Article

Structures of fungal and plant

acetohydroxyacid synthases

https://doi.org/10.1038/s41586-020-2514-3 Thierry Lonhienne1,7 ✉, Yu Shang Low1,7, Mario D. Garcia1, Tristan Croll2, Yan Gao3, Quan Wang3,
Lou Brillault4, Craig M. Williams1, James A. Fraser1, Ross P. McGeary1, Nicholas P. West1,
Received: 12 September 2019
Michael J. Landsberg1, Zihe Rao3,5,6, Gerhard Schenk1 & Luke W. Guddat1 ✉
Accepted: 1 June 2020

Published online: xx xx xxxx

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin
Check for updates adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme
that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the
target for more than 50 commercial herbicides2. AHAS requires both catalytic and
regulatory subunits for maximal activity and functionality. Here we describe
structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and
dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory
subunits of these AHAS complexes form a core to which the catalytic subunit dimers
are attached, adopting the shape of a Maltese cross. The structures show how the
catalytic and regulatory subunits communicate with each other to provide a pathway
for activation and for feedback inhibition by branched-chain amino acids. We also
show that the AHAS complex of Mycobacterium tuberculosis adopts a similar
structure, thus demonstrating that the overall AHAS architecture is conserved across
kingdoms.

AHAS (enzyme classification code, EC 2.2.1.6) is the first enzyme in
the biosynthesis pathway for branched-chain amino acids (BCAAs)1
that leads to the production of l-valine, l-leucine and l-isoleucine. It
is present in plants and microorganisms, but not animals. Owing to its
importance in plants, AHAS has been used as a target for 58 commercial
herbicides2, and is an emerging target for antimicrobial agents3. How-
ever, with concerns over the spread of resistance to such herbicides4, it
is important to improve our understanding of the functional features
of this enzyme.
AHAS consists of two subunits: a catalytic subunit (CSU) that
contains flavin adenine dinucleotide (FAD), thiamine diphosphate
(ThDP) and magnesium (Mg2+)5,6, and a regulatory subunit (RSU)
that enhances the activity of the CSU1,7,8. The RSUs also contain the
binding sites for BCAAs, which are feedback inhibitors 1. Notably, the
mechanism by which the RSUs exert their function(s) has remained
unclear.
The CSUs consist of three domains—α, β and γ—that have highly con-
served amino acid sequences and polypeptide lengths (Supplementary
Fig. 1). By comparison, the RSUs are variable (Supplementary Fig. 2),
with sizes ranging from 11 to 60 kDa1. RSUs have ACT domain(s)9 that
contain the BCAA-binding site9–11. In plant AHASs, there are two ACT
domains in a single polypeptide, whereas microbial AHASs have only
one (Supplementary Fig. 2).
Structures of the CSUs from the AHASs of Arabidopsis thaliana
(AtAHAS)2,12, Saccharomyces cerevisiae (ScAHAS)13 and Candida
albicans3, and the RSUs from four bacterial AHASs10,14,15 have been deter-
mined. In addition, a model has been suggested to show how the RSUs
and CSUs of Escherichia coli could combine16. However, the structure
of the CSU–RSU complex has not yet been determined.
Here, the crystal structure of ScAHAS (~800 kDa) and the cryo-electron
microscopy (cryo-EM) structures of AtAHAS (~710 kDa) in the presence
or absence of l-valine have been determined (Extended Data Tables 1,
2). In addition, we determined a crystal structure of a fungal hybrid
AHAS complex that consists of the CSU of Cryptococcus neoformans
(CnAHAS) and the ScRSU in the presence of l-valine (Extended Data
Table 1). The ScAHAS complex was purified (Supplementary Figs. 3a,
4a) in 50 mM phosphate (Supplementary Fig. 3b) and crystals were
obtained in the presence of ATP and the herbicide bensulfuron methyl
(BSM)2,17. The ScAHAS structure has eight CSUs and eight RSUs arranged
in a Maltese cross shape, in which the four CSU dimers are attached at
the extremities and the eight RSUs form the central core to which pairs
of ATP are bound (Fig. 1a). A similar subunit arrangement occurs in
AtAHAS (Fig. 1b, c), except that the eight RSUs of ScAHAS are replaced
by four AtAHAS RSUs, each of which contains two pseudo-repeats of the
ScAHAS sequence (Supplementary Fig. 2). Furthermore, negative-stain
microscopy illustrates that the AHAS complex of Mycobacterium tuber-
culosis has a similar shape (Supplementary Fig. 5), thus demonstrating
that this overall arrangement of subunits is common across kingdoms.
The hexadecameric ScAHAS complex
In the ScAHAS complex, N- and C-terminal extensions of the RSUs,
the ACT domains within the RSUs and the ATP-binding site provide
major contributions to the assembly of the complex. Each RSU interacts

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia. 2Cambridge Institute for Medical Research, University of Cambridge, Cambridge,
1

UK. 3Shanghai Institute for Advanced Immunochemical Studies, School of Life Science and Technology, ShanghaiTech University, Shanghai, China. 4Centre for Microscopy and Microanalysis, The
University of Queensland, Brisbane, Queensland, Australia. 5State Key Laboratory of Medicinal Chemical Biology, College of Life Science, Nankai University, Tianjin, China. 6Laboratory of Structural
Biology, Tsinghua University, Beijing, China. 7These authors contributed equally: Thierry Lonhienne, Yu Shang Low ✉e-mail: t.lonhienne@uq.edu.au; luke.guddat@uq.edu.au

