# Structural Insights into Ciona intestinalis Septins: Complexes Suggest a Mechanism for Nucleotide-dependent Interfacial Cross-talk

Deborah Mendonça, Sinara T.B. Morais, Heloísa Ciol, Andressa P.A. Pinto, Diego Leonardo, Humberto D'muniz Pereira, Napoleão Valadares, Rodrigo Portugal, Bruno P. Klaholz, Richard Garratt, et al.

► To cite this version:

Deborah Mendonça, Sinara T.B. Morais, Heloísa Ciol, Andressa P.A. Pinto, Diego Leonardo, et al. Structural Insights into Ciona intestinalis Septins: Complexes Suggest a Mechanism for Nucleotide-dependent Interfacial Cross-talk. Journal of Molecular Biology, 2024, 436 (16), pp.168693. 10.1016/j.jmb.2024.168693. hal-04792055

HAL Id: hal-04792055

https://hal.science/hal-04792055v1

Submitted on 19 Nov 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
Structural Insights into *Ciona intestinalis* Septins: complexes suggest a mechanism for nucleotide-dependent interfacial cross-talk

Deborah C. Mendonça¹⁹, Sinara T.B. Morais¹⁹, Heloísa Ciol<fnref n="1" />, Andressa P.A. Pinto<fnref n="1" />, Diego A. Leonardo<fnref n="1" />,
Humberto D'Muniz Pereira<fnref n="1" />, Napoleão F. Valadares<fnref n="2" />, Rodrigo V. Portugal<fnref n="3" />,<fnref n="4" />, Bruno P. Klaholz<fnref n="5" />,<fnref n="6" />,<fnref n="7" />,<fnref n="8" />, Richard
C. Garratt<fnref n="1" />*, Ana P.U. Araujo<fnref n="1" />*

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹São Carlos Institute of Physics, USP, São Carlos, SP, Brazil</footnote>

<footnote n="2">²Department of Cellular Biology, University of Brasília, Brasília, Brazil</footnote>

<footnote n="3">³Brazilian Nanotechnology National Laboratory, CNPEM, Campinas, SP, Brazil</footnote>

<footnote n="4">⁴Biotechnosciency Program, Federal University of ABC, Santo André, SP, Brazil</footnote>

<footnote n="5">⁵Centre for Integrative Biology (CBI), Department of Integrated Structural Biology, IGBMC (Institute of Genetics and of Molecular and Cellular Biology), 67404 Illkirch, France</footnote>

<footnote n="6">⁶Centre National de la Recherche Scientifique (CNRS) UMR 7104, 67404 Illkirch, France</footnote>

<footnote n="7">⁷Institut National de la Santé et de la Recherche Médicale (INSERM) U964, 67404 Illkirch, France</footnote>

<footnote n="8">⁸Université de Strasbourg, 67081 Strasbourg, France</footnote>

<footnote>§ These authors contributed equally to this work.</footnote>

<footnote>* Corresponding author: anapaula@ifsc.usp.br; richard@ifsc.usp.br</footnote>
**Abstract**

Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. *Ciona intestinalis*, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that *C. intestinalis* septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had *CiSEPT2* occupying the terminal position were able to assemble into filaments via NC-interface association. The *CiSEPT7-CiSEPT9* tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7Å) provides a well-defined view of the central NC-interface. On the other hand, the *CiSEPT7-CiSEPT9* G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that *C. intestinalis* may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.

**Keywords:** Septin complexes, *Ciona intestinalis*, GTPase, Cryo-EM, protein structure
Introduction

In mammals, during the course of evolution, the genes for a group of filamentous
cytoskeletal proteins called septins underwent significant gene expansion. ¹,² This led not
only to a greater diversity of the possible combinations of septins that are capable of
assembling into viable heterocomplexes, but also to an increase in the number of
cellular processes in which the resulting filaments participate. These filaments are
formed by the polymerization of the heterocomplexes (hexamers or octamers,
composed of three or four different septins respectively) by end-to-end association.³⁻⁶
The termini of both oligomers are identical leading to the possibility of coexisting in
mixed filaments, giving rise to yet further structural diversity.⁷

Besides mammalian septins, which have been the most studied to date in terms of 3D
structure, there is now growing structural information for many other species.⁸⁻¹² The
conserved and divergent aspects of septin structure which these studies reveal have
shed light on septin assembly and enable a structure-based approach to study their
evolution.¹³ One largely conserved characteristic is their ability to form the
aforementioned filaments, which seem to be decisive for most of their functions. This
depends on the successful self-assembly of alternating G- and NC- interfaces along the
filament which may have been one of the drivers for the conservation of the structural
elements involved.²

Septin heterocomplexes and filaments play roles in cytokinesis and in many other
important cellular processes including ciliogenesis and neurogenesis.<sup>14-19</sup> Studies have
also attributed a role for septins in innate immunity and during infection, including in
bacterial entrapment.<sup>20-22</sup> In cells that do not undergo division, septins may act as
scaffolds for the recruitment of other proteins or even form diffusion barriers that
compartmentalize membranes into specific cellular domains.<sup>14,23-26</sup> Not surprisingly
therefore, several septin functions appear to be related to their capacity to interact with
both the plasma membrane and to other cytoskeletal components, such as actin
filaments and microtubules, possibly simultaneously.<sup>27,28</sup> These interactions may involve
both curvature sensing or curvature induction of the cellular components involved and
a bewildering array of models has been proposed for such septin-membrane
association, not all of which appear to be mutually compatible.²⁷,²⁹⁻³⁹ For example,
different authors have suggested different polybasic regions and/or amphipathic helices
to be involved.

