact forms
were also similar, including their curvatures. Thus, the overall structural difference
between the relaxed and compact forms can be explained by the rigid-body motions of
the LRR and the other regions, in which the IL2H4 helix in the IL2 loop serves as a
hinge. Interestingly, a similar structural difference is also observed between the α and β
subunits in the compact form (Fig. 2, Extended Data Fig. 4). These structural
differences suggest the structural flexibility around the IL2H4 helix in the IL2 loop,
which might be involved in the important structural transition for the channel function
(Fig. 5c).
Structural comparison with gap junction channels
Consistent with the previous bioinformatics analysis13, the extracellular, TM, and
intracellular regions of HsLRRC8A share several structural features with those of the
CeInnexin-6 and human connexin 26 (HsConnexin-26) gap junction channels (Extended
Data Fig. 7)14,15, although they have low sequence identity (~15% with CeInnexin-6;
~18% with HsConnexin-26). In the TM region, they share the same topology of four
TM helices, in which TM1 is the innermost helix. In the extracellular region, they
commonly possess conserved disulfide bonds between the EL1 and EL2 loops, and
common secondary structure elements, including helix EL1H, as well as the β-sheet
bridging the EL1 and EL2 loops (Extended Data Fig. 7a-c). In the intracellular region,
helices IL1H1 and IL1H3 in the IL1 loop are also conserved in CeInnexin-6 (Extended
Data Fig. 7a, b). In contrast, HsLRRC8A possesses several distinct structural features as
compared to the gap junction channels. First, the subunit interfaces of HsConnexin-26
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and CeInnexin-6 are tightly packed and the large crevice is not observed (Extended
Data Fig. 7d-f)14,15, suggesting that the crevice of LRRC8 is involved in its specific
function. Second, the HsConnexin-26 and CeInnexin-6 structures revealed that their
short N-terminal extension (~15 residues) forms a short α helix (NTH), which is
involved in the channel pore formation (Extended Data Fig. 7g-i). This NTH was
speculated to be important for the ion permeation and gating in the gap junction
channels14,15. HsLRRC8A also possesses a short extension at its N terminus; however,
its density was not observed in the present cryo-EM structure. The amino-acid sequence
of the N-terminal extension of HsLRRC8A is quite different from those of both
HsConnexin-26 and CeInnexin-6, and is not predicted to form a helix, unlike the cases
of HsConnexin-26 and CeInnexin-6 (Extended Data Fig. 7j). Thus, the N-terminal
extension of LRRC8 would not form a helix and is likely to play a different role from
those of the gap junction channels. Third, in the TM region, while TM1 in HsLRRC8A
is straight and faces toward the intracellular side, those in the gap junction channels are
bent at the middle, and consequently the N-termini of the gap junction channels face
toward the extracellular side to form the channel pore (Extended Data Fig. 7b, c).
Fourth, in the intracellular region, the IL1 and IL2 loops of HsLRRC8A (144 residues
in IL1; 67 residues in IL2) are longer than those of CeInnexin-6 (54 residues in IL1; 59
residues in IL2) (Extended Data Fig. 7a, b). As a result, in HsLRRC8A, the long linker
between the IL1H2 and IL1H3 helices in IL1, as well as the IL2H4 helix in IL2, directly
interacts with the LRR region. Particularly, the IL2H4 helix, which connects the
intracellular and LRR regions, only exists in the LRRC8 isoforms. Fifth, in the
extracellular region, EL1H in HsLRRC8A (14 residues) is longer than those in the gap
junction channels (10 residues), and the N-terminal tip of the EL1H helix in
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HsLRRC8A forms the channel pore with the TM1 helix (Extended Data Fig. 7a-c, g-i).
In addition, the sizes and structures of the EL1 and EL2 loops of the gap junction
channels are different from those of HsLRRC8A (Extended Data Fig. 7a-c). Indeed, the
extracellular regions of HsConnexin-26 and CeInnexin-6 were shown to interact with
each other to form the gap junction channel, in which the EL1 and EL2 loops provide
the interaction interface14,15. Therefore, these structural differences in the subunit
interfaces, as well as the extracellular, TM, and intracellular regions, contribute to the
functional diversity between the volume-regulated channels and the gap junction
channels.
Discussion
In this study, we present the homomeric human LRRC8A cryo-EM structure, the first
structure of a member of the LRRC8 protein family responsible for the VRAC function.
The overall structure, reconstructed at 4.25 Å resolution (~3.8 Å in the TM region),
exhibited a hexameric assembly with a trimer of dimers, rather than a symmetrical
hexamer, revealing the architecture of this unique class of ion channels (Fig. 1) The
channel pore observed along the central axis is open toward both the extra- and
intracellular sides, suggesting that the present structure represents the channel-open
form (Fig. 3).
Although LRRC8A is an essential component of the VRAC activity, overexpression of
the LRRC8A gene in the quintuple-knockout LRRC8−/− cells or quadruple-knockout
LRRC8(B/C/D/E)−/− cells did not rescue the VRAC activity, suggesting that the
homomer of LRRC8A does not form the active VRAC pore in vivo. Nevertheless,
interestingly, the homomer of LRRC8A reconstituted into lipid droplet bilayers still
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retains VRAC characteristics, including hypotonicity-induced currents and sensitivity to
a specific VRAC inhibitor, DCPIB
(4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid)17. We also
confirmed that the homomeric HsLRRC8A protein used in this study has
hypotonicity-induced channel activity upon osmotic change, using the reconstituted
liposomes (Extended Data Fig. 1b,c). Furthermore, a previous biochemical experiment
showed that the overall assembly, including the oligomeric state, of the LRRC8A
homomer is similar to those of the LRRC8A-containing heteromers. The present
structure of the homomeric HsLRRC8A provides important structural insights into the
LRRC8A-containing heteromers. To visualize the conserved and diversified sites
among the LRRC8 gene family members, we calculated the conservation score using
the program ConSurf27, and mapped the score on the molecular surface of HsLRRC8A
(Extended Data Fig. 8a). The result showed that the subunit interface and the channel
pore in the TM, extracellular, and intracellular regions, except for the constriction site,
are highly conserved among the LRRC8 gene family members. In contrast, the
membrane side of the TM region, the overall LRR region, and the constriction site of
the pore exhibited relatively low conservation scores. Next, based on the present
structure of HsLRRC8A, we constructed a model structure of the HsLRRC8 heteromer
containing HsLRRC8D (54% sequence identity with HsLLRC8A; Extended Data Fig.
8b). Although the stoichiometry of the heteromeric LRRC8 is still unknown, this
(LRRC8A)5·LRRC8D heteromer may be one of the oligomeric states present in the
cellular context17. In this model structure, no large steric clashes are observed at the
subunit interfaces (Extended Data Fig. 8b), suggesting that the formation of the
hexamer, as well as the crevice between the subunits, is plausible in the
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LRRC8A-containing heteromers. In contrast, the structure of the pore constriction site,
which is mainly formed by the helix EL1H, is different from that of the homomeric
LRRC8A (Extended Data Fig. 8b). These results again underscore the notion that the
sequence diversity in the pore constriction site is important for the functional diversity
of the LRRC8A-containing heteromers.
In the subunit interface of the TM region, we observed the lateral crevice formed
between the subunits, which is opened toward the membrane side (Fig. 2a). Although it
is currently unknown whether the activity of LRRC8 proteins is influenced by the lipid
environment, it is possible that the LRRC8 proteins might directly interact with the lipid
molecules through these crevices, in a similar manner to other channels, such as the
mechanosensitive two-pore domain K+ channels28. A recent electrophysiological
analysis using purified LRRC8 proteins demonstrated that low ionic strength alone is
sufficient for their activation, and it was speculated that the sensor for the ionic strength
exists in the IL1 loop, as well as in LRRCT17. Indeed, the long linker between the
IL1H2 and IL1H3 helices, which is largely disordered in the present structure, contains
several charged residues that are conserved among LRRC8 isoforms (Extended Data
Fig. 2). Although the question of whether the ionic strength directly modulates the
channel activities of the LRRC8 proteins remains to be answered, these observations
lead us to speculate that the interaction of the IL1 loop with ions affects its
conformation, thereby changing the arrangement of the LRR and other regions, as
observed in the present relaxed form (Fig. 5a, c), to regulate the channel activity. To
understand the gating mechanism of the LRRC8 protein depending on the cellular
volume change, the structural analysis of the closed form of LRRC8 is required.