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Article
a ScCSU dimer b AtCSU dimer
ScRSU
octamer
ScCSU dimer
ScCSU dimer
AtCSU dimer
ATP ×2
AtCSU dimer
ScCSU dimer
c AtCSU dimer
AtRSU
tetramer
90°
AtCSU dimer
AtCSU dimer
AtCSU dimer
76 Å
Fig. 1 | The structure of the ScAHAS and AtAHAS complexes. a, ScAHAS. Eight
RSUs in different colours—each of which contains a single ACT domain—form a
central core to which four CSU dimers are attached. Four ATP pairs (red
transparent surface) are bound to the RSU dimers. b, AtAHAS. Four RSUs
(magenta and green)—each of which contains two pseudo-repeats of the ACT
domain—form the central core. c. The cryo-EM map of AtAHAS.
with six RSUs and three CSUs (Supplementary Table 1). Both the N
and C termini of the RSUs adopt tentacle-like structures that extend
beyond the core regions of the RSUs and establish interactions with
neighbouring subunits (Extended Data Fig. 1a). Four pairs of ATP also
contribute to the association between RSUs (Figs. 1a, 2). The RSUs
alone form dimers, which assemble into an octameric conformation
that is dependent on the presence of ATP (Extended Data Fig. 1b). The
ATPs form pairs linked by a Mg2+ ion (Fig. 2a and Extended Data Fig. 1c,
d), while the adenine and ribosyl moieties associate with all four RSUs
to complete a network of interactions (Fig. 2a and Extended Data
Fig. 1d). There are five key residues in ScAHAS that interact with ATP.
Of these, three (R159, F232 and R280) are invariant (Supplementary
Fig. 2), and one (R254) is highly conserved. Neither ATP nor the RSUs
alone can enhance AHAS activity (Fig. 2b). However, in combination
ATP and the RSUs cooperatively assemble and activate the AHAS
complex (Fig. 2b).
Comparison of CSU structures with or without RSUs
The fold of the CSUs, the conformation of the active site, and the loca-
tion of ThDP, Mg2+ and FAD are unaltered in the presence or absence
of RSUs17 (Extended Data Fig. 2a). Accordingly, the Michaelis–Menten
constant (KM) for the substrate (pyruvate) is similar in the presence
and absence of the RSUs (3.2 and 3.0 mM, respectively; Extended Data
Fig. 2b). However, a substantial structural change occurs in one of the
regions that involves the linker between the α- and β-domains (α–β
linker) of the CSUs13 (residues 258–279) when the RSUs bind. In the
structure of the CSU alone, or in complex with BSM, this linker is ran-
dom coil, in which the six central residues are completely disordered.
In the complex, this region has a helical fold (Extended Data Fig. 2c)
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that interacts with several RSUs (see ‘Interactions between CSUs and
AtRSU RSUs’), thus stabilizing the complex.
tetramer
Conformation of the RSUs in the ScAHAS complex
AtCSU dimer
The RSUs in ScAHAS have four distinct regions: an N-terminal extension
and domain, and a C-terminal extension and domain (Supplementary
Fig. 6a). The N-terminal and C-terminal domains have similar topolo-
gies to those observed in the bacterial RSU homodimers14,15, and have
an N-terminal domain formed by two α-helices and four β-strands that
are arranged in the βαββαβ arrangement (Supplementary Fig. 6b) that
is characteristic of ACT domains9,18. The conformation of the C-terminal
domain shares a high level of structural similarity with its counterpart
in bacteria, except that there is an extra sequence inserted in the ScRSU
that forms a protruding ‘contact’ loop (Supplementary Fig. 6c). This
loop interacts with the α–β linker of the CSU and with the N-terminal
extension of neighbouring RSUs.
195 Å
Interactions between CSUs and RSUs
The main region of the RSUs that interacts with the CSUs is the ACT
domain (Extended Data Fig. 3a and Supplementary Fig. 7), which
is consistent with a previously proposed model for an E. coli AHAS
complex16. Notably, the first α-helix of the ACT domain (α1; residues
90–101) forms a salt bridge (R101 (RSU)–D199 (CSU)) with the ‘active
site’ Q-loop (191–204)19 (Extended Data Fig. 3b). F201 and Q202, located
at the top of the Q-loop, are essential for enzymatic activity20. Q202 has
an important role in the stabilization of the substrate and/or intermedi-
ate within the active site19,20, and F201 has a role in the stabilization of
FAD and the substrate20,21. The pairing of R101 (RSU) and D199 (CSU) is
highly conserved in AHAS complexes (Supplementary Figs. 1, 2), which
emphasizes the importance of this interaction. In addition, the second
α-helix of the ACT domain (α2, residues 129–140 (RSU)) interacts with
the β-domain of one of the two CSUs, principally using N134 (CSU)
and R137 (CSU) (Extended Data Fig. 3b). Furthermore, two β-strands
of the ACT domain, the contact loop and the N-terminal extension—all
of which are from different RSUs—form a cluster of residues that inter-
act with and stabilize the α–β linker of the CSUs (Extended Data Fig. 3c).
Activation of the ScAHAS by the RSUs
Two distinct factors contribute to the enhancement of ScAHAS activity:
the dimerization of CSUs and allosteric communication within a dimer.
First, dimerization is essential to form fully functional active sites13,
and dimer stabilization is strongly promoted by the presence of RSUs
and ATP (their presence decreases the dissociation constant (Kd) of the
CSUs by approximately 20-fold; Extended Data Fig. 4a). Second, the
conformation of the CSU dimer ensures that the movements in the two
active sites are complementary and synchronized13,20,22; when the first
step (decarboxylation of pyruvate) occurs in one active site, the second
step (carbon–carbon bond formation) proceeds in the other in a con-
certed manner20. Complex formation triggers an approximately fourfold
increase in activity that is largely entropy-driven (Extended Data Fig. 4b, c)
and is mediated by a bridge formed by the RSUs that directly connects
the two active sites of the CSU dimer (Fig. 3). This involves an interaction
between an α-helix (α1) from the two ACT domains and the Q-loops of
the CSUs (that is, the R101 (RSU)–D199 (CSU) salt bridge; Fig. 3b). The
importance of this salt bridge for the regulation of the CSU by the RSUs
was confirmed by assessing the interactions between the CSU and the
extended ScRSU form with an R101A mutation (ScRSUE(R101A)). Kinetic
assays found two marked effects: the affinity between ScRSUE(R101A)
and CSU is around 20 times weaker than the affinity of the native com-
plex (Extended Data Fig. 4d) and the R101A mutation decreases the
catalytic rate constant (kcat) twofold (Extended Data Fig. 4e). These
results suggest that the role of the α1-bridge (Fig. 3b) is important to
 a
Chain H