It is therefore clear that human septins are multi-functional proteins which, for activity,
must assemble in the form of hetero-oligomeric particles. These are linear rod-like
structures which present a specific, palindromic order for the different septins
involved.³,⁷,³⁶,⁴⁰,⁴¹ The 13 recognized human septins can be clustered into four subgroups
represented by septins 2, 3, 6 and 7⁴,⁴²,⁴³ and it is now widely accepted that the
arrangement of the hexamers and octamers are respectively 2-6-7-7-6-2 and 2-6-7-3-3-
7-6-2. This is considered to be the “standard” or “canonical” model. Kinoshita proposed
that septins from the same subgroup should be interchangeable at a given position
within the heterocomplexes leading to the existence of many different combinations (20
and 60 respectively for human hexamers and octamers) and increasing the complexity
of attempts to understand correct oligomeric assembly.⁴

In a recent report⁴⁴, structural data was provided that lends support to Kinoshita's
conjecture suggesting that it merits being considered a general principle for septin-
septin interactions and filament assembly. For this reason, it has recently been dubbed
“Kinoshita's rule”.¹⁹,⁴¹ Nevertheless, there are many aspects of the molecular
recognition involved that still remain to be clarified and understanding the relationship
between filament composition and function is one of the most challenging. Searching
for a minimalist but representative study model for septin heterocomplexes in Chordata,
we choose *Ciona intestinalis*, a solitary sea squirt sessile, member of Urochordata. This
animal has been widely used as a model system to explore the evolutionary origins of
the chordate lineage.⁴⁵,⁴⁶ Phylogenetic analyses, conducted by Cao et al (2007), led to
the identification of only four orthologous septin-coding genes, one representative for
each of the four human septin subgroups, corresponding to a complete set of chordate
septins without redundancy.⁴³,⁴⁷ Based on this observation, it is supposed that these
would be organized similarly to that observed in mammals and other animals, fulfilling
the requirements for a complete but simpler model for septin complex and filament assembly.

In this work, we describe the heterologous production of *C. intestinalis* septins in *E. coli*, in different combinations, arrangements and oligomeric states, including hetero-complexes and filaments. These were used for structural analyses by cryo-EM of a tetrameric sub-complex (composed of septins 7 and 9) at 2.7Å as well as the full hexameric and octameric particles at 3.3 and ~10 Å respectively. The richness of the accumulated structural information available, together with these new data, provides insight into how polymerization depends on the presence of SEPT2 and the potential mechanism by which communication between the NC and G interface may occur.

# Results and Discussion

## Characterisation and biophysical properties

The main objective of the present work was the production of stable and physiologically relevant complexes of *Ciona intestinalis* septins, composed of the expected combinations of subunits, with a view to their structural characterization. For this purpose, some basic studies of the monomers were also necessary. Based on sequence similarity, each of the *C. intestinalis* septins can be readily attributed to one of the four human sub-groups (SEPT2, SEPT6, SEPT7 and SEPT9). The equivalent septins from *C. intestinalis* share 73% (SEPT2), 71% (SEPT6 and SEPT7), and 48% (SEPT9) sequence identity with their human orthologues. The reduced value observed in the case of SEPT9 is largely due to significant differences in the N- and C-terminal domains. More specifically, *CiSEPT6* lacks a critical threonine in switch I, implying that it is catalytically inactive as is its human counterpart.⁴⁸ Knowing the order of subunits within the human hexameric and octameric particles made it possible to choose appropriate combinations for the *C. intestinalis* septins and predict their expected oligomeric state.⁷,⁴⁰ When referring to complexes we will occasionally simplify the nomenclature by using only the numbers (for example the canonical octamer would be simply 2-6-7-9-9-7-6-2).

Recombinant proteins corresponding to each of the isolated septins were successfully expressed and purified and shown to be soluble and stable, except for
CiSEPT9, which expressed only poorly and tended to precipitate, rendering certain biophysical studies impossible. Regarding the complexes, we used strategies to either co-express or to combine cells after expression to produce the following septin combinations: CiSEPT2-6, CiSEPT7-9, CiSEPT2-6-7, CiSEPT6-7-9 and CiSEPT2-6-7-9. The assembly of the expected complexes was achieved in all cases independent of the strategy used, as indicated by an increase in the apparent molecular mass during size exclusion chromatography when compared with the individual components. To evaluate more accurately the size of the complexes as well as their homogeneity we used SEC-MALS, which showed mostly tetramers for combinations of two septins and hexamers for those containing three (Fig. S1). Since the combinations had been chosen on the basis of the standard model and with this specific goal in mind (namely the production of 6-2-2-6 and 7-9-9-7 tetramers and 2-6-7-7-6-2 and 6-7-9-9-7-6 hexamers) these results were not unexpected. In the case of CiSEPT2-6-7-9 SEC-MALS indicated the assembly of an octamer, likely 2-6-7-9-9-7-6-2, as intended.

Septins are proteins that bind, and often hydrolyse GTP, and the presence and, in some species, the nature of the bound nucleotide has been shown to influence the conformation of the dimerization interfaces.⁴⁹ Therefore, we performed analysis of the bound nucleotide in each sample (Fig. S2). CiSEPT6 when expressed in isolation purified free of nucleotide. This is consistent with previous studies that have reported that septins belonging to the catalytically inactive sub-group are unable to bind nucleotide in the absence of their physiological G-interface partner.⁵⁰,⁵¹ This also agrees with the monomeric behaviour of CiSEPT6 (Fig. S1), since homodimers using G interfaces are dependent on the nucleotide binding to both subunits involved.⁴⁸ What was slightly more surprising was that CiSEPT2, which belongs to a catalytically active sub-group, also purified as a monomer and in the apo form. This is different to what is observed for human SEPT2 which is normally GDP-bound and dimeric.³,⁴⁸ The absence of the physiological G-interface partner (CiSEPT6) may explain this behaviour. More important is what we observe for the different complexes. These were composed of two to four different septins and corresponded to full hexamers/octamers or sub-complexes thereof. The CiSEPT2-6 complex contains both GTP and GDP, implying, as expected, that both are indeed competent for nucleotide binding when in the form of a heterocomplex and with the GTP expected to be bound to CiSEPT6 (Fig. S2B). Furthermore, the
remaining heterocomplexes which include these two septins also present a mixture of GDP and GTP (Fig S2B). However, in the case of *CiSEPT2-6-7* and *CiSEPT2-6-7-9* the GTP/GDP ratio was lower than that of *CiSEPT2-6* due to the expected catalytic activity of both *CiSEPT7* and *CiSEPT9*. This is consistent with the result for the *CiSEPT7-9* complex which contained exclusively GDP (Fig S2A). In this case attempts to exchange GDP for GTP or an analogue thereof, under several different conditions, were unsuccessful. On the other hand, when *CiSEPT2* was absent from a complex containing *CiSEPT6* (which is the case for *CiSEPT6-7-9*, for example), we observed only GDP released during the nucleotide content assay (Fig. S2A). This is additional evidence that *CiSEPT6* remains in the apo form in the absence of its G-interface binding partner (*CiSEPT2*). *CiSEPT6-7-9* forms hexamers in solution (Fig S1) which, according to the standard model, would have *CiSEPT6* at their termini where they expose a G-interface unable to secure GTP in the absence of *CiSEPT2*.