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Methods
Protein expression, purification and sample preparation for cyro-EM
The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2)
was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an
C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was
expressed in HEK293S GnTI- (N-acetylglucosaminyl-transferase I-negative) cells
(ATCC, cat. no. CRL-3022)29. Cells were collected by centrifugation (6000×g, 10 min,
4 °C) and disrupted by sonication in buffer (50 mM Tris, pH 8.0, 150 mM NaCl)
supplemented with 5.2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1.4 µg/ml pepstatinA
(all from Calbiochem). Cell debris was removed by centrifugation (10,000×g, 10 min,
4 °C). The membrane fraction was collected by ultracentrifugation (138,000×g, 1 h,
4 °C). The membrane fraction was solubilized for 1 h at 4 °C in buffer (50 mM Tris, pH
8.0, 150 mM NaCl, 5 mM dithiothreitol (DTT), 1% digitonin (Calbiochem)). Insoluble
materials were removed by ultracentrifugation (138,000×g, 1 h, 4 °C). The
detergent-soluble fraction was incubated with CNBr-Activated Sepharose 4 Fast Flow
beads (GE Healthcare) coupled with an anti-GFP nanobody (GFP enhancer)30, and
incubated for 2 h at 4 ºC. The beads were washed with SEC buffer (50 mM Tris, pH 8.0,
150 mM NaCl, 5 mM DTT, 0.1% digitonin), and further incubated overnight with TEV
protease to remove the GFP-His8 tag. After TEV protease digestion, the flow-through
was collected, concentrated, and purified by size-exclusion chromatography on a
Superose 6 Increase 10/300 GL column (GE Healthcare), equilibrated with SEC buffer.
The peak fractions of the protein were collected and concentrated to 15 mg/ml, using a
centrifugal filter unit (Merck Millipore, 100 kDa molecular weight cutoff). A 3 µl
portion of the concentrated HsLRRC8A protein (15 mg/ml) was applied to a
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glow-discharged Quantifoil R1.2/1.3 Cu/Rh 300 mesh grid (Quantifoil), blotted using
Vitrobot Mark IV (FEI) under 4ºC and 100% humidity conditions, and then frozen in
liquid ethane.
EM image acquisition and data processing
The grid images were obtained with a Tecnai Arctica transmission electron microscope
(FEI) operated at 200 kV, and recorded by a K2 Summit direct electron detector (Gatan)
operated in the super-resolution mode with a binned pixel size of 1.490 Å. The dataset
was acquired with the SerialEM software31. Each image was dose-fractionated to 40
frames at a dose rate of 6-8 e-/pixel/sec, to accumulate a total dose of ~50 e-/Å2. In total,
5,305 super-resolution movies were collected. The movie frames were aligned in 5 x 5
patches, dose weighted and binned by two in MotionCor2 (ref. 32), and down-sampled
by two. Defocus parameters were estimated by CTFFIND 4.1 (ref. 33). First,
template-based auto-picking was performed with 2D class averages of a few hundred
manually picked particles as templates34. A total of 1,421,314 particles were extracted
in 3.25 Å/pix. These particles were divided into four batches and subjected to three
rounds of 2D classification in RELION 2.1 (refs. 34,35). The initial model was
generated in RELION. Subsequently, 664,971 good particles were further classified in
3D without symmetry. As indicated in Extended Data Fig. 3b, some classes exhibited
very weak density for one or two of the six subunits. Therefore, 164,749 particles in
classes with six intact subunits were re-extracted in the original pixel size of 1.49 Å/pix
and refined in C3 symmetry. The overall gold-standard resolution was 4.25 Å, with the
local resolution in the core TM region extending to 3.8 Å and that in the peripheral
region extending to 6.5 Å (Extended Data Fig. 3c-d).
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Model building
The extracellular, transmembrane, and intracellular regions of HsLRRC8A were built
de novo into the electron density map in COOT36, using the Innexin-6 (PDB ID: 5H1Q)
structure as a guide. The LRR region was initially generated by creating a homology
model based on the LRR protein from L. interrogans (PDB ID: 4U08), using the Phyre2
server 24. The initial model was then refined, using PHENIX37 with secondary structure
restraints. For cross-validation, the final model was refined against one of the half maps
generated in RELION, and FSC curves were calculated between the refined model and
the half map 1, and between the refined model and the half map 2 (Extended Data Fig.
3e).
Liposome reconstitution
The purified HsLRRC8A protein was incorporated into artificial liposomes, using a
modified sucrose procedure38. Briefly, a 200 µl aliquot of a 25 mg/ml solution of
azolectin in chloroform (Avanti Polar Lipids) was dried in a glass test tube under a
stream of N2 while rotating the tube to form a homogeneous lipid film. After the lipid
was dried, 5 µl of pure water was added to the bottom (pre-hydration), followed by 1 ml
of 0.4 M sucrose. The solution was incubated for 3 h at 50 °C until the lipid was
resuspended. After cooling the solution to room temperature, the purified HsLRRC8A
protein was added to achieve the desired protein-to-lipid ratio (protein: lipid = 1:1000,
w/w). The glass tube containing the protein-lipid solution was shaken gently on an
orbital mixer for 3 h at 4 °C. After this procedure, the sample was ready for patch
clamping.
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Patch clamping recordings
An aliquot of liposomes (2-4 µl) was taken from the liposome cloud and introduced to
the recording bath. All recordings were performed with the excised liposome patched.
For recording under asymmetric conditions, the pipette buffer (70 mM KCl, 1.5 mM
CaCl2, 1 mM MgCl2, 10 mM dextrose, and 10 mM HEPES, pH 7.4) and the bath buffer
(500 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM dextrose, and 10 mM HEPES, pH
7.4) were used. For recording under symmetric conditions, the bath and pipette buffers
were identical and consisted of a solution of 70 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2,
10 mM dextrose, and 10 mM HEPES, pH 7.4. The pH of buffers was adjusted by KOH.
For patch pipette preparation, the borosilicate glass pipettes (BF150-86-10, Sutter
Instrument, Novato) were pulled using a Flaming/Brown micropipette puller (P-97,
Sutter Instrument, Novato) and polished to a diameter corresponding to a pipette
resistance in the range of 4.0-6.0 MΩ. The recordings were performed when the pipette
was tightly sealed with the liposome membrane and the patch resistance increased
toward ~2 GΩ. The data were acquired at 50 kHz with a 0.5-kHz filter and a 50 Hz
notch filter, using an EPC-10 amplifier (HEKA, Lambrecht, Germany). All experiments
were performed at room temperature (21-24 ºC). All electrophysiological data were
analyzed by using the pCLAMP10 software (Axon Instruments, Foster City, CA).
Cell line formation for measurements of VRAC activity HEK293A cells (Invitrogen) and LRRC8A-knockout HEK293A cells were cultured in
DMEM-high glucose (Sigma-Aldrich, Cat. #D5671), supplemented with 10% fetal
bovine serum (FBS; BioWest, Cat. #S1560-500) and 100 units/ml penicillin G (Meiji
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Seika, Cat. #6111400D2039). All cells were cultured in a 5% CO2 atmosphere at 37ºC
and verified to be negative for mycoplasma. LRRC8A-knockout HEK293A cells were
established using the CRISPR-Cas9 system39. Briefly, the targeting sequence for human
LRRC8A, 5′-GCACAACATCAAGTTCGACGTGG-3′, was designed using the
CRISPR design tool (http://crispr.mit.edu/), and oligo DNAs for its sense and anti-sense
strands were purchased from Eurofins Genomics. The sgRNA expression construct was
generated by inserting the annealed guide oligo duplex with BbsI into
pSpCas9(BB)-2A-Puro (PX459) V2.0, a gift from Dr. Feng Zhang (Addgene plasmid
#62988). The targeting sequence of the generated constructs was verified by sequencing.
Subsequently, HEK293A cells were transfected with the LRRC8A sgRNA expression
plasmid, using Lipofectamine 2000 (Invitrogen, Cat. #11668-019). After 29 h, the cells
were selected by 1 µg/ml puromycin for 44 h, followed by limiting dilution to establish
clonal cell lines. The generated LRRC8A-knockout cell lines were validated by checking
for the depletion of the LRRC8A proteins and the absence of novel truncated LRRC8A
proteins by western blotting.
Expression plasmids for this study were constructed by standard molecular biology
techniques, and all constructs were verified by sequencing. The human LRRC8A cDNA
(CDS of NM_019594.3 with the silent mutation c.1509C>T) was cloned from a cDNA
pool derived from HEK293A cells and subcloned into pcDNA3. LRRC8A mutants
(D102A, R103A, H104A, H105A, and Y106A) were constructed from LRRC8A
wild-type (WT) and subcloned into pcDNA3 (Invitrogen). A halide-sensitive YFP
(H148Q/I152L)26 mutant was generated from YFP (WT) and subcloned into pcDNA3.