F232
Chain G ATP
R254
K251
R159

Mg2+ R280
R280

ATP
R254 K251

R159

Chain D
F232

Chain C
b
25 Kd = 1.25 ±0.02 μM
25 Kd = 36.7 ± 0.6 μM
Specific activity (U mg–1)

Specific activity (U mg–1)

20
20
ATP 1 mM ATP
15 15
ADP No ATP

10 10

5 5

0 0
0 20 40 60 80 100 120 140 0 1 2 3 4 5
ATP/ADP (μM) ScRSU (μM)

Fig. 2 | The role of ATP in assembling the ScAHAS complex. a, Binding mode
of ATP. Four RSUs (chains C, D, G and H) interact with an ATP pair coordinated
by Mg 2+. The electron density (2Fo − Fc map) is shown in grey. b, Activation of
ScAHAS by ATP and the RSU. The specific activity of AHAS was measured at
30 °C by monitoring pyruvate consumption. Left, the specific activity of
ScAHAS (0.13 μM in the assay) was measured with increasing ATP
concentrations and a saturating RSU concentration (8.5 μM). ATP binds
cooperatively to the RSU (Hill coefficient = 3.0 ± 0.1; Kd = 36.7 ± 0.6 μM). ADP
has no effect on AHAS activity. Right, the specific activity of ScAHAS (0.13 μM
in the assay) was measured with increasing RSU concentrations and a
saturating concentration of ATP (1 mM). RSU binding is cooperative (Hill
coefficient = 3.1 ± 0.1; Kd = 1.25 ± 0.02 μM). The RSU in the absence of ATP had no
effect on activity. Data are mean ± s.e.m. of technical triplicates. Data were
fitted to the Hill equation to obtain the Kd and the Hill coefficient.

increase the turnover of the enzyme, presumably by improving the com-
munication between the active sites of CSU dimers. Other regions of the
RSUs (that is, the other α-helix (α2) and two of the β-strands of the ACT
domain) interact with the α- and β-domains of a CSU dimer (Extended
Data Fig. 3b, c). These interactions rigidify regions of the dimer
that are not directly involved in the communication between the
active sites. Therefore, the apparent effect of the RSU is to decrease
the entropy of the enzyme–substrate complex before activation, ena-
bling a more-efficient channelling of the energy that is transmitted from
one active site to the other. The structural and kinetic data show that
the increase in AHAS activity after the formation of the AHAS complex
is due to contacts that restrict the population of unfavoured conforma-
tional states for catalysis and enhance the allosteric communication23
between catalytic centres. We therefore proposed that the primary role
of the RSUs is to enhance the efficiency of the allosteric communication
between active sites (Extended Data Fig. 5).
The dodecameric AtAHAS complex
The cryo-EM structure of the AtAHAS complex was obtained without
herbicide (BSM) or ATP (Fig. 4b and Supplementary Fig. 8). The cryo-EM
map (Fig. 1c and Supplementary Fig. 9) revealed that most of the poly-
peptide in the two subunits is visible (Fig. 1b), except for the region that
corresponds to the linker between the RSU repeats (Extended Data
Table 2). The overall arrangement of the RSUs and CSUs is similar to
the ScAHAS complex, except that there is no contact loop in AtAHAS
(Supplementary Fig. 10). Most features that have roles in the activation
of ScAHAS are also preserved in the AtAHAS complex. In comparison
with the CSU alone (Protein Data Bank (PDB) code 5K6Q), the complex
shows that a contraction of the CSU dimer occurs when the RSU binds
(Supplementary Fig. 11). Thus, the RSU stabilizes the CSU in AtAHAS
complex in the same manner as proposed for the ScAHAS complex.
Analogous to ScAHAS (Fig. 3), R110 (RSU repeat one) and R343 (RSU
repeat two) each form a salt bridge with D204 (Q-loop) from opposing
CSUs (Supplementary Fig. 12). The weak affinity of the R110A and R343A
double mutant for the CSU prevented the isolation of the complex
(Supplementary Fig. 13a). This confirms, as in the ScAHAS complex
(Extended Data Fig. 4d), that the R–D salt bridge is essential for the
stabilization of the complex.
The most notable difference between the ScAHAS and AtAHAS com-
plexes is the presence and absence of ATP, respectively. However, visual
inspection shows that there is room to accommodate an ATP pair in the