Taking together, our results in terms of nucleotide content imply a similar behaviour for *C. intestinalis* septins to that of their human orthologs. The cryo-EM studies described below allowed the structures of the oligomeric states to be observed more directly.

## Oligomeric states and filament formation

After verifying that *C. intestinalis* septins interact with each other to form heterocomplexes, the next step was to establish how they are organized into oligomeric structures and if they follow the expected canonical arrangement (the standard model) seen in humans. Therefore, septin complexes were analysed by negative stain transmission electron microscopy (TEM), initially at high salt concentration (300mM NaCl) with the intention of preventing polymerization into filaments. Under these conditions, the *CiSEPT2-6* and *CiSEPT7-9* complexes form oligomers with a length of approximate 20 nm (Figure S3a and b). This length is compatible with the size expected for a tetrameric complex composed of approximately 5 nm wide monomers and agrees well with the SEC-MALS results (Fig. S1). Interestingly, the *CiSEPT2-6-7-9* complex under the same conditions (300 mM NaCl) shows oligomers as well as long filaments, indicating that this salt concentration is insufficient to completely prevent polymerization via homomeric NC interfaces involving *CiSEPT2* monomers occupying the terminal positions
of adjacent octamers (Fig. S3C). This further suggests that the tetramers we observe in
the case of CiSEPT2-6 sub-complex would also be expected to utilize this same interface.
These are therefore stabilized by two types of physiological interface (those expected to
be present within oligomers and filaments), a homomeric 2-2 NC interface at the centre
together with two heteromeric 2-6 G-interfaces leading to a 6-2-2-6 tetrameric
arrangement imitating the inter-oligomeric contacts which arise during filament
formation. Due to this observation, we increased the salt content of the buffer up to
500mM NaCl in all subsequent assays to obtain a more homogenous sample and avoid
polymerization.

SEC-MALS analysis of the CiSEPT2-6-7 and CiSEPT6-7-9 complexes revealed some heterogeneity (Fig. S1), with the experimentally determined values for the molecular masses differing slightly from those expected for hexamers. Nevertheless, these appeared to be the dominant species. This was confirmed by TEM, where oligomers of different sizes were observed in both cases under high salt conditions (500 mM NaCl), with lengths consistent with tetramers and hexamers. The greater instability of the CiSEPT6-7-9 complex at high salt concentration was evident from the greater variation observed in particle size (Fig 1). Most noticeable, however, was that there was a marked contrast in the behaviour of the two complexes at low salt concentration (40 mM NaCl) where long filamentous bundles were observed in the case of CiSEPT2-6-7 versus no filaments at all for CiSEPT6-7-9 (Fig. 1A and 1B). The fact that CiSEPT6-7-9 was unable to form filaments under the same salt conditions as CiSEPT2-6-7 (or CiSEPT2-6-7-9, Fig. 1C), highlights the essential role played by CiSEPT2 in enabling polymerization. This result indicates that, when present, CiSEPT2 is positioned at the end of the oligomers, as observed for the orthologous human septin complex.<sup>7,40,52</sup> Therefore, in complexes lacking CiSEPT2, such as CiSEPT6-7-9, it is expected that the subunit arrangement involves CiSEPT6 located at the termini of the hexamer (6-7-9-9-7-6). Within this configuration, the apo form of CiSEPT6 obstructs potential interactions through its G interface, consequently hindering polymerization.
## Overall cryo-EM structure of the *C. intestinalis* complexes

Here we provide a description of the organization of the *CiSEPT2-6-7-9* octamer (2-6-7-9-9-7-6-2), the *CiSEPT2-6-7* hexamer (2-6-7-7-6-2) and the *CiSEPT7-9* tetrameric subcomplex (7-9-9-7) which forms the central part of the octamer. The latter two are reported at higher average resolutions than that reported previously for the human hexameric complex³⁶ and were determined using particles derived from two distinct samples and datasets (Table S2). The first dataset corresponds to the production of *CiSEPT2-6-7-9*, initially designed to observe the complete octameric particle, resulting in both octameric and hexameric maps. The second dataset corresponds to the *CiSEPT7-9* sample, elaborated specifically to study tetramers.

The presence of the four septins that compose the octamer was confirmed by SDS-PAGE (Fig. S4). However, the slight underestimation of the molecular mass of the *CiSEPT2-6-7-9* octamer by SEC-MALS is the first indication of the sample's inherent heterogeneity (Fig. S1 and Table S3). This observation was confirmed by processing the cryo-EM dataset corresponding to *CiSEPT2-6-7-9*, which exhibited tetramers and hexamers as well as octamers, all within the same sample (Fig. 2). These were sorted by particle selection during image processing and classification followed by individual refinement. These additional oligomeric states were not initially expected given that the dual affinity tag strategy, including the streptag attached to *CiSEPT9*, had been designed to exclusively isolate octamers during the purification process. This behaviour reveals the fragility of the octameric complex, paralleling the findings observed for human septins.⁵ Through the application of 2D classification to segregate particles corresponding to each of these distinct oligomers, it became feasible to generate a low-resolution map for the octamer (approximately 10 Å average resolution) and a 3.3 Å resolution map for the hexamer (Fig. S5a), both from the same dataset. The latter allowed for atomic model building.