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Western blotting and antibodies
Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM
EDTA, 1% sodium deoxycholate, and 1% Triton X-100), supplemented with 1 mM
phenylmethylsulfonyl fluoride (PMSF) and 5 µg/ml leupeptin. The cell extracts were
clarified by centrifugation, and aliquots of the supernatants were mixed with 2x SDS
sample buffer (80 mM Tris-HCl, pH 8.8, 80 µg/ml bromophenol blue, 28.8% glycerol,
4% SDS, and 10 mM dithiothreitol). After boiling at 98ºC for 3 min, the samples were
resolved by SDS-PAGE and electroblotted onto an Immobilon-P membrane (Millipore,
Cat. #IPVH00010). The membrane was blocked with 2.5% skim milk in TBS-T (50
mM Tris-HCl, pH 8.0, 137 mM NaCl, and 0.05% Tween 20) and probed with the
appropriate antibodies. Antibody-antigen complexes were detected on X-ray films
(FUJIFILM, Cat. #47410-07523, Cat. #47410-26615 or Cat. #47410-07595) using the
ECL system (GE Healthcare). Antibodies against LRRC8A (Cat. #A304-175A, 1:5,000),
YFP-tag (YFP; Clone #1E4, Cat. #M048-3, 1:10,000), and actin (Cat. #A3853, 1:5,000)
were purchased from Bethyl Laboratories, MBL, and Sigma-Aldrich, respectively.
HRP-conjugated secondary antibodies against rabbit IgG (Cat. #7074, 1:2,000) and
mouse IgG (Cat. #7076, 1:10,000–1:20,000) were purchased from Cell Signaling
Technology.
Cell-based measurements of VRAC activity The VRAC activity in LRRC8A-rescued LRRC8A-knockout HEK293A cells was
evaluated by the efficiency of iodide influx in the halide-sensitive YFP quenching
assay9,10,26. LRRC8A-knockout HEK293A cells were transfected with YFP
(H148A/I152L) and/or LRRC8A mutant expression plasmids, using Lipofectamine
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2000. After 4 h, the cells were reseeded in 96-well black imaging plates (Falcon, Cat.
#353219) and cultured for 65 h. Prior to measurements, the culture medium was
–1 exchanged with 50 µl/well of isoosmotic buffer (332 mOsm (kg H2O) , pH 7.4; 145
mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES).
The initial YFP fluorescence (Ex: 500 ± 6 nm, Em: 545 ± 6 nm) was measured 5 times
at 10-sec intervals using a Varioskan Flash luminometer (Thermo Fisher Scientific).
After the addition of 125 µl/well of iodide-containing hypoosmotic buffer (190 mOsm,
pH 7.4; 70 mM NaI, 5 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
glucose, 10 mM HEPES) or isoosmotic buffer (332 mOsm, pH 7.4; 70 mM NaI, 5 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 140
mM mannitol), subsequent measurements were performed 45 times at 10-sec intervals.
For the data analysis, each fluorescent value was subjected to background subtraction
by the fluorescent values of non-YFP (H148Q/I152L)-transfected cells, followed by
normalization with the fluorescent value at the 1st time point after the addition of the
iodide-containing buffer. To evaluate the efficiency of iodide influx, the normalized
fluorescence values obtained after adding the iodide-containing buffer from sextuplicate
wells were fitted to a one-phase exponential decay curve y = A exp(-t/t) + B by
nonlinear least squares on R, using RStudio. Using the estimated parameters, the speed
of YFP quenching at time t was calculated as -dy/dt = A/t exp(-t/t), and the maximum
speed of YFP quenching was defined as the speed of YFP quenching at the 1st time
point after the addition of the iodide-containing buffer: A/t. Finally, the mean of the
maximum speed of YFP quenching was computed from 4 independent experiments, for
the comparisons between the samples.
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Immunocytochemistry LRRC8A-knockout HEK293A cells were transfected with the LRRC8A mutant
expression plasmids, using Lipofectamine 2000. After 5 h, the cells were reseeded on a
1% gelatin-coated cover slip (Matsunami Glass, Cat. #C015001) and cultured for 65 h.
The cells were fixed by 4% formaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 8.1
mM Na2HPO4•12H2O, 1.47 mM KH2PO4) for 15 min at room temperature,
permeabilized with 1% Triton X-100 in PBS for 15 min at room temperature, blocked
with 5% skim milk in TBS-T for 30 min at room temperature, and incubated with the
primary antibody for LRRC8A overnight at 4ºC. The samples were incubated with an
Alexa Fluor 594-conjugated secondary antibody against rabbit IgG (Molecular Probes,
Cat. #R37117) for 2 h at room temperature, followed by nuclear staining with Hoechst
33258 (Dojindo, Cat. #343-07961) for 30 min at room temperature. The cover slip was
mounted on a microscope slide (Matsunami Glass, Cat. #S2444) with Fluoromount
(Diagnostic BioSystems, Cat. #K024). The images were collected with a confocal
microscope, TCS-SP5 (Leica), with an HCX PL APO 63x/1.40–0.60 oil objective lens.
Statistical analysis The data are summarized as the mean ± s.e.m. No statistical method was utilized to
predetermine sample size. Homoscedasticity was assumed unless P < 0.01 in Levene’s
test based on the absolute deviations from the median. Statistical tests and sample size n
are indicated in the figure legends. Statistical tests were performed using R with
RStudio (http://www.rstudio.com/), and P < 0.05 was considered statistically significant.
No samples were excluded from statistical tests. The investigators were not blinded to
allocation during experiments and outcome assessment. The experiments were not
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randomized.
Data availability
The raw image of HsLRRC8A after motion correction has been deposited in the
Electron Microscopy Public Image Archive (EMPIAR), under the accession number
XXXX. The cryo-EM density map of HsLRRC8 has been deposited in the Electron
Microscopy Data Bank (EMDB), under the accession number YYYY. The molecular
coordinates of HsLRRC8A have been deposited in the RCSB Protein Data Bank (PDB),
under the accession number ZZZZ.
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Acknowledgements
We thank the members of the Nureki lab, the Kikkawa lab, and the RIKEN Center for
Life Science Technologies, especially Dr. Mizuki Takemoto and Dr. Reiya Taniguchi
(Nureki lab) for assistance with the manuscript preparation and Dr. Hideki Shigematsu
(RIKEN) for microscope maintenance. We also thank Dr. Yuhei Araiso (Kyoto Sangyo
University) for instructions regarding the digitonin preparation; Mr. Kouki Touhara
(The Rockefeller University) for advice about the anti-GFP nanobody preparation; and
Dr. Kenji Iwasaki, Dr. Naoyuki Miyazaki, and Dr. Akihiro Kawamoto (Osaka
University) for optimization of the cryo-EM experiment. This work was supported by a
grant from the National Key R&D Program of China (2016YFA0502800); by the
RIKEN Pioneering Project “Dynamic Structural Biology”; by the Platform for Drug
Discovery, Informatics and Structural Life Science from Japan Agency for Medical
Research and Development (AMED) to O.N.; by AMED (Grant No. JP17gm5010001)
to H.I.; by a MEXT Grant-in-Aid for Specially Promoted Research (Grant No.
16H06294) to O.N.; by the National Natural Science Foundation of China (Grant No.
31571083) to Z.Y.; by the Program for Professor of Special Appointment (Eastern
Scholar of Shanghai, TP2014008) to Z.Y.; by the Shanghai Rising-Star Program
(4QA1400800) to Z.Y.; by the Young 1000 Talent Program of China to Z.Y.; and by
JSPS KAKENHI (Grant Nos. 17J06101 to G.K.; 17K15086 to K.W.; 25221302 to H.I.;
17H03640 to R.I.; 24227004 to O.N.). This research was also supported by the Platform
Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting
Innovative Drug Discovery and Life Science Research (BINDS)) from AMED.
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Author contributions
G.K. and O.N. conceived and designed the project. G.K. screened and purified the
protein, and prepared cryo-EM samples. N.D. and R.N. assisted with the preparation of
cryo-EM samples. G.K., T.Nakane, T.Y., and M.S. performed cryo-EM data collection
and processing. T.Nishizawa, T.K., N.M., A.T., H.Y., and M.K. assisted with the
optimization of the cryo-EM data collection. G.K. and R.I. performed the model
building, with assistance from T.Nishizawa. Y.J., M.H., and Z.Y. performed the patch
clamping recordings. M.I., K.W., and H.I. performed the cell-based quenching assay.
G.K., R.I., and O.N. wrote the manuscript. O.N. supervised all of the research.
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Figure Legends
Figure 1. Overall structure and subunit interactions of the HsLRRC8A
hexamer.
(a) Local resolution of the HsLRRC8A structure, estimated by RELION. (b)
Representative density maps of the transmembrane and LRR regions. The cartoon
models of the TM1, EL1 and the following TM2, and LRR regions were fitted to the
density maps (colored gray). (c) Overall structure of the HsLRRC8A hexamer, viewed
parallel to the membrane (left). Overall structure of the HsLRRC8A hexamer with
molecular envelope, viewed from the extracellular side (right, upper) and the
intracellular side (right, lower). (d) The subunit structures of HsLRRC8A, viewed
parallel to the membrane. Each region is colored as follows: TM1, purple; EL1, blue;
TM2, light blue; IL1, cyan; TM3, green; EL2, light green; TM4, yellow; IL2, orange;
LRRNT, red; LRR1-15, pink; and LRRCT, brown. The N- and C- termini are indicated
by ‘N’ and ‘C’, respectively. (e) Schematic representation of the HsLRRC8A membrane
topology, colored as in (d). The dotted lines indicate the disordered linkers. The
molecular graphics were illustrated with CueMol (http://www.cuemol.org/).