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Article
a a Q-loops
ThDP ThDP
L-Valine (site 1)
FAD FAD
D204 D204
R343 R110
α1 α1
α2 α2
90°

CSU α- and ACT domains

CSU α- and L-Valine (site 2)
β-domain β-domain
stabilization
stabilization b
V485 V485
ThDP ThDP
b C2 C2
FAD 7.15
5.07 FAD 7.15
5.07
He-ThDP ThDP 5.96 5.52 5.96 5.52
4.39 6.56 4.39 6.56
G121 G121
FAD FAD F206 F206

Q207 Q207

D204 D204

N217 N217
D199 D199
Q-loop
R101 R93 R93 R101 Q-loop
α1 α1
α1-bridge
Fig. 3 | The ScRSU influences the conformation of the ScCSU. a, The two CSU
dimer polypeptides are shown in dark grey and light brown. The γ subunit is
omitted for clarity. The two chains of the RSU dimer are shown in green and
magenta. The two α 1 helices of the ACT domains in the RSUs establish a direct
path between the two catalytic Q-loops of the CSU dimer. The remaining
regions of the ACT domains are involved in the stabilization of the α- and
β-domains of the CSU dimer (enclosed in dashed grey ellipses). b, Communication
paths between the Q-loops of the CSU dimer in the ScAHAS complex. The
structure of ScAHAS (CSU alone) in complex with pyruvate (shown in green
ball-and-stick format) that represents the enzyme state during catalysis
(PDB code 5INU)20 is superimposed on a CSU dimer of the ScAHAS (CSU + RSU)
complex. The model displays the asymmetry that occurs between catalytic
centres, each of which has a different conformation to accomplish the two
half-cycles of the reaction at the same time. The representation of the CSU
dimer is restricted to the Q-loops and polypeptide extensions on both sides.
The polypeptide extensions from both Q-loops interact with each other to
form the path through which the active sites communicate. Thus, the α 1-bridge
of the RSUs binds to the Q-loops (that is, R101–D199 and R93–N217) and
enhances the communication between active sites.
AtAHAS complex (Supplementary Fig. 14). Thus, the precise role of ATP
in the AtAHAS complex should be investigated further.
The AtAHAS complex with bound l-valine
A cryo-EM map of the AtAHAS complex obtained in the presence of
l-valine (Extended Data Table 2 and Supplementary Fig. 15) shows
two densities in adjacent ACT domains where l-valine could be fit-
ted (Extended Data Fig. 6a, b). The effect of l-valine is to induce bend-
ing of the α1 helices (Fig. 4a and Extended Data Fig. 6c). This triggers
distancing (around 5 Å) between R110 (RSU) and D204 (CSU), which
causes the disruption of the α1-bridge that links the Q-loops of the CSU
Fig. 4 | The effects of l-valine binding on the AtAHAS complex.
a, Superimposition of the ACT domains of the free AtAHAS complex and the
complex with bound l-valine. The RSUs are shown in dark green and magenta
for the repeats in the structure of the complex in the absence of l-valine, and
cyan and coral for the repeats in the structure of the complex with bound
l-valine. l-Valine (ball and stick representation) is shown with a light-yellow
transparent surface around the outside. The Q-loops of the CSU dimer are
shown as stick models. The binding of l-valine triggers the disruption of the
R110/R343–D204 salt bridge and the bending (black arrows) of the α-helices in
the ACT domains. b, Stereoimage of the superimposed active sites. The
displayed residues are involved in substrate binding (green and light blue for
the complexes in the absence and presence of l-valine, respectively). In the
structure of the complex without l-valine, FAD and ThDP (ball and stick models)
are shown in yellow and coral, respectively. In the structure of the complex with
l-valine, FAD and ThDP have similar but lighter-colour shades. The image shows
that the active site expands after the binding of l-valine. C2 denotes the
carbanion of the ylid that nucleophilically attacks the carbonyl of the substrate.
All distances are stated in angstroms.
dimer in the free AtAHAS complex (Fig. 4a and Extended Data Fig. 7a).
Another important feature is that the bending of the α1 helix—which is
transmitted to the α2 helix (Fig. 4a and Extended Data Fig. 7b)—induces
a reorganization of the interactions between the ACT domain of the
RSUs and the β-domain of the CSUs (Supplementary Fig. 16). This
causes distancing (around 3 Å) between the β-domain and ACT domain
(Supplementary Fig. 16a) and leads to the expansion of the CSU dimer
(Extended Data Fig. 6c and Supplementary Fig. 11). A destabilization
of the CSUs is suggested as there is a decrease in the interface area
between CSU subunits (by 7%) and between the RSU and CSU dimer
(by 14%) (Supplementary Fig. 17). These changes probably account for
the partial dissociation of the CSU from the complex in the presence
of 5 mM l-valine (Supplementary Fig. 13b). Furthermore, there is an
expansion of the active sites as the distances between the reactive
carbon of ThDP and key residues that are involved in the binding of
the substrate20 increase by around 1.5 Å (Fig. 4b).
Taken together, these structural observations provide several mecha-
nisms by which feedback inhibition by BCAAs occurs. First, the altera-
tion of the communication between the active sites, coupled with the
destabilization of the CSU dimer, is expected to reduce kcat. This hypoth-
esis is supported by data obtained for the ScAHAS complex, in which