Despite its limited resolution, the shape of the octamer clearly shows the G and NC interfaces alternating along the complex (Fig. 2). Furthermore, the clearly recognizable surface topography of both the monomers and the interfaces between them leaves no doubt that it is an NC interface which is exposed at the termini of the particle, consistent with the notion that the difference between hexamers and octamers
is that the latter includes two additional subunits (SEPT9) at its center.⁷,³⁶,⁴⁰ The enhanced quality of the second map allowed a detailed structural elucidation of the hexamer (Fig. 2 and S5a). This included the identification of characteristic residues contributing to interface formation (described below) and the determination of the bound nucleotides. The subunit order (2-6-7-7-6-2, in accordance with the standard model) was inferred from previous negative stain TEM images using an MBP-fused construct for CiSEPT2 (Fig. S6) similar to that reported by Mendonça et al. (2019) for the human hexamer. This clearly showed that CiSEPT2 occupies the terminal position of the hexamer which, together with the sequence identity between orthologues allowed us to infer the correct subunit order.

The hexamer differs from the octamer in lacking the two central (SEPT9) subunits. In order to recover this missing information (given the low resolution of the octamer itself) a second dataset was acquired for the central tetrameric sub-complex composed of CiSEPT7 and CiSEPT9 (Table S2 and Fig. 2). The preparation of this oligomer exhibited much greater homogeneity and primarily consisted of 7-9-9-7 tetramers. The high-resolution map of this complex (2.7 Å, Fig.2 and S5b), combined with the information from the hexamer, allowed us to build a structural model of the complete octameric particle which could be fitted into the Coulomb potential density map (Fig. 2). Fourier Shell Correlation (FSC) and local resolution information is presented in Figure S5.

All septins within the complexes show the established canonical fold, based on an αβα three-layered sandwich architecture, as previously described in other organisms.⁴¹ The structural details of all the complexes reinforce the findings from the nucleotide-content analyses, confirming that CiSEPT2, CiSEPT7 and CiSEPT9 contain GDP as their natively bound nucleotide, whereas CiSEPT6 is associated with GTP. Notably, all three oligomeric states terminate with an NC interface, a pattern consistent with observations previously reported for the human hexamer under high salt conditions. The fact that hexamers and octamers expose the same polymerization interface at their extremities would be expected to permit the formation of mixed filaments such as have been described for human septins.⁷
## The structures

Much has already been described in the literature concerning the details of septin structures and the nature of their interfaces.<sup>3,8,9,12,41</sup> A very detailed description of the structures determined in the present work would therefore be largely redundant and, for the most part, will not be presented here. Instead, we will concentrate on some important differences and on aspects which have attracted less attention so far. Some of these may be relevant to protein structure in general and not limited specifically to septins.

## The G-interface: Conserved characteristics

The cryo-EM structures of the hexamer and tetramer provide insights into the characteristics of the physiological G interfaces CiSEPT2-CiSEPT6, CiSEPT7-CiSEPT7 and CiSEPT7-CiSEPT9. Previous descriptions of this interface have divided it up into upper, middle, and lower sections.<sup>8,9,44</sup> This has demonstrated overall structural conservation across species as well as specific features which stabilize individual interfaces.

In the upper section, the previously reported β-bridge formed by the switch II regions of both septins involved is also observed in *Ciona intestinalis* (Fig. 3A). The β-bridge is stabilized by main chain intermolecular hydrogen bonds involving residues before and after Asx(Sw2), Asx being a conserved Asn or Asp (Fig. 3A).<sup>41</sup> The nomenclature Asx(Sw2) (ie, the Asp/Asn from Switch II) was proposed by Cavini et al (2021) and will be used here. The same authors suggest that the β-bridge is a characteristic motif in septins (differentiating them from other small GTPases) and that the unusual φ/ψ angles of the main chain of Asx(Sw2) are important to maintain this peculiarity. In order to investigate the potential importance of these torsion angles further, we initially performed a stereochemical analysis of a non-redundant database of 11,600 structures with resolutions < 2Å extracted from the Protein Data Bank (PDB). The main chain torsion angles (φ and ψ) were extracted for all residues in the dataset to generate an updated version of the distribution profile of a generic Ramachandran plot (Fig. 3B). This could then be compared with a similar plot specific for asparagine or aspartic acid residues, Asx (Fig. 3C). The (φ, ψ) coordinates of all of the crystallographically independent Asx(Sw2) residues observed in solved septin structures (Table 3) were then compared with these two distributions. Asx(Sw2) residues are observed to concentrate outside the
allowed regions of the generic Ramachandran plot (Fig. 3B, Asx(Sw2)), where they fall
below and between the two main peaks of the β-region. However, in the case of the Asx-
specific plot we observe that the values of Asx(Sw2) in septins now lie largely within
allowed regions (Fig. 3C). This is a consequence of the unique, but rarely discussed,
Ramachandran plot for Asx, despite having been recognized for many years.⁵³,⁵⁴ Mainly
region 2 (Fig. 3B and 3C) and specifically close to the coordinates φ~−110° and ψ~100°
(where Asx of septins are mostly concentrated), is a region allowed exclusively for
aspartate or asparagine.⁵³ Over the years this region has been included within an
overarching β-region[54] or considered part of a less-populated region which has been
given different names by different authors.⁵⁵⁻⁵⁸ However, the study of Hovmoller et al.
(2002), and repeated here with a larger dataset, clearly shows this region to be
effectively unique to Asx. The justification for the conservation of Asx(Sw2) in septins
appears to be the need to maintain the orientation of the β-bridge in switch II in a flat
arrangement, with little or no twist, for which the unusual φ/ψ angles are a prerequisite.