Figure 2. Subunit Interface.
(a) The tight and loose subunit interfaces (left and right panels, respectively) between
the three neighboring subunits (α, β, and ζ) of HsLRRC8A. The structures of the
subunits are shown as the solvent-accessible surface models. (b) Two adjacent subunits
(α and β subunits) of HsLRRC8A are superimposed on each other, using only the TM
region. The RMSD value for the 553 Cα atoms from the subunits is 3.79 Å. (c)
Close-up view of the superimposed intracellular IL2 regions from two adjacent subunits.
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The residues involved in helix formation are depicted in stick representations. The
RMSD value for the 66 Cα atoms from the subunits of the IL2 regions is 0.88 Å.
Figure 3. Ion channel pore.
(a) Cross-sections of the surface representation for the N-terminal half of the
HsLRRC8A subunits, showing the ion channel pore. The four subunits are colored
according to Fig. 1d, and are shown in cartoon representations. (b) The pore radius for
the HsLRRC8A structure along the pore center axis. The pore size was calculated with
the program HOLE40. (c-e) Close-up views of the channel pore forming regions. In the
left views, only two diagonal subunits viewed parallel to the membrane are shown, for
clarity. The side chains of the pore-lining amino residues are depicted in stick
representations. The distances between the residues involved in the pore constrictions
are shown. In the right views, all six subunits viewed from the extracellular side are
shown.
Figure 4. Pore constriction site.
(a) Close-up view of the neighboring EL1 helices forming the pore constriction site.
The side chains are depicted by stick models. (b) Sequence alignment around the pore
constriction site, depicted according to Extended Data Fig. 2. (c) VRAC activity in the
LRRC8A-rescued LRRC8A-knockout HEK293A cells. Each line indicates the mean ±
s.e.m. of the normalized YFP fluorescence (n = 4). Hypotonicity after the addition of
iodide: 231 mOsm. WT (+) and WT (++++) represent the different expression levels of
the LRRC8A wild-type protein detected by western blotting, according to Extended
Data Fig. 6c. (d) Maximum speed of hypotonicity-induced YFP quenching. Data are
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presented as the mean ± s.e.m. with each individual light grey point (n = 4). ** P < 0.01,
*** P < 0.001 by Dunnett’s test.
Figure 5. Compact and relaxed structures of HsLRRC8A.
(a,b) Overall structure of the HsLRRC8A hexamer in the relaxed form (a), and in the
high-resolution compact form (b), viewed parallel to the membrane (left), and from the
extracellular side (right). The structures are depicted in cylinder representations. (c)
Schematic drawing depicting the structural transition between the compact and relaxed
forms of HsLRRC8A.
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Extended Data Figure Legends
Extended Data Figure 1. Functional properties of the HsLRRC8A protein.
(a) FSEC profiles on a Superose 6 Increase 10/300 GL column (GE Healthcare) for the
EGFP-fused HsLRRC8A expressed in HEK293S GnTI- cells. The arrows indicate the
estimated elution positions of the void volume, the EGFP-fused HsLRRC8A, and the
free EGFP. (b) Single-channel current recordings of HsLRRC8A after reconstitution in
liposomes under asymmetric salt conditions (500 mM KCl in bath, 70 mM KCl in
pipette), and the normalized all-points amplitude histogram analysis of HsLRRC8A in
the open and closed states at 100 mV. The gray dashed lines indicate the closed (C) and
open (O) states. For the plot, the bin width is set at 0.1 pA/bin and the total count of
events is normalized to 1.0. The distribution data were fit by the sum of two Gaussians,
and the peaks correspond to the closed (C) and open (O) states. (c) Single-channel
current recordings of HsLRRC8A after reconstitution in liposomes under symmetric salt
conditions (70 mM KCl in bath, 70 mM KCl in pipette), and the normalized all-points
amplitude histogram analysis of HsLRRC8A at 100 mV.
Extended Data Figure 2. Sequence alignment of LRRC8 isoforms
The amino acid sequences of LRRC8 isoforms were aligned using Clustal Omega41, and
are shown using ESPript3 (ref. 42). The secondary structure elements and the colors of
the domains from HsLRRC8A are labeled above the alignments. For the sequence
alignment, the human and mouse LRRC8 isoforms were used: human (HsLRRC8A,
NCBI Reference sequence number: NP_062540.2; HsLRRC8B, NP_056165.1;
HsLRRC8C, NP_115646.2; HsLRRC8D, NP_060573.2; HsLRRC8E, AAH70089.1)
and mouse (MmLRRC8A, NP_808393.1; MmLRRC8B, NP_001028722.1;
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MmLRRC8C, NP_598658.1; MmLRRC8D, NP_848816.3; MmLRRC8E,
NP_082451.2).
Extended Data Figure 3. Cyro-EM analysis of HsLRRC8A
(a) A representative cryo-EM micrograph of HsLRRC8A. Red circles indicate
individual particles. The white scale bar represents 50 nm. (b) Flow chart of cryo-EM
data processing of the HsLRRC8A structure, including particle picking, classification,
and 3D refinement. (c) Angular distribution plot of particles included in the final 3D
reconstruction of HsLRRC8A, with C3 symmetry imposed. (d) Fourier Shell
Correlation (FSC) curve of the final 3D reconstruction model calculated using
“relion_postprocess” with masked marked 4.25 Å resolution, corresponding to the FSC
= 0.143 gold standard cut-off criterion. (e) Cross-validation FSC curves for the refined
model versus half maps (Half maps 1 and 2), and versus summed map.
Extended Data Figure 4. Structural comparison of two adjacent subunits.
(a) Two adjacent subunits (α and β subunits) of HsLRRC8A are superimposed on each
other. The RMSD value for the 553 Cα atoms from the subunits is 3.79 Å. For clarity,
the overall subunits superimposed using only the TM region are presented. (b) The
extracellular regions from two adjacent subunits are superimposed on each other. The
RMSD value for the 68 Cα atoms from the two extracellular regions is 0.39 Å. (c) The
transmembrane regions from two adjacent subunits are superimposed on each other.
The RMSD value for the 110 Cα atoms from the two extracellular regions is 0.40 Å. (d)
The intracellular regions from two adjacent subunits are superimposed on each other.
The RMSD value for the 119 Cα atoms from the two extracellular regions is 0.98 Å. (e)
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The LRR regions from two adjacent subunits are superimposed on each other. The
RMSD value for the 395 Cα atoms from the two extracellular regions is 0.47 Å.
Extended Data Figure 5. Subunit interfaces between two adjacent
subunits.
(a,b) Close-up views between two adjacent subunits at the extracellular region, showing
the tight (a) and loose (b) interactions. (c,d) Close-up views between two adjacent
subunits at the transmembrane region, showing the tight (c) and loose (d) interactions.
(e,f) Close-up views between two adjacent subunits at the intracellular region, showing
the tight (e) and loose (f) interactions. (g,h) Close-up views between two adjacent
subunits at the LRR region, showing the tight (g) and loose (h) interactions. A surface
model with the side chains of the residues lining the subunit interfaces depicted by stick
models is presented for each panel. Three adjacent subunits (α, β, and ζ subunits) of
HsLRRC8A are colored according to Fig. 1d.
Extended Data Figure 6. Validation of LRRC8A mutants and YFP
quenching assay.
(a) Subcellular localization of LRRC8A mutants rescued in LRRC8A-knockout
HEK293A cells. The white dashed square indicates a region around the cell membrane;
the magnified image of the LRRC8A-derived fluorescent channel is located in the
corner. The white scale bar indicates 25 µm. (b) FSEC profiles on a Superose 6 Increase
10/300 GL column (GE Healthcare) for the EGFP-fused HsLRRC8A mutants expressed
in HEK293S GnTI- cells. The arrows indicate the estimated elution positions of the void
volume, the EGFP-fused HsLRRC8A, and the free EGFP. The analysis of the mutants
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showed symmetric and monodisperse peaks, similar to those of the wild-type channel,
confirming the proper structural integrities of these mutant channels. (c) Amounts of
HsLRRC8A expression in rescued LRRC8A-knockout HEK293A cells. † indicates
non-specific bands. (d) Comparison between individual time courses and the fitting
curve of the normalized YFP fluorescence. Grey lines and light blue lines indicate
individual time courses of normalized YFP fluorescence (n = 4). Blue lines indicate the
mean of one-phase exponential decay-fitting curves. I–-iso: 332 mOsm, I–-hypo: 231
mOsm.
Extended Data Figure 7. Structural similarity between HsLRRC8A, innexin,
and connexin.
(a-c) The N-terminal halves of the HsLRRC8A subunit (a), the CeInnexin-6 (PDB ID:
5H1Q) subunit (b), and the HsConnexin-26 (PDB ID: 2ZW3) subunit (c) are presented.