4 | Nature | www.nature.com
the increase in the stability of the CSU dimer (Extended Data Fig. 4b, c)
and the connection between active sites made by the α1-bridge of the
RSUs (Extended Data Fig. 4e) are central features that are involved
in the upregulation of kcat. Second, the deformation of the active site
after the addition of l-valine (2 mM) leads to an increase in the KM for
pyruvate (from 12.6 ± 1.9 to 24.2 ± 3.9 mM; Supplementary Fig. 18),
and potentially also decreases the catalytic rate. Third, the weakening
of the interaction between the CSU dimer and the RSU promotes the
dissociation of the CSUs from the complex. Although only a partial
dissociation is observed in the presence of 5 mM l-valine, it is possible
that this dissociation is more complete under physiological conditions.
These interpretations are corroborated by a crystal structure
obtained for a fungal hybrid AHAS complex that consists of the CnCSU
and ScRSU in the presence of l-valine (Extended Data Fig. 8a and
Extended Data Table 1). Although it is a hybrid from two different spe-
cies, kinetic studies show that like the ScAHAS complex, this complex
is activated by ATP and inhibited by l-valine (Extended Data Fig. 8b).
Density for l-valine is observed and its location is similar to that in
the AtAHAS complex (Extended Data Fig. 8c). Notably, the structural
changes observed after l-valine binding correlate with the observa-
tions observed for AtAHAS (Extended Data Fig. 8d). This includes the
disruption of the α1-bridge, the presence of which is essential for the
regulatory mechanism.
Previously, a minimal subunit arrangement for the AHAS complex
had been proposed that involved only two to four RSUs and CSUs8,16.
Here we demonstrate that AHAS adopts a higher-order structure that
is conserved across three different kingdoms. The formation of a RSU
multimeric core platform is proposed to have two advantages. First,
it contributes to the stabilization of both the ACT domains and CSUs.
In ScAHAS, this is achieved by the N- and C-terminal extensions that
interact with distal ACT domains (Extended Data Fig. 1a). The linker
between repeats in AtAHAS may have a similar role. The stabilization
of the CSUs is a major factor for the regulation of activity. The second
advantage of the RSU core structure—at least in ScAHAS—is that it ena-
bles a higher level of regulation through the creation of a binding site
for ATP. The BCAA biosynthetic pathway is an anabolic process that
requires energy, and it is therefore logical that downregulation occurs
in response to low levels of energy (ATP) in the cell. The formation of
the core formed by four RSU dimers has created an opportunity for
a pocket to accommodate two paired ATPs, suggesting a previously
unknown function for ATP. Overall, the active and regulatory sites
(that is, Q-loop and ACT domain) in AHAS are shaped and positioned
for functional simplicity, with only minimal movements required for
their interaction. By contrast, when the feedback inhibitor—l-valine—
binds to the RSUs it triggers considerable structural changes, which
result in the perturbation of interactions between the CSUs and lead
to a reduction in catalytic activity.
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availability are available at https://doi.org/10.1038/s41586-020-2514-3.
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Article
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
The structures of the ScAHAS complex, CnCSU–ScRSU–valine complex
and AtAHAS in the presence and absence of l-valine have been depos-
ited in the Protein Data Bank with accession numbers 6U9D and 6WO1,
6VZ8 and 6U9H, respectively. The cryo-EM map has been deposited in
the Electron Microscopy Data Bank under accession numbers EMDB-
21487 and EMDB-20700. Plasmids, strains and raw data are available
from the corresponding author upon reasonable request.
24. Lonhienne, T., Gerday, C. & Feller, G. Psychrophilic enzymes: revisiting the
thermodynamic parameters of activation may explain local flexibility. Biochim. Biophys.
Acta 1543, 1–10 (2000).
Acknowledgements Crystallization was performed at the University of Queensland
Remote-Operation Crystallization and X-ray diffraction facility (UQROCX). Data collection was
carried out at the Australian Synchrotron, part of ANSTO and made use of the Australian
Cancer Research Foundation (ACRF) detector. This work was supported by funds from the
National Health and Medical Research Council (grant numbers 1087713 and 1147297), and the
Australian Government Department of Agriculture and Water Resources (CT-06). We thank the
Bio-Electron Microscopy facility (iHuman Institute) at ShanghaiTech University and the
Cryo-EM Facility Center of Southern University of Science and Technology (Shenzhen) for
providing support and acknowledge the facilities and scientific and technical assistance of the
Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and
Microanalysis, The University of Queensland. T.C. is supported by a Wellcome Trust grant
(209407/Z/17/Z), awarded to R. Read. We thank R. Duggleby for initiating the early functional
and structural studies on AHASs.
Author contributions L.W.G., T.L., G.S., Z.R., C.M.W., R.P.M., J.A.F. and N.P.W. conceived the
project. T.L. and Y.S.L. established the protein-purification protocols, generated mutants and
performed kinetic experiments. M.D.G. performed the initial purification and cryo-EM
experiments with AtAHAS. Y.S.L., Y.G., Q.W. and M.J.L. collected and processed the cryo-EM
data. T.L. crystallized and solved the structures of the fungal AHASs. T.L. and L.W.G. collected
the X-ray data for the fungal AHASs. T.L., Y.S.L., L.W.G., G.S. and M.J.L. interpreted the data. L.B.
assisted with cryo-EM and ReFyn analysis. T.C. performed the final refinement steps for the
cryo-EM structure of AtAHAS in complex with l-valine. T.L. and Y.S.L. prepared the figures. T.L.,
Y.S.L., L.W.G. and G.S. wrote the paper and T.L., Y.S.L., L.W.G., G.S., C.M.W., M.J.L., Z.R., N.P.W.,
J.A.F., R.P.M. and T.C. edited the paper.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2514-3.
Correspondence and requests for materials should be addressed to T.L. or L.W.G.
Peer review information Nature thanks Robert Sammons, Kai Tittmann and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.
Extended Data Fig. 1 | See next page for caption.
Article
Extended Data Fig. 1 | The assembly of the ScAHAS complex. a, General view
of the interactions that stabilize the ScAHAS complex. The 16 chains in the
complex are signified by the letters A–P. Each RSU has N-terminal and
C-terminal extensions that span through the core of the structure. As an
example, two regions of the N-terminal extension of the RSU (chain L) form
interactions with four neighbouring subunits (chains D (top inset), O, P (middle
inset) and M (bottom inset)). b, The assembly of the ScRSU octamer monitored
by mass photometry (ReFeyn, see Supplementary Methods). For this
experiment, the extended form of the RSU was used (RSUE; see Supplementary
Methods). In the absence of ATP (left), the majority of the RSUs (97%) have an
average molecular mass of 63 kDa, corresponding to the RSUE (31.4 kDa) dimer.
In the presence of 1 mM ATP (right), there are three peaks that correspond to
the dimer (34%), the tetramer (13%) and the octamer (49%). Partial assembly of
the octamer was observed because the RSU could only be used at a maximum
of 130 nM in this experiment due to saturation of the detector (Supplementary
Methods). c, d, Stereo views of the binding of ATP to the ScAHAS complex.
c, The coordination of the six phosphates by Mg 2+ and the stabilization of their
negative charges by six arginine and two lysine residues from two different
RSUs. d, Stabilization of the adenine and ribosyl moieties of one of the ATPs by
three RSUs. Dashed black lines represent electrostatic interactions. Thick
dashed green lines represent π–π interactions.
Extended Data Fig. 2 | Structure of a catalytic centre in the CSU dimer of the
ScAHAS complex. a, Stereo view of the superimposition of the catalytic
centres of a CSU dimer in the CSU–RSU complex (light brown) and the
CSU dimer in the absence of the RSU (light blue) (PDB code 5FEM). The
conformation of the active site and the location of ThDP, Mg 2+ and FAD are
unaltered, as well as the binding mode of the herbicide, BSM. FAD, ThDP and
BSM are represented as magenta and orange ball-and-stick models in the
structures of the ScAHAS CSU–RSU complex and of the ScAHAS CSU alone,
respectively. b. KM for pyruvate for the only CSU of ScAHAS (left; assayed using
0.28 μM CSU) and for the ScAHAS complex (right; assayed using 0.14 μM CSU,
1 mM ATP and 8.5 μM RSU). These results appear to contradict previous studies
that show that the KM of pyruvate increases in presence of the ScRSU (2.1 mM for
CSU alone and 13.3 mM for the complex)7. However, this discrepancy can be
explained by the different buffers used in the assay reported here and those
performed previously7 (Supplementary Methods). Data are mean ± s.e.m. of
technical triplicates. Data were fitted to the Michaelis–Menten equation to
obtain the KM values. c, Stereo view of the superimposition of the α–β linker and
nearby regions in the structures of the ScAHAS complex (light brown) and CSU
alone (light blue). The dashed line indicates residues that are disordered (not
visible) in the structure of CSU alone.
Article