The middle section of the interface is characterized by the presence of a bifurcated salt
bridge that contributes to its stabilization. De Freitas et al. (2022) showed that Glu58 of
the P-loop in human SEPT7 and Lys173 of the β4-α3 loop are essential for the formation
of the bifurcated salt bridge. They also showed that by restoring these residues in Pnut
(the homolog of SEPT7 in *Drosophila melanogaster*), which normally lacks them,
increases the stability of the interface. *Ci*SEPT2, *Ci*SEPT6 and *Ci*SEPT7 all present Glu in
the P-loop and Lys in the β4-α3 loop in positions homologous to human SEPT7. Hence,
the G interface would be expected to present the bifurcated salt bridge. However, when
analyzing the *Ci*SEPT2-*Ci*SEPT6 and *Ci*SEPT7-*Ci*SEPT7 G interfaces in the hexameric
complex, this region is seen to present poor density making detailed interpretation
impossible. On the other hand, *Ci*SEPT9 has a serine in the position homologous to
Glu58. The absence of glutamate in the P-loop eliminates the possibility of full formation
of the salt bridges at the *Ci*SEPT7-*Ci*SEPT9 G interface (Fig. S7), leading to a potentially
weaker interface which would facilitate the transformation of octamers into hexamers
as observed with human septins.

The structures from *C. intestinalis* septins have also allowed for a comparative analysis
of aspects of the lower section of the G-interface which have not previously been
emphasized. It is in this region that the interdigitation of the β-meanders from the two subunits are observed.⁸,⁹ This motif is important for the stabilization of the G interface since it is the last contact region to dissociate when opposite forces are applied to separate the subunits in molecular simulations.⁸,⁵⁹ Its relevance for stability has recently been commented on by Hussain et al. (2023).¹³

A conserved tyrosine at the end of the βb strand (Fig. 4E, secondary structure nomenclature according to Cavini et al, 2021) is important for nucleotide binding and stabilization by using its side chain hydroxyl to form a weak interaction with the exocyclic NH₂ on the 2-position of the guanine base. It is interesting to note that septins of the SEPT3 subgroup have a threonine instead of this otherwise conserved tyrosine and are characterized by being monomeric and typically purified in their apo form.⁶⁰,⁶¹ However, restitution of tyrosine in human SEPT3 (T282Y) led to a mutant which purified instead as a GDP-bound homodimer.⁶⁰ Furthermore, Rosa et al. (2020) mention the difficulty in obtaining crystals of the human SEPT7-SEPT3 complex and that it was only possible to solve the structure by using the SEPT3T282Y mutant bound to SEPT7 (6UQQ).⁴⁴ Taken together, the currently available data suggest a significant role for the tyrosine in both stabilizing the interface and aiding in nucleotide association, two phenomena which are strongly interrelated.

The tyrosine is part of an interdigitated aromatic network within the β-meanders of the G interfaces of the CiSEPT2-6-7 hexamer, as is often observed in other crystal structures. Besides the tyrosine from the βb strand, a tryptophan from the β-turn connecting βb and βc and a histidine from the βc-α5 loop complete the network (Fig. 4A-B). The tryptophan is part of the conserved S4 motif¹ and the tyrosine serves to orient it in a parallel arrangement (without clear signs of π-π stacking) such that it forms an edge-to-face (T-shaped) aromatic interaction⁶² with the histidine from the neighbouring subunit. For example, Tyr305 in CiSEPT2 orients Trp307 to interact with His317 of CiSEPT6 and a similar cluster is observed at the CiSEPT7 homodimeric interface (Fig. 4A-B). CiSEPT9, like its human homologues of the SEPT3 subgroup, does not have this tyrosine. However, in the case of CiSEPT9 it is replaced by an alanine rather than a threonine (Fig. 4E). Nevertheless, in the structure of the C. intestinalis SEPT7-SEPT9 tetramer this substitution appears to make little difference as the tryptophan is still oriented to
interact in an identical fashion with the histidine of the other subunit (CiSEPT7) (Fig. 4C).
It is therefore interesting to ask if there is any expected difference in the consequences
of the alanine substitution seen in C. intestinalis and the threonine observed in the SEPT3
subgroup. Because the only structural information for a G-interface heterodimer
involving a human SEPT3 subgroup member is the SEPT3T282Y-SEPT7 mutant complex, in
which the threonine has been returned to a tyrosine, we are unable to shed light on this
directly.

However, a similar situation exists in the yeast Cdc3-Cdc10 heterodimer (Fig. 4D) which
serves as a model for the native SEPT3-SEPT7 interface. Cdc10 is the yeast septin which
is most closely related to the SEPT3 subgroup (group 1a according to the nomenclature
proposed by Shuman & Momany, 2022² and it also has a threonine at the tyrosine
position. In this case the absence of the tyrosine leads to a flip in the orientation of the
tryptophan (a ~180° rotation about x₂), which is maintained by a hydrogen bond
between the hydroxyl group of the threonine and the Nε₁H of the tryptophan (Fig. 4D),
leading to a disruption of the normal contact with the histidine. This could destabilize
the lower section of the heteromeric G interface between septins of the SEPT3 and
SEPT7 subgroup facilitating dissociation of the SEPT3 NC-dimer during octamer-to-
hexamer exchange. Since a similar observation is not made in the case of the CiSEPT7-
CiSEPT9 interface, it is tempting to speculate that it is the ability of the threonine
sidechain to form a direct hydrogen bond with the tryptophan sidechain which drives
the conformational change of the latter and thereby the reduced stability of the
interface. This appears to be an example of how subtle changes at the inter-septin
interfaces may have relevant functional consequences.¹³ However, our observations
should not be interpreted to imply anything about the cdc10-cdc3 G-interface in yeast
since this presents many differences with respect to SEPT3-SEPT7 and appears to be
relatively stable. The yeast structure was used here merely to model the impact of a
single point mutation at the SEPT3-SEPT7 interface in human septins.
The NC-interface: the hook-loop and α0 helix stability

The structured region of the N-terminal extension that includes the domain-swapped loop (or hook-loop (HL)) followed by the α0 helix (including its polybasic region, PB1), have been described in crystal and cryo-EM structures of human and yeast septins.<sup>3,9,12,36,41</sup> The α0 helix has been recognized as an important motif for the stabilization of the NC interface where it contributes significant hydrophobic contacts between the two subunits.<sup>9,36</sup> Furthermore, the basic residues of the PB1 region of α0 interact with a series of elements in the neighbouring monomer including the polyacid region (PAR) present in the region of the α5' helix.<sup>9,36</sup>