The subunits of CeInnexin-6 and HsConnexin-26 are colored according to the
HsLRRC8A coloring. The N- and C- termini are indicated by ‘N’ and ‘C’, respectively.
In each structure, the Cys residues forming disulfide bonds at the extracellular region
are depicted by stick models. In each panel, a close-up view of the disulfide bonds is
shown in the inset. (d-f) The solvent-excluded molecular surfaces of HsLRRC8A (d),
CeInnexin-6 (e), and HsConnexin-26 (f). The surfaces are colored according to the
subunits. For HsLRRC8A, the extracellular, TM, and intracellular regions are shown.
(g-i) The channel pore and N-terminal region of HsLRRC8A (d), CeInnexin-6 (e), and
HsConnexin-26 (f). The protein main chains are shown as cylinder models. (j) The
secondary structure prediction of the N-terminal regions of HsLRRC8A, CeInnexin-6,
and HsConnexin-26. The prediction was performed by the program PSIPRED43. The
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actual locations of the corresponding helices are indicated by the red and pink boxes for
NTH and TM1, respectively.
Extended Data Figure 8. Model of the heteromeric LRRC8 protein.
(a) The conservation score mapped on the molecular surface of HsLRRC8A. The score
was calculated by the program CONSURF27, based on the multiple-sequence alignment
of the A-E isomers in Extended Data Fig. 2. The molecular surface is colored according
to the normalized conservation score, from 0.752 (pink) to -0.983 (purple). (b) The
model structure of the (LRRC8A)5·LRRC8D heterohexamer, viewed parallel to the
membrane (left), and from the extracellular side (right). The homology model of human
LRRC8D was generated by the program MODELLER44, using the alignment in
Extended Data Fig. 2, and then the α subunit of the present structure was replaced by
this model. In the inset, a close-up view of the channel pore forming regions from the
extracellular side is shown, and the side chains of pore-lining amino residues are
depicted in stick representations. The five LRRC8A subunits are colored light blue,
while the LRRC8D isoform is colored light brown.
Extended Data Table 1. Data collection, refinement and validation
statistics.
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— 42 — bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A B D102 T48 R103
3.8 Å
4.2 Å
4.7 Å I135 W23 5.2 Å TM1 EL1-TM2 LRRNT,LRR1-15,LRRCT 5.7 Å (W23-T48) (D102-I135) (L413-D806)
C
Extracellular region (35 Å) Out
TM region (40 Å) 90°
Intracellular In region (35 Å)
α subunit LRR region (60 Å) β subunit 90° γ subunit δ subunit ε subunit 130 Å ζ subunit
D Extracellular E Extracellular region region EL1H EL1 EL1β EL2β2 EL2 EL2β1
Out Out TM region TM region TM3 TM1 TM2 TM1 TM1 TM3 TM3 TM4 TM4 TM2
In N In N Intracellular Intracellular N region region
IL2H2 IL2H3 IL1 IL2H3 IL1H1 IL2H1 IL2 180° IL1H2 IL1H3
IL2H4 LRRNT
LRR region LRR1 LRR2 LRR3 LRR4 LRR5 LRR6 LRR7 LRR8 LRR9 LRR10
C C LRRNT C
LRRCT LRR1-15 LRRCT LRR15 LRR14 LRR13 LRR12 LRR11
Kasuya et al. Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A Tight interface Loose interface
60°
α subunit β subunit ζ subunit
B C
N IL2H2
IL2H3 IL2H1
60° IL2H4 ~11º
LRRNT α subunit β subunit C
~22º~22°
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A C Extracellular region Extracellular EL1H region
EL1H D102 R103 14.8 Å
TM region N107 D110 7.6 Å 180° R103 Y114 TM1 Intracellular IL1H1 region D TM region IL1H3 TM1 T48 T44 13.7 Å V40 M37 I33 180° T48 B Y30 V26 140
Extracellular 50.8 Å region 120 D102 R103 Constriction 100 T48 Intracellular region T44 TM region E 80 I33 IL1H1 S150 60 R18 Intracellular
region E154 D165 40 S158 Distance along pore (Å) K235 D165 K235
20 LRR region D414 K235
0 IL1H3 22.5 Å 180° 0 4 8 12 16 Pore radius (Å)
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A B H104 (α) EL1H R103 (α) 100 110
H104 (β) HsLRRC8A K YD L D RH QY NYV DA VCY E N R103 (β) D102 (α) MmLRRC8A K YD L D RH QY NYV DA VCY E N HsLRRC8B Q N DL HR QQY SYI DA VCY E K MmLRRC8B Q N DL HR QQY SYI DA VCY E K HsLRRC8C KT DL D L QQY SFI NQ MCY E R Q105 (α) MmLRRC8C KT DL D L QQY SFI NQ MCY E R L101 (α) Y106 (α) HsLRRC8D KT NL D F QQY V FI NQ MCY HL MmLRRC8D KT NL D F QQY V FI NQ MCY HL Q105 (β) HsLRRC8E KN NL D L QQY SFI NQ LCY E T D100 (α) MmLRRC8E KN NL D L QQY SFI NQ LCY E T
K98 (α) Y99 (α)
C Iodide-containing buffer D 0.006 ***
1.0 0.005 *** **
- 0.004 *** Y106A 0.8 Q105A 0.003 WT (+) D102A H104A 0.002 0.6 WT (++++)
R103A 0.001 Normalized YFP fluorescence (a.u.) Maximum speed of YFP quenching [a.u.] 0 100 200 300 400 0.000 - WT WT D102A R103A H104A Q105A Y106A Time after adding iodide-containing buffer (Sec) (+) (++++)
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A Relaxed form Extracellular region (35 Å) Out
TM region (40 Å)
Intracellular In region 90° (33 Å)
LRR region (52 Å)
150 Å B Extracellular Compact form region (35 Å) Out
TM region (40 Å)
Intracellular In region 90° (35 Å)
LRR region (60 Å)
130 Å
C Compact form Relaxed form Out
TM
In IL1 and IL2
LRR
Kasuya et al. Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B 1.0
0.8 Count (N) HsLRRC8A-EGFP O 0.6 C C 5 pA 0.4 0.5 s 0.2 O
-2 -1 0 1 2 3 4 5 EGFP Amplitude (pA) C 1.0 Void
0.8 Count (N)
0.6 10 pA Normalized fluorescence (a.u.) 0 1 s 0.4 0 5 10 15 20 25 0.2 Volume (ml)
-0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 Amplitude (pA)
Kasuya et al. Figure S1 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
TM1 EL1β α1 β1 . 1 10 20 30 40 50 60 70 HsLRRC8A M I P V TE L R YFA D TQ P A YRI LKPWWDV F T DY ISIV ML MI A VF GG TL Q VTQDKMI .CLP C K WVTKD S CNDSFRGWA A .... . MmLRRC8A M I P V TE L R YFA D TQ P A YRI LKPWWDV F T DY ISIV ML MI A VF GG TL Q VTQDKMI .CLP C K WVTKD S CNDSFRGWA A .... . HsLRRC8B M I T L TE L K CLA D AQ SS YHI LKPWWDV F WY Y ITLI ML LV A V LAGA LQ LTQ S RVL CCLP C K VEFD N HCAVPWDILK A .... . MmLRRC8B M I T L TE L K CLA D AQ SS YHI LKPWWDV F WY Y ITLI ML LV A V LAGA LQ LTQ S RVL CCLP C K VEFD NQ CAVPWDLLKG.... . HsLRRC8C M I P V TE F R QFS E QQ P A FRV LKPWWDV F T DY LSVA ML MI G VF GC TL Q V M QDKII .CLP K R VQPA QN HSSLSNVSQ A .... . MmLRRC8C M I P V TE F R QFS E QQ P A FRV LKPWWDV F T DY LSVA ML MI G VF GC TL Q V M QDKII .CLP K R VQPA QN HSSVPNVSQ A .... . HsLRRC8D M FT L AE VASLN D IQ P T YRI LKPWWDV F M DY L A VV ML MV A IF AG TM Q LT K D Q VV .CLP VLPSPV NS KAHTPPGNAEVTTN I MmLRRC8D M FT L AE VASLN D IQ P T YRI LKPWWDV F M DY L A VV ML MV A IF AG TM Q LT K D Q VV .CLP VLPSPA NS KAHTPPGNADITTE V HsLRRC8E M I P V AE F K QFT E QQ P A FKV LKPWWDV LA EY LTVA ML MI G VF GC TL Q VTQDKII .CLP N H ELQE N L...... SE A .... . MmLRRC8E M I P V AE F K QFT E QQ P A FKV LKPWWDV LA EY LTVA ML MI G VF GC TL Q VTQDKII .CLP S H ESRE N I...... SG A .... .