Extended Data Fig. 3 | See next page for caption.

Extended Data Fig. 3 | Interactions between a ScCSU dimer and
neighbouring RSUs in the ScAHAS complex. a, Stereo view of a CSU dimer
and interacting RSUs. The CSU dimer is represented by chains A (dark grey)
and B (light brown), and includes the cofactors FAD (yellow transparent
surface) and ThDP (orange transparent surface) and the herbicide BSM
(blue transparent surface). The RSU dimer is represented by chains C (green)
and D (magenta). The two bound ATP pairs are shown as a red transparent
surfaces. Chain G (cyan) and chain P (violet) are RSUs that interact with chain B
and chain A, respectively, through their N-terminal extensions. b. Stereo view
and magnification of the α-helixes of the ACT domain of RSU (chain C). The
colours of the chains and cofactors are as in a. BSM has been omitted for clarity.
Dashed black lines represent electrostatic bonds. The first α-helix (α 1) of the
ACT domain in the RSU interacts with the Q-loop of the CSU (chain B) (that is,
R101 interacts with Q202) whereas the second α-helix (α 2) interacts with the
β-domain of the CSU (chain A) (that is, N134 and R137 interact with I413 and
P410, respectively). c, Stereo view and magnification of the α–β linker of a CSU.
The chains are coloured as in a. Dashed black lines represent electrostatic
bonds and white thick dashed lines represent hydrophobic interactions. The
α–β linker of CSU (chain B) interacts with the ‘contact loop’ of the RSU (chain C),
two β-strands of the ACT domain of the RSU (chain D) and the N-terminal
extension of the RSU (chain G).
Article

Extended Data Fig. 4 | See next page for caption.