Marques et al. (2023) recently described the importance of a conserved glutamine of the α₀ helix (Glnα₀) to keep the helix buried within the NC interface. The glutamine side chain points towards the kink of about 50° (the elbow) formed by the α5 and α6 helices of the neighboring subunit, where it recomposes the broken hydrogen bonding pattern of the main chain. In *C. intestinalis*, Glnα₀ is conserved in all septins where it fulfills the same role at the CiSEPT6-CiSEPT7 and CiSEPT9-CiSEPT9 NC-interfaces (Fig. S8). To explore whether this restitution of the hydrogen bonding pattern by the side chain of a glutamine residue is a unique characteristic of septin NC-interfaces or a feature of protein structures in general, we performed a search for helical kinks with angles between 50° and 80° using the KinkFinder 1.01 software<sup>63</sup> within the database described above (11,600 proteins from the PDB). Within the identified kinks, we searched for the presence of a glutamine side chain recomposing the hydrogen bonding pattern of the main chain. Approximately 1100 proteins with kinks of 50° - 80° were identified, however, only two presented a complete reconstruction of the main chain hydrogen bonding pattern as observed in septins (Fig. S9). Some examples that presented glutamines pointing towards the kink used only one side of the amide group of the side chain (N or O) to form hydrogen bonds and other examples used different amino acids altogether or other ligands such as ions (Fig. S9). These results suggest that the specific interaction between Glnα₀ and the α5-α6 elbow is a rather unusual structural motif characteristic of septins and is of importance both for keeping the α0 helices buried and for the stability of the NC interface as a whole.
The ordered hook-loop (HL) motif was first properly observed in the cryo-EM structure
of the human SEPT2-SEPT6-SEPT7 hexameric complex where it embraces the adjacent
subunit.³⁶ The HL participates in the domain-swapping of the structured region of the N-
terminal extension prior to α₀, burying itself between strands β1-β3 and the C-terminal
ends of helices α1 and α5 of the neighboring subunit, thereby aiding in stabilizing the
NC interface.⁴¹ At the CiSEPT6-CiSEPT7 NC interface this pattern is retained.
Furthermore, the conserved hook-loop phenylalanine (baptized PheHL, Cavini et al.,
2021) is buried in the hydrophobic cavity formed by β2, β3, α1, and α5 of the other
subunit (Fig. 5A, 5D). In CiSEPT7 and CiSEPT6, a hydrophobic amino acid two positions
upstream of PheHL (valine in both septins) also penetrates the hydrophobic cavity,
implying that both PheHL and PheHL-2 are important anchor positions for maintaining the
hook-loop embrace of the NC-partner (Fig. 5A, 5D). The higher resolution cryo-EM
structure of the CiSEPT7-CiSEPT9 tetramer also shows the hook-loop of CiSEPT9 buried
at the CiSEPT9-CiSEPT9 NC interface in a similar manner (Fig. 5B). However, in this case,
the conserved PheHL is replaced by an isoleucine, as in all members of the SEPT3
subgroup (Fig. 5D). Despite the PheHL substitution, IleHL fits well into the hydrophobic
cavity together with IleHL-2 and IleHL-6, maintaining a stable interaction (Fig. 5C).
Nevertheless, this subtle difference between SEPT3 group members and other septins
may be of functional significance, given that the concertina mechanism for opening and
closing the NC interface and thereby exposing α₀, has thus far only been seen in this
group. Additional mobility to α₀ might be the result of a weaker hook-loop anchor due
to the substitution of the PheHL by Ile. In this context, Grupp et al. (2024), using yeast
septins, have provided useful biochemical information concerning the importance of a
series of apolar residues at the NC-interface (the hydrophobic crest). Amongst these
residues, a mutation changing PheHL to Ala was not only the most destabilizing for the
interface according to theoretical energy calculations but was also lethal for yeast cells.¹²
In summary, the nature of the amino acid occupying the PheHL position could be critical
for defining the properties of the NC-interface.
Communication between the interfaces: a new hypothesis

A concertina movement for the opening and closing of the NC-interface has been described previously. This was most dramatically noticed when soaking pre-formed GDP-bound crystals of human SEPT9 with excess GTPyS, leading to the closure of an originally open interface.⁶¹ Since the nucleotide binds to the G-interface rather than the NC-interface, this immediately implied cross-talk between them. Several speculative proposals have been raised in attempts to explain this communication.¹¹,⁴¹,⁶¹,⁶⁴ Initially, strand slippage of β3 was proposed to lead to ordering/disordering of the Switch II regions on toggling between GTP/GDP bound states.¹¹,⁶⁴ However, this was discarded when further crystal structures were solved and the switch II loop was seen to be ordered in physiological G interfaces with GDP bound. An alternative proposal was that helix α2 served as the conduit by moving as a rigid body or a spring between interfaces mediated by a cluster of aromatic residues.⁶¹ However, no evidence for exceptional mobility of this helix has ever been found.

The cryo-EM structure of the tetramer described here allows us to explore in detail the open physiological NC-interface between the two copies of *Ci*SEPT9 which reside at the center of the octamer and thereby to propose a more robust, evidence-based, mechanism for communication between the two interfaces. This involves helix α1, switch I, the hook-loop, its hydrophobic binding cavity and α0 (Fig. 6). Comparison of the structure of human SEPT3 bound to the GTP analogue GMppNp (PDB ID: 4Z51) with that of *Ci*SEPT9 bound to GDP, allows us to describe conformational changes to helix α1/switch I on GTP hydrolysis. These allow for the capture of the α0 helix into the NC interface and insertion of IleHL of the hook-loop into the hydrophobic cavity, thereby adopting the conformation shown in Figure 6C.