EL1H α2
80 90 100 110 HsLRRC8A ...... PGPEPTYPN S TIL P T P D...... TGPT GIK YD L D RH QY NYV DA VCY E NR L MmLRRC8A ...... SSPEPTYPN S TVL P T P D...... TGPT GIK YD L D RH QY NYV DA VCY E NR L HsLRRC8B ...... SMNTSS.....NP G T P L...... PLPLR I Q N DL HR QQY SYI DA VCY E KQ L MmLRRC8B ...... SENASS.....NS G LLL...... PLPLR I Q N DL HR QQY SYI DA VCY E KQ L HsLRRC8C ...... VAS T TPL P P P K....PSPANPITVEMK GLKT DL D L QQY SFI NQ MCY E RA L MmLRRC8C ...... VIS T TPL P P P K....PSPTNPATVEMK GLKT DL D L QQY SFI NQ MCY E RA L HsLRRC8D PKMEAATNQDQDGRT.TNDISFGTSAVTPDIPLRATY P RTDFALPNQEAKKEKKDPT G R KT NL D F QQY V FI NQ MCY HLA L MmLRRC8D PRMETATHQDQNGQTTTNDVAFGTSAVTPDIPL Q ATH P HAESTLPNQEAKKEKRDPT G R KT NL D F QQY V FI NQ MCY HLA L HsLRRC8E ...... PC Q QLL P R G I....P.EQIGALQEVK GLKN NL D L QQY SFI NQ LCY E TA L MmLRRC8E ...... PC Q QLL P Q G I....S.EQMGGLRELS GLKN NL D L QQY SFI NQ LCY E TA L TM2 IL1H1 IL1H2
α3 α4 α5 α6
120 130 140 150 160 170 180 190 HsLRRC8A HW FA K YFPYL VLL HT LIFLACSN FW FKF P R TS S KL EHF VS IL LKCF DSPWTT RALSE TVV E E S DPKPAFSKMNGS.MDK K MmLRRC8A HW FA K YFPYL VLL HT LIFLACSN FW FKF P R TS S KL EHF VS IL LKCF DSPWTT RALSE TVV E E S DPKPAFSKMNGS.MDK K HsLRRC8B HW FA K FFPYL VLL HT LIFAACSN FW L HY P S TS S RL EHF V AIL HKCF DSPWTT RALSE TVA E Q S VRPLKLSKSKIL...L S MmLRRC8B HW FA K FFPYL VLL HT LIFAACSN FW L HY P S TS S RL EHF VS IL HKCF DSPWTT RALSE TVA E Q S VRPLKLSKSKTL...L S HsLRRC8C HW YA K YFPYL VLI HT LVFMLCSN FW FKF P G SS S KI EHF IS IL GKCF DSPWTT RALSE VSG E D S EEKDNRKNNMNRSNTI . MmLRRC8C HW YA K YFPYL VLI HT LVFMLCSN FW FKF P G SS S KI EHF IS IL GKCF DSPWTT RALSE VSG E D S EEKDNRKNNMNRSGTI . HsLRRC8D PW Y SK YFPYL ALI HT II L MV S SN FW FKY P K T CS KV EHF VS IL GKCF ESPWTT KALSE TAC E D S EENKQRITGAQTLPKH V MmLRRC8D PW Y SK YFPYL ALI HT II L MV S SN FW FKY P K T CS KV EHF VS IL GKCF ESPWTT KALSE TAC E D S EENKQRITGAQTLPKH V HsLRRC8E HW Y TK YFPYL VVI HT LIFMVCTS FW FKF P G TS S KI EHF IS IL GKCF DSPWTT RALSE VSG E N Q KGPAATERAAATIVAM A MmLRRC8E HW YA K YFPYL VVI HT LIFMVCTS FW FKF P G TS S KI EHF IS IL GKCF DSPWTT RALSE VSG E NHKGPASG.RAMVTTVTT T IL1H3 TM3
α7 α8
200 210 220 230 240 250 260 270 HsLRRC8A S S TVSEDVEA.....TVPMLQRTKSRI E QG IVD RSETG VLDKK EGEQAKA LFEKV KK FR THVE E GD IV Y R LY MR Q TII K V MmLRRC8A S S TVSEDVEA.....TVPMLQRTKSRI E QG IVD RSETG VLDKK EGEQAKA LFEKV KK FR THVE E GD IV Y R LY MR Q TII K V HsLRRC8B S S GCSADIDS.....GKQSLPYPQPGL E SAG IE SPTSS VLDKK EGEQAKA IFEKV KR FR MHVE QK D II Y R VY LK Q I IV K V MmLRRC8B T S GGSADIDA.....SKQSLPYPQPGL E SPG IE SPTSS VLDKK EGEQAKA IFEKV KR FR LHVE QR D II Y R VY LK Q I IV K V HsLRRC8C Q S GP...EGS...... LVNSQSLKSIP E KF VVD KSTAG ALDKK EGEQAKA LFEKV KK FR LHVE E GD IL Y A MY VR Q TVL K V MmLRRC8C Q S GP...EGN...... LVRSQSLKSIP E KF VVD KSAAG ALDKK EGEQAKA LFEKV KK FR LHVE E GD IL Y A MY VR Q TVL K V HsLRRC8D ..STSSDEGSPSASTPMINKTGFKFSA E KP VIE VPSMT ILDKK DGEQAKA LFEKV RK FR AHVE D SD LI Y K LY V VQ TVI K T MmLRRC8D ..STSSDEGSPSASTPMINKTGFKFSA E KP VIE VPSMT ILDKK DGEQAKA LFEKV RK FR AHVE D SD LI Y K LY V VQ TLI K T HsLRRC8E G T GP...GKA.....GEGEKEKVLAEP E KV V T E PPVVT LLDKK EGEQAKA LFEKV KK FR MHVE E GD IL Y T MY IR Q TVL K V MmLRRC8E GAGS...GKV.....GEGEKEKVLIEP E KV V S E PPVVT LLDKK EGEQAKA LFEKV KK FR VHVE E GD IL Y S MY IR Q TVL K V EL2β1 EL2β2 TM4
β2 β3 α9
280 290 300 310 320 330 340 350 HsLRRC8A IK F IL I I CY T VY YV HN I K F D V DC T VDI ES LT GY RT Y RC A H P LA T LF KI L A SF Y I SL V IF YG L I CM Y TL WW M L R RS L K KY S MmLRRC8A IK F AL I I CY T VY YV HN I K F D V DC T VDI ES LT GY RT Y RC A H P LA T LF KI L A SF Y I SL V IF YG L I CM Y TL WW M L R RS L K KY S HsLRRC8B I LF VL I I TY VPY FL TH IT LE I DC S VDV QAF TGY KR Y QC VY SL A E IF KV L A SF Y V IL V I LYG L TSS Y SL WW M L R SS L K QY S MmLRRC8B I LF VL I I TY VPY FL SY IT LE I DC S IDV QAF TGY KR Y QC VY SL A E IF KV L A SF Y V IL V M LYG L TSS Y SL WW M L R SS L K QY S HsLRRC8C IK F LI I I AY N SAL V SK VQF T V DC N VDI QD MT GY KN F SC N HTM A H LF SK L SFC Y L CF V S IYG L T CL Y TL YW L FYRS L R EY S MmLRRC8C IK F LI I I AY N SAL V SK VQF T V DC N VDI QD MT GY KN F SC N HTM A H LF SK L SFC Y L CF V S IYG L T CL Y TL YW L FYRS L R EY S HsLRRC8D AK F I FI L CY T AN FV NA ISF EHV C KPK V EH L IGY EV F EC T HNM A Y M LKK L L IS Y I SI I C VYG FI CL Y TL FW L F R IP L K EY S MmLRRC8D AK F I FI L CY T AN FV NA ISF EHV C KPK V EH LT GY EV F EC T HNM A Y M LKK L L IS Y I SI I C VYG FI CL Y TL FW L F R IP L K EY S HsLRRC8E C KF LA I L VY N LV YV EK ISF L V AC R VE TSE VT GY AS F CC N HT KA H LF SK L A FC Y I SF V C IYG L T CI Y TL YW L F H RP L K EY S MmLRRC8E C KF F AI L VY N LI YV EK ISF L V AC R VE TSE IT GY AS F CC N HT KA H LF SK L A FC Y I SF V C VYG I T CL Y TL YW L F H RP L K EY S IL2H1 IL2H2 IL2H3 IL2H4 LRRNT LRR1
α10 α11 α12 α13 α14 β4
360 370 380 390 400 410 420 430 HsLRRC8A F E S IR EES SY SDIPDVKNDFAF MLH LI DQYD PLYSKRF A VFLSEVSE NK L RQLN LN NEWT L DK L RQRLTK N A QD KL EL HL MmLRRC8A F E S IR EES SY SDIPDVKNDFAF MLH LI DQYD PLYSKRF A VFLSEVSE NK L RQLN LN NEWT L DK L RQRLTK N A QD KL EL HL HsLRRC8B F E A LR E K S NY SDIPDVKNDFAF ILH LA DQYD PLYSKRF S IFLSEVSE NK L KQIN LN NEWT V EK L KSKL V KN A QD KI EL HL MmLRRC8B F E A LR E K S NY SDIPDVKNDFAF ILH LA DQYD PLYSKRF S IFLSEVSE NK L KQIN LN NEWT V ER L KSKL V KN SQD KV EL HL HsLRRC8C F E Y VR Q ET GID DIPDVKNDFAF MLH MI DQYD PLYSKRF A VFLSEVSE NK L KQLN LN NEWT P DK L RQKLQ TN A HN RL EL PL MmLRRC8C F E Y VR Q ET GID DIPDVKNDFAF MLH MI DQYD PLYSKRF A VFLSEVSE NK L KQLN LN NEWT P DK L RQKLQ TN A HN RL EL PL HsLRRC8D F E K VR EES SF SDIPDVKNDFAF LLH MV DQYD QLYSKRF G VFLSEVSE NK L R E IS LN HEWT F EK L RQHISR N A QD K QEL HL MmLRRC8D F E K VR EES SF SDIPDVKNDFAF LLH MV DQYD QLYSKRF G VFLSEVSE NK L R E IS LN HEWT F EK L RQHVSR N A QD K QEL HL HsLRRC8E F RS VR EET GMG DIPDVKNDFAF MLH LI DQYD SLYSKRF A VFLSEVSE SR L KQLN LN HEWT P EK L RQKLQR N A AG RL EL AL MmLRRC8E F RS VR EET GM NDIPDVKNDFAF MLH LI DQYD SLYSKRF A VFLSEVSE SR L KQLN LN HEWT P EK L RQKLQR N M RG RL EL SL