Extended Data Fig. 4 | Regulatory mechanism of ScAHAS. a, The specific
activity of ScCSU measured at 30 °C as a function of the concentration of
ScCSU, in the presence (blue data, right y axis) of saturating concentrations of
ScRSU (8.5 μM) and ATP (1 mM) or in their absence (black data, left y axis).
Data (technical triplicates) were fitted using Supplementary equation (3)
(Supplementary Methods), yielding a Kd of 0.033 ± 0.005 μM for the CSU in the
presence of the RSU and ATP, and Kd = 0.63 ± 0.05 μM for the CSU alone. Data are
mean ± s.e.m. b, Measurement of the energy of activation (Ea) of ScAHAS.
Arrhenius plots were generated for the CSU alone (black data, technical
triplicates) and for CSU in the presence of saturating concentrations of RSU
and ATP (blue data, technical triplicates) (Supplementary Methods). Data
points between 24 and 36 °C (7 temperatures) were used to perform a linear
regression calculation to obtain an Ea value (the absolute value of the slope
multiplied by the gas constant value24 (R = 8.314 kJ/mol)) of 51.7 ± 1.5 kJ/mol for
the CSU in presence of the RSU and ATP, and Ea = 51.5 ± 2.1 kJ/mol for the CSU
alone. Data are mean ± s.e.m. (s.e.m. of the slope value multiplied by R).
c, Measurement of the thermodynamic parameters of the activation at 30 °C.
Left, the kcat of ScCSU at 30 °C was measured in the presence (blue data, right
y axis) of saturating amounts of ScRSU (33 μM) and ATP (1 mM) or in their
absence (black data, left y axis). Data points are technical replicates (n = 5). Data
are mean ± s.e.m. of the five replicates. Right, values for the thermodynamic
parameters of the activation at 30 °C. The values of the change in Gibbs free
energy (ΔG#), enthalpy (ΔH#), and temperature and entropy (TΔS #) were
calculated using the equations derived from the transition state theory24. The
comparison of ΔH# (49.2 versus 48.9 kJ/mol) and TΔS # (−16.23 versus −20 .3 kJ/
mol) values (of CSU + RSU versus CSU, respectively) that account for the value
of ΔG# (ΔG# = ΔH# − TΔS #) revealed that the decrease in ΔG# (which is directly
associated with the increase in kcat) is largely entropy-driven. Data are
mean ± s.e.m. of the kcat (left) for ΔG# and of the Ea for ΔH# and TΔS #, according to
the literature24. d, e, Effect of the R101A mutation on the activity of ScRSU.
d, Activation of ScAHAS by ScRSUE(WT) or ScRSUE(R101A). The specific activity
of ScAHAS (130 nM in the assay) was measured at different RSU concentrations
with a saturating concentration of ATP (1 mM). The data (technical triplicates)
were fitted to the Hill equation, showing that the mutation increases the Kd
between the CSU and the RSU from 0.27 ± 0.02 (ScRSUE(WT)) to 6 ± 0.3 μM
(ScRSUE(R101A)). Data are mean ± s.e.m. e, The specific activity of the ScCSUs
as a function of their concentration, alone (black data) and in the presence of
1 mM ATP and 2.6 μM ScRSUE(WT) (blue data, saturating concentration) or
20 μM ScRSUE(R101A) (red data, saturating concentration). The data (technical
triplicates) were fitted to Supplementary equation (3) (Supplementary
Methods), yielding a Kd of 0.04 ± 0.005 and 0.036 ± 0.007 μM for the CSU in the
presence of RSUE(WT) and RSUE(R101A), respectively. For the CSU alone, the Kd
is 0.62 ± 0.04 μM confirming the value shown in a. The values of kcat—which are
also derived from Supplementary equation (3)—are 11.1 ± 0.2, 40.9 ± 1.1 and
19.5 ± 0.8 s−1 for the CSU alone, the CSU with RSUE(WT) and the CSU with
RSUE(R101A), respectively, showing that the R101A mutation strongly affects
catalysis. Data are mean ± s.e.m.
Article

Extended Data Fig. 5 | See next page for caption.

Extended Data Fig. 5 | Model for the regulation of the ScAHAS reaction.
A hybrid image showing the catalytic centres taken from an asymmetric ScCSU
dimer in the presence of pyruvate (grey and brown) (PDB code 5INU) and
the α 1-helices (pink and green) of the RSU from the complex. The different
conformations of FAD and the Q-loops, and the He-ThDP intermediate or ThDP
are shown. The Q-loops and their N-terminal and C-terminal extensions have
been drawn in brown and dark grey for catalytic centre 1 and 2, respectively.
The model shows how the conformational change of the active sites during the
catalytic cycle20 triggers a movement of the Q-loops that is complementary
between the catalytic centres (red arrows). This feature facilitates the
synchronization of the reactions that occur in the two catalytic centres of a
CSU dimer13. In the ScCSU dimer, direct contact between the Q-loops is made
through their N- and C-terminal extensions, which transmit the movements
between catalytic centres (red arrows). When the complex is formed, the RSU
works at two levels. (1) The RSU creates a ‘rigid scaffold’ (magenta) that is
formed by interactions of the ACT regions (that is, the α 2-helix and two
β-strands of the ACT domain) with the α- and β-domains and the α–β linker of
the CSUs (Extended Data Fig. 3). This restrict movements of the CSU that is not
involved in the communication between active sites. (ii) The α 1-helices of the
RSUs create a bridge (α 1-bridge) between Q-loops (curve line in magenta) that
increases the efficiency of the mechanical transmission between catalytic
centres. Both aspects are involved in decreasing the entropy of the enzyme–
substrate complex before activation, leading to an increase in kcat.
Article

Extended Data Fig. 6 | See next page for caption.