In the description that follows, it should be kept in mind that the structures of human SEPT3 are excellent models for *Ci*SEPT9 for this particular purpose since the sequence corresponding to helix α1 is identical in both proteins with the exception of a single hydrophobic residue (a leucine for an isoleucine). In the pre-hydrolysis state (represented by the structure of SEPT3 bound to GMppNp), switches I and II are oriented to keep magnesium and the catalytic water in positions to initiate nucleophilic attack on the γ-phosphate of GTP. In this state, helix α1 is longer than that observed in
CiSEPT9/GDP and is formed of 3 turns. The final turn would overlap with part of the
hook-loop in the GDP-bound state (blue arrow in Fig. 6A). Clearly this is untenable, and
something must give. In fact, what is observed in the GDP-bound state is that α1 is
shorter due to unwinding of its final turn alleviating the steric clash and permitting
insertion of IleHL into its hydrophobic binding cavity. Indeed, these differences result in
alterations to the internal topography of the cavity, due to the different positions
assumed by Phe49, Ile77 and Leu84 in the two states (as inferred from their homologues
Phe82, Ile112 and Met119 in SEPT3/GMppNp (Fig. 6A). Effectively, no binding cavity
exists in the GTP-bound state. These observations imply that the hook-loop will be
dislodged from the NC interface when GTP is bound. This is consistent with the closure
of the NC interface in the presence of GTP, which also demands that the α0 helix and the
hook-loop be forced out from between the two subunits.

After hydrolysis of the nucleotide and the consequent release of the γ-phosphate and
magnesium, switch I moves towards the G-interface (black arrow in Fig. 6A) which is
coupled to a smaller shift to Switch II. Helix α1 accompanies this movement and its final
turn unwinds, as mentioned above, allowing for the formation of the hook-loop binding
cavity which becomes occupied by IleHL. This forces the entry of the α0 helix into the NC
interface, which is possible due to the concertina movement, changing from the closed
to the open state. Val53 appears to be particularly important for maintaining Switch I in
its new position. Its side chain occupies a cavity which is filled by Phe49 prior to
hydrolysis and appears to act as an anchor aiding in dragging switch I away from the
hook-loop of the neighbouring subunit.

This hypothesis, summarized in Fig. 6B, connects the hydrolysis of the nucleotide at the
G-interface with conformational change at the adjacent NC-interface, a mechanism that
depends on the movement of the switches towards the G interface together with partial
unwinding of α1 thereby unblocking the hook-loop binding cavity. This allows for the
correct positioning of the structured N-terminal region (hook-loop and helix α0) of the
neighboring subunit within the NC interface, hiding the polybasic region (PB1) such that
it would be unable to interact with membranes in the GDP-bound state.
Conclusions

The significant increase over recent years in our understanding of septin structure
thanks to the success of X-ray crystallography and more recently cryo-EM, allied to
predictions from AlphaFold2 and its derivatives, has generated the potential for reliable
hypotheses for experimental validation. Nevertheless, many doubts still remain
particularly concerning the true role of nucleotide binding and hydrolysis and why the
distribution of catalytically active/inactive subunits varies so greatly from species to
species. This justifies the insistence on accumulating yet more structural data. Here,
*Ciona intestinalis* septins have proven to be a valuable model system for the study of
septin complexes, sub-complexes and filaments. For example, this has allowed for the
determination of a tetrameric complex by cryo-EM for the first time. The higher
resolution of the structure in this case is almost certainly related to the reduced
flexibility of the particles compared to the hexamers and octamers. The viability of
studying such small particles is also a proof of concept. Since this tetramer corresponds
to the central portion of the octamer, this opens up the possibility of testing directly the
proposed concertina hypothesis for the NC-interface (allied to the communication
mechanism put forward here) by cryo-EM in the near future.

The visualization of the octameric particle of CiSEPT2-6-7-9 by cryo-EM is the first of its
kind to be reported in the literature. Although clearly indicating an exposed NC-interface
it's limited resolution precluded the unambiguous identification of the terminal subunit.
However, polymerization data for the octamer and the two hexamers (CiSEPT2-6-7 and
CiSEPT6-7-9) clearly established that this is dependent on the presence of CiSEPT2,
implying it to be located at the extremities of the particle as expected from the standard
model.⁷,³⁶ The determination of both the structure of the CiSEPT2-6-7 complex and the
CiSEPT7-9 sub-complex has allowed us to overcome the limitations of the resolution of
CrSEPT2-6-7-9 and to assemble a structure for the octamer.

Finally, the new structures reported here have stimulated some thoughts concerning
protein structure in general. One example is the completion of the missing hydrogen
bonds of kinked α-helices by a glutamine side chain. Although rather elegant, this turns
out to be very rare in protein structures in general and is effectively a unique feature
shown only by septin NC-interfaces. A second example is the justification for the
conservation of the Asx residue at the top of the β-bridge formed between the switch II
regions of both G-interface subunits. This has its origins in the unusual φ/ψ angles of the
Asx, which occupy a region of the Ramachandran plot inaccessible to most amino acids.
This region of φ/ψ space corresponds to a value of n=2⁶⁵, necessary to generate the
flattened β-bridge whose hydrogen bonds unite the two subunits. The subtleties of the
amino acid specific Ramachandran plots should probably be more appreciated in the
future as we strive to understand more fully the intricacies of protein structure and their
relationship to function.⁵³

**Materials and Methods**

Plasmid constructs

All the DNA coding sequences (CDS) for *Ciona intestinalis* septins were obtained
as synthetic genes, purchased from GenScript USA Inc. The CDSs were based on GenBank
access numbers XM_002125416.2, XM_002123318.1, XM_0002131313.2, and
XM_002129160.2 for *CiSEPT2*, *CiSEPT6*, *CiSEPT7* and *CiSEPT9*, respectively. The vector
constructs used for expression and/or co-expression are detailed in Table S1.