Kasuya et al. Figure S2 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
LRR2 LRR3 LRR4 η1 β5 η2 β6 α15 β7 TT TT 440 450 460 470 480 490 500 510 HsLRRC8A FML SG IPD TVF DL V ELEV L KLE LI P DV T IP PS I AQ L TG L KEL W L Y H TA AKI EAP A LA FL RE NL RALHI K F T DI K EI P LW I MmLRRC8A FML SG IPD TVF DL V ELEV L KLE LI P DV T IP PS I AQ L TG L KEL W L Y H TA AKI EAP A LA FL RE NL RALHI K F T DI K EI P LW I HsLRRC8B FML NG LPD NVF ELTEMEV L SLE LI P EV K LP SA V SQ L V NL KEL R V Y H SS L V V DHP A LA FL E E NL KILRL K F T EM GK IP RW V MmLRRC8B FML NG LPD NVF ELTEMEV L SLE LI P EV K LP AA V AQ L V NL REL H V Y H SS L V V DHP A LA FL E E NL RILRL K F T EM GK IP RW V HsLRRC8C IML SG LPD TVF EITEL QS L KLE II KN V M IP AT I AQ L D NL QEL S L HQCS VKI HSA A LS FL KE NL KVL S VK F D DM R EL P PW M MmLRRC8C IML SG LPD TVF EITEL QS L KLE II KN V M IP AT I AQ L D NL QEL C L HQCS VKI HSA A LS FL KE NL KVL S VK F D DM R EL P PW M HsLRRC8D FML SG VPD AVF DLTDLDV L KLE LI P EA K IP AK I SQ M T NL QEL H L C H CP AKV EQT A FS FL RD HL R C LHV K F T DV A EI P AW V MmLRRC8D FML SG VPD AVF DLTDLDV L KLE LI P EA K IP AK I SQ M T NL QEL H L C H CP AKV EQT A FS FL RD HL R C LHV K F T DV A EI P AW V HsLRRC8E CML PG LPD TVF ELSEVE SL RLE AI C DI TF P PG L SQ L VH L QEL S L L H SP ARL PFS L QV FL RD HL KVMRV K CE EL R EV P LW V MmLRRC8E CML PG LPD TVF ELSEVEA L RLE AI C DI SF P PG L SQ L V NL QEL S L L H SP ARL PFSSQI FL RD RL KVI C VK F E EL R EV P LW V LRR5 LRR6 LRR7 β8 α16 η3 β9 η4 β10 α17 TT TT TT TT 520 530 540 550 560 570 580 590 HsLRRC8A Y SL KT L EEL HL TG N L SA E NN R Y I V ID G LRE L K RL KV L R LK S NLSKL P Q V VT D V GV HL QKL S I NN EG TK L IV LN SLKK MA N MmLRRC8A Y SL KT L EEL HL TG N L SA E NN R Y I V ID G LRE L K RL KV L R LK S NLSKL P Q V VT D V GV HL QKL S I NN EG TK L IV LN SLKK MV N HsLRRC8B F HL KN L KEL YL SG C V LP E QLST M Q LE GFQ DL K NL R TL Y LK S SLSRI P Q V VT D L LPS L QKL S L DN EG SK L VV LN NLKK MV N MmLRRC8B F HL KN L KEL YL SG C V LP E QLSS L H LE GFQ DL K NL R TL Y LK S SLSRI P Q V VT D L LPS L QKL S L DN EG SK L VV LN NLKK MV N HsLRRC8C Y GL RN L EEL YL VG S L SH D IS R N V T LE S LRD L K SL KI L S IK S NVSKI P Q A V VD V SS HL QKM C I HN DG TK L VM LN NLKK M T N MmLRRC8C Y GL RN L EEL YL VG S L SH D IS K N V T LE S LRD L K SL KI L S IK S NVSKI P Q A V VD V SS HL QKM C V HN DG TK L VM LN NLKK M T N HsLRRC8D Y LL KN L REL YL IG N L NS E NN K M I G LE S LRE L R HL KI L H VK S NLTKV P S N IT D V AP HL TKL V I HN DG TK L LV LN SLKK MM N MmLRRC8D Y LL KN L REL YL IG N L NS E NN K M I G LE S LRE L R HL KI L H VK S NLTKV P S N IT D V AP HL TKL V I HN DG TK L LV LN SLKK MM N HsLRRC8E F GL R GL EEL HL EG LFPQ E LA R A A T LE S LRE L K QL KV L S LR S NA G KV P AS VT D V AG HL QRL S L HN DG A RL VA LN SLKK LA A MmLRRC8E F GL R GL EEL HL EG LFPP E MA R G A T LE S LRE L K QL KV L S LR S NA G KV P AS VT D V AG HL QRL S L HN DG A RL LA LN SLKK LA V LRR8 LRR9 LRR10 LRR11
β11 η5 β12 α18 β13 η6 β14 TT TT 600 610 620 630 640 650 660 670 HsLRRC8A L T ELEL I RC DLERIPH S IFSL H NL QE IDL KD N NLKTI EEI ISFQH LHR L T CL KLW YN HI AYI P IQ I GN L T NLE RL Y L NR N MmLRRC8A L T ELEL I RC DLERIPH S IFSL H NL QE IDL KD N NLKTI EEI ISFQH LHR L T CL KLW YN HI AYI P IQ I GN L T NLE RL Y L NR N HsLRRC8B L KS LEL I SC DLERIPH S IFSL N NL HE LDL RE N NLKTV EEI ISFQH L QN L S CL KLW HN NI AYI P AQ I GA L S NLE QL SLD HN MmLRRC8B L KS LEL L SC DLERIPH S IFSL N NL HE LDL KE N NLKTV EEI ISFQH L PS L S CL KLW HN NI AYI P AQ I GA L S NLE QL F LG HN HsLRRC8C L T ELEL V HC DLERIPH A VFSL L SL QE LDL KE N NLKSI EEI VSFQH LRK L T VL KLW HN SI T YI P EH I KK L T SLE RL SF SH N MmLRRC8C L T ELEL V HC DLERIPH A VFSL L SL QE LDL KE N NLKSI EEI VSFQH LRK L T VL KLW YN SI AYI P EH I KK L T SLE RL F F SH N HsLRRC8D V A ELEL QN C ELERIPH A IFSL S NL QE LDL K SN NIRTI EEI ISFQH LKR L T CL KLW HN KI V T IP PS I TH V K NLE SL Y F S NN MmLRRC8D V A ELEL QN C ELERIPH A IFSL S NL QE LDL K SN NIRTI EEI ISFQH LKR L T CL KLW HN KI V A IP PS I TH V K NLE SL Y F S NN HsLRRC8E L R ELEL V AC GLERIPH A VFSL GA L QE LDL KD N H LRSI EEI LSFQH C RK L VT L RLW HN QI AYV P EH V RK L R SLE QL Y L S YN MmLRRC8E L R ELEL V AC GLERIPH A IFSL GA L QE LDL KD N H LRSI EEI LSFQH C RK L VT L RLW HN QI AYV P EH V RK L R SLE QL Y L SH N LRR12 LRR13 LRR14
η7 β15 η8 β16 η9 β17 η10 TT TT TT 680 690 700 710 720 730 740 750 HsLRRC8A KI E K IP T Q LF Y C R KLR YLD LS HN N L TF LP A DIGL L QN LQ NLA IT AN R IE TLP PE L F QC RKL R AL H LG NN V LQS L PS RVG E MmLRRC8A KI E K IP T Q LF Y C R KLR YLD LS HN N L TF LP A DIGL L QN LQ NLA VT AN R IE ALP PE L F QC RKL R AL H LG NN V LQS L PS RVG E HsLRRC8B N IE N LP LQ LF L CTKLH YLD LS YN H L TF IP E EI QY L SN LQ YFA VT NN N IE MLP DG L F QC KKL QC L L LG KN S L M NL SP HVG E MmLRRC8B N IE S LP LQ LF L CTKLH YLD LS YN H L TF IP E EI QY L TN LQ YFA VT NN N IE MLP DG L F QC KKL QC L L LG RN S LT DL SPL VG E HsLRRC8C KI E V LP S H LF L CNKIR YLD LS YN D I RF IP P EIGV L QS LQ YFS IT CN K VE SLP DE L Y FC KKL K TL K IG KN S LS VL SP KIG N MmLRRC8C KV E V LP S H LF L CNKIR YLD LS YN D I RF IP P EIGV L QS LQ YFS IT CN K VE SLP DE L Y FC KKL K TL K IG KN S LS VL SP KIG N HsLRRC8D KL E S LP VA VF SL QKLR CLD VS YN N I SM IP I EIGL L QN LQ HLH IT GN K VD ILP KQ L F KC IKL R TL N LG QN C ITS L PE KVG Q MmLRRC8D KL E S LP T A VF SL QKLR CLD VS YN N I ST IP I EIGL L QN LQ HLH IT GN K VD ILP KQ L F KC VKL R TL N LG QN C I A SL PE KI S Q HsLRRC8E KL E T LP S Q L GL CS G LR LLD VS HN G L HS LP P EVGL L QN LQ HLA LS YN A LE ALP EE L F FC RKL R TL L LG DN Q LSQ L SP HVG A MmLRRC8E KL E T LP T Q L GQ C FG LR LLD LS HN G L RS LP P ELGL L QS LQ HLA LS YN A LE SLP DE L F FC HKL R TL L LG YN H LTQ L SPD V A A LRR15 LRRCT