Extended Data Fig. 6 | Binding site interactions, cryo-EM map for l-valine in
AtRSU and consequences of l-valine binding. a, Cryo-EM densities of the two
BCAA-binding sites (site 1 and 2) occupied by l-valine. l-Valine is shown as a
green stick model. The cryo-EM map (5.5σ) is overlaid. b, Stereo view of the
interaction of l-valine in site 1 with the α 1-helices of the ACT domains. Dashed
lines represent hydrogen bonds (black), van der Waals interactions (blue) and
hydrophobic interactions (yellow). c, Superimposition of the free AtAHAS
complex and the l-valine-bound complex, representing one of the four CSU
RSU pairs that interact with each other. The CSU dimers are shown in green and
light blue for structures of the complex in the absence and presence of l-valine,
respectively. The RSUs are shown in dark green and magenta for the repeats in
the structure of the complex in the absence of l-valine, and cyan and coral for
the repeats in the structure of the complex with l-valine bound. The cofactors
FAD (yellow) and ThDP (orange) are shown in the structure of the l-valine free
complex only. l-Valine (ball and stick representation) has a light-yellow
transparent surface around the outside. The comparison of the structures
shows that the CSU dimer expands when l-valine is bound to the ACT domains
of the RSUs.
Article
Extended Data Fig. 7 | Structural changes in the ACT domains of AtRSU that
are triggered by the binding of l-valine. a, Stereo view of the translocation of
R110 induced by the binding of l-valine in site 1 of the ACT domain. When
l-valine is present the backbone of the α 1-helix of repeat 1 is shifted (black
arrows), causing a marked translocation of R110 and the breaking of the
R110–D204 salt bridge. b, Stereo view showing the conformational change in α 2
caused by the bending of α 1. The conformational change of α 1 induced by
l-valine triggers new interactions to occur between α 1 and α 2 (that is, the
interaction of V138 (α 2) with R110 (α 1) and L139 (α 2) with Y112 (α 1)) leads to the
bending of α 2). The colour scheme is as described in Extended Data Fig. 6c.
Extended Data Fig. 8 | See next page for caption.
Article
Extended Data Fig. 8 | The structure of the fungal hybrid complex formed
by the CnCSU and ScRSU in the presence of l-valine and comparisons with
the AtAHAS complexes. a, The overall structure of the hybrid complex
resembles the ScAHAS and AtAHAS complexes, possessing the characteristic
Maltese cross shape. b, The addition of ATP leads to the activation of the fungal
hybrid complex, whereas the addition of l-valine inhibits its activity. This
experiment was performed once. c, Left, the cryo-EM map at 3.4 Å resolution
(8σ level) in the AtAHAS complex with l-valine overlaid and optimally fitted.
Middle, the hybrid Cn–ScAHAS complex with the Fo − Fc electron density
(3.5σ level) with l-valine overlaid and optimally fitted. Right, the binding mode
of l-valine to E. coli RSU (structure determined in the absence of the CSU at 2.3 Å
resolution; PDB code 5YPW). In all three complexes, the carboxylate group
of l-valine forms three hydrogen bonds to backbone amides of the RSUs (I114,
V332, L333 in AtAHAS; I105, V90, L91 in Cn–ScAHAS; V38, V23, M24 in the E. coli
RSU alone). The amide group of l-valine forms either two or three hydrogen
bonds to the RSU (N113 side chain, I114 carbonyl oxygen, D328 side chain in
AtAHAS; N104 side chain, I105 carbonyl oxygen in Cn–ScAHAS; N37 side chain,
V38 carbonyl oxygen, Q19 side chain in E. coli RSU alone). The side chain
of l-valine is stabilized by hydrophobic and/or van der Waals interactions with
nearby hydrophobic side chains. The similarity of the network of interactions
involving the binding of l-valine that is observed in all three complexes,
and the fact that these three sites align when the sequences are compared
(Supplementary Fig. 2) shows that the binding mode of l-valine is conserved in
bacterial, fungal and plant AHASs. d, Comparison of the conformational
changes that occur in the AtAHAS and in the Cn–ScAHAS complexes on l-valine
binding. These changes are highly conserved in the two complexes as
emphasized by the arrows.
Extended Data Table 1 | Data collection and refinement statistics for ScAHAS complexes

Data collection and refinement statistics, including molecular replacement analyses, for the ScAHAS CSU–RSU and CnCSU–ScRSU (AHAS) + valine complexes. One crystal was used to obtain
each dataset.
*Values in parentheses are for the highest-resolution shell.
Article
Extended Data Table 2 | Cryo-EM data collection, refinement and validation statistics for the AtAHAS complexes
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