Production of recombinant septin complexes for biophysical characterisation

Expression of septins was performed using several different vectors. To make it
clear, all the vector constructs were numbered and are detailed in Table S1. The
heterocomplexes were expressed using bicistronic vectors and, in most cases, by
combining different vectors. The strategy to combine these was as follows: construct #1
(or alternatively #2 or #3) was co-expressed with #5 to produce the *CiSEPT2-6*
complexes; construct #8 alone was used to co-express the *CiSEPT7-9* complex; constructs
#3 and #5 were co-expressed and the cells were mixed with those expressing construct
#7 prior to purification in order to assemble the *CiSEPT2-6-7* complex; a mixture of cells
(1:1) from the expression of construct #5 and construct #8 was used to produce *CiSEPT6-7-9*;
finally, construct #2 was co-expressed with #5 and these cells were mixed (1:1) with
cells expressing construct #8 to produce the *CiSEPT2-6-7-9* complex.
All *C. intestinalis* septins and heterocomplexes followed similar expression and purification protocols. Briefly, *E. coli* Rosetta (DE3) cells were transformed with the chosen expression plasmids. As a general rule, protein expression was induced by 0.2 mM isopropyl β-D-1-thiogalactopyranoside at OD<sub>600nm</sub> = 1.0, for 20 hours, at 18°C, whilst shaking. Cells were then harvested by centrifugation (5,000 g for 10 min at 4 °C) and the pellets were resuspended with 20 mL of buffer A (25 mM HEPES pH 8.0, 500 mM NaCl, 5 mM MgCl<sub>2</sub>, 5 mM β-mercaptoethanol, 10% glycerol), and disrupted by sonication. The suspension was centrifugated and the supernatant was loaded onto an equilibrated affinity column HisTrap HP 5 mL (Cytiva). Contaminating protein was eluted by washing with 4 volumes of buffer A plus 0.1% Triton X-100 and, subsequently, 6 volumes of buffer A with 35 mM imidazole. Septins and their complexes were eluted by an imidazole gradient up to 500 mM (in buffer A). When applicable, the SUMO-tag was subsequently cleaved using SUMO protease for 2 hours at 4°C either before or after purification.

A second purification step was performed using size exclusion chromatography on a Superdex 200 10/300 GL column (Cytiva), with buffer B (25 mM Hepes pH 8.0, 500 mM NaCl). Purification of the complexes containing an MBP tag followed the purification protocol described in Mendonça et al. (2019).

The purity of septin complexes was analysed on SDS-PAGE 12% and protein quantification was performed using Bradford. The purified samples were preferentially used fresh. Otherwise, they were flash frozen and kept at -80°C.

## Production of recombinant septin complexes for cryo-EM

Aiming to achieve enhanced uniformity in complex formation for the cryo-EM data acquisition, the purification approaches used to produce both the CISEPT2-6-7-9 octamer and the CISEPT7-9 tetramer were distinct from those described above. For both complexes, a SEPT9 construct including a strep-tag was employed in order to allow for an additional affinity chromatography step using a 1 mL StrepTrap HP column (Cytiva) following the initial purification with the HisTrap HP 5 mL column. For this purpose, constructs #3, #5, #7 and #9 were employed to produce the CISEPT2-6-7-9 complex and constructs #7 and #9 for CISEPT7-9 (Table S1). In the case of CISEPT2-6-7-9 SUMO protease (as described above) was used prior to purification to remove SUMO and its
associated His-tag from CiSEPT7 and the IMAC step therefore took advantage solely of
the His-tag attached to CiSEPT2. In the case of CiSEPT7-9, the SUMO protease was
employed after both affinity steps to take advantage of the SUMO associated His-tag on
CiSEPT7. The final purification step was performed using size exclusion chromatography
on Superdex 200 10/300 (Cytiva) as described above.

**Size-exclusion chromatography coupled with multi-angle light scattering assay (SEC-MALS).**

The polydispersity and oligomeric state of each individual septin and septin
complex were determined by size-exclusion chromatography coupled with multi-angle
light scattering. Aliquots of 20 µM of the pure proteins were loaded onto a Superdex 200
5/150 column (Cytiva) and analysed by HPLC using a Waters 600 Controller. The
chromatography was performed at a continuous flow rate of 0.3 mL/min in buffer
composed of 50 mM HEPES pH 8.0 and 300 mM NaCl. Coupled with the chromatographic
separation, the DAWN TREOS miniature system (Wyatt Technology) was used to
determine mass distribution, size and composition, and the OptiLab T-REX system (Wyatt
Technology) the differential refractive index. The ASTRA7 software (Wyatt Technology)
was used to process the data.

**Nucleotide binding and hydrolytic activity assay.**

In order to verify the presence and nature of nucleotides bound to the C.
Intestinalis septins, nucleotide content analysis was performed according to the method
described by Seckler et al. (1990)⁶⁶ with some modifications. Briefly, 10 µM of purified
protein was incubated with ice-cold HClO₄ (1.5 M) for 10 min to unfold the protein and
release the bound nucleotides. After a centrifugation step, K₂HPO₄ (1 M), KOH (3 M) and
acetic acid (5 M) were added to precipitate the KClO₄ and neutralize the pH. The samples
were kept at -20°C for 1 h and then centrifuged. The nucleotides were analysed by anion
exchange chromatography using a Protein Pack DEAE 5-PW, 7.5 mm x 7.5 cm column
(Waters) driven by a Waters 2695 chromatography system. The absorbance was
monitored at 253 nm. As a reference, 10 µM of GTP and GDP were used as standards in
the same conditions as the nucleotides released from the protein. For the hydrolytic
activity assay, samples were incubated with a 3-fold excess of GTP and aliquots were
removed at different time intervals (0, 40, 80 and 120 min), fast frozen in liquid nitrogen
and the nucleotides analysed as described above.

## Negative stain electron microscopy

Purified septin complexes were diluted to 20-80 µg/mL using buffer to maintain
an NaCl concentration of 25-40 mM (for favouring filament formation) or 500-800 mM
(for favouring the maintenance of oligomeric complexes without polymerization).
Samples were adsorbed for 1 min onto glow-discharged ultrathin carbon film supported
by lacey carbon on a copper grid (Ted Pella). The samples were washed in a drop of
deionized water and stained in two drops of 2% uranyl acetate. Micrographs of the
complexes were taken on a Talos F200C microscope (Thermo Fisher Scientific) at 200kV
and recorded using a Ceta camera.

## Cryo-EM of the CiSEPT2-6-7-9 octamer

3 µL of the freshly purified septin octameric complex at a concentration of 1.4 mg/mL was applied to a glow-discharged holey-carbon grid (R2/2 300 mesh, Quantifoil). Subsequently, the grid was