β18 η11 η12 α19 α20
760 770 780 790 800 810 HsLRRC8A L T NL T Q IELR GN R LE CLP V EL G EC PL LK RSG L VVE E DL F N TLP P EV KE R L WRADKEQA.... MmLRRC8A L T NL T Q IELR GN R LE CLP V EL G EC PL LK RSG L VVE E DL F S TLP P EV KE R L WRADKEQA.... HsLRRC8B L S NL T H LEL IGN Y LE TLP P EL EG C QS LK RN CL IVE E NL LN TLP LP V T E R L QTCLDKC..... MmLRRC8B L S NL T H LEL TGN Y LE TLP V EL EG C QS LK RS CL IVE D SL LN SLP LP V A E R L QTCLDKC..... HsLRRC8C L LF L S Y LDVK GN HF E ILP P EL G DC RA LK R A GL VVE D AL F E TLP S DV RE Q M KTE...... MmLRRC8C L LF L S Y LDIK GN HF E VLP P EL G DC RA LK R A GL VVE D AL F E TLP S DV RE Q M KAD...... HsLRRC8D L S QL T Q LELK GN C LD RLP AQ L G QC RM LK KSG L VVE D HL F D TLP L EV KE A L NQDINIPFANGI MmLRRC8D L T QL T Q LELK GN C LD RLP AQ L G QC RM LK KSG L VVE D QL F D TLP L EV KE A L NQDVNVPFANGI HsLRRC8E L RA L S R LELK GN R LE ALP E EL G NC GG LK K A GL LVE D TL Y QG LP A EV RD K M EEE...... MmLRRC8E L Q AL S R LELK GN R LE TLP E EL G DC KG LK KSG L LVE D TL Y EG LP A EV RE K M EEE......
Kasuya et al. Figure S2 (continued) bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A
50 nm
B 2D classification Manual screening Auto picking (3 rounds, 4 batches) 5,304 micrographs 5,284 micrographs 1,421,314 particles
3D classification (8 classes)
664,971 particles
Class #1 Class #2 Class #3 Class #4 Class #5 Class #6 Class #7 Class #8 (14.2%) (10.5%) (8.6%) (17.8%) (9.5%) (12.0%) (16.7%) (10.6%)
90° 90° 90° 90° 90° 90° 90° 90°
3D auto refinement 3D auto refinement
Overall (4.25 Å) C Overall (9.17 Å)
Kasuya et al. Figure S3 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. D E
1.0 1.0 Model vs. Sum Model vs. Half map 1 Model vs. Half map 2 0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2 FSC = 0.143
Fourier Shell Correlation 0.0 Fourier Shell Correlation 0.0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Resolution (1/Å) Resolution (1/Å)
Kasuya et al. Figure S3 (continued) bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B C region Extracellular TM region
Extracellular TM region region
D E region Intracellular LRR region
α subunit β subunit Intracellular LRR region region
Kasuya et al. Figure S4 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A Tight interface B Loose interface TM2 (β) T316 TM2 (α) T316
Y124 I320 Y124 I320 Y127 F324 Y127 F324 TM region L131 L131
L134 V34 L134 V34
Y30 A138 Y30 A138 TM4 (α) TM4 (ζ) F27 F27 F142 F142 W23 W23 F146 F146 TM1 (α) P147 TM1 (ζ) C P147 D IL1H1 (β) IL2H2 (α) Y382 IL1H1 (α) S151 Y382 IL2H2 (ζ) E154 S151 D383 E154 H155 D383 H155 L385 L385 R389 Intracellular
R389 region K249 K249 T170 T170 E245 E245 S174 IL2H3 (α) S174 IL2H3 (ζ) E238 E238 IL1H2 (α) IL1H3 (ζ) IL1H3 (α) IL1H3 (β) D233 D233
E F
LRR (ζ) LRR region
LRR (α) LRR (α) LRRCT LRR (β)
G H
Y106 H104 L302 C57 EL2 (β) EL2 (α) C57
Y106 Extracellular H104 L302 region R309 R309 Y108 Y108
A111 A311 A111 A311 EL2 (α) P313 EL2 (ζ) P313 EL1H (β) E115 EL1H (α) E115
Tight interface Loose interface
Kasuya et al. Figure S5 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A WT D102A R103A H104A Q105A Y106A
Hoechst LRRC8A
DIC
B C YFP D102A (H148Q/I152L) R103A WT HsLRRC8A-EGFP LRRC8A: D102A R103A H104A Q105A Y106A H104A Q105A Y106A † 100 IB: LRRC8A 75
EGFP
IB: YFP 28 Void Normalized fluorescence
48 0 IB: Actin 0 5 10 15 20 25 (kDa) Volume (ml)
D WT (+) WT (++++) D102A 1.4 - I--iso I--hypo 1.2 Regression curve
1.0
0.8
0.6
0.4 R103A H104A Q105A Y106A 1.4
1.2
1.0 Normalized YFP fluorescence (a.u.)
0.8
0.6
0.4 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 Time after adding iodide-containing buffer (Sec)
Kasuya et al. Figure S6 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A HsLRRC8A B CeInnexin-6 C HsConnexin-26 c EL1 EL2 C310 C54 EL1 EL2 EL2 C295 EL1
N C113 TM3 TM3 TM3 TM1 TM1 TM1 NTH N TM4 TM4 TM2 TM2 TM4 NTH N C TM2
IL IL2 IL1 IL1 C IL2
C
D E F
HsLRRC8A CeInnexin-6 HsConnexin-26 G H I J HsLRRC8A EL1H EL1H Pred: TM1 EL1H AA: MIPVTELRYFADTQPAYRILKPWWDVFTDYISIVMLMIAV 10 20 30 40 N
TM1 TM1 TM1 CeInnexin-6 Pred: N NTH TM1 NTH AA: MASQVGAINSVNALISRVFVQPKGDLADRLNSRVTVVILA N 10 20 30 40 NTH HsConnexin-26 Pred: NTH TM1 AA: MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAA 10 20 30 40 HsLRRC8A CeInnexin-6 HsConnexin-26
Kasuya et al. Figure S7 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A
Out
In Conserved
180° Diversified
B
Out
In 90°
F143 (LRRC8D)
LRRC8A LRRC8D
Kasuya et al. Figure S8 bioRxiv preprint doi: https://doi.org/10.1101/331207; this version posted May 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Table S1. Data collection and model statistics Data collection Microscope FEI Tecnai Arctica Voltage (kV) 200 Detector Gatan K2 Summit Pixel size (Å)* 1.490 Nominal magnification* 23,500x Electron dose (e-/Å2)* 50 Defocus range (mm) -0.5 ~ -3.5 Number of collected micrographs 5304 Number of selected micrographs 5284
Reconstitution Number of used particles 164,749 Symmetry imposed C3 Resolution 4.25
Refinement Number of protein residues 4,227 R.m.s. deviations Bond lengths (Å) 0.006 Bond angles (°) 1.124 Ramachandran (%) Favored 88.74 Allowed 11.26 Outlier 0.00 * Calibrated pizel size at the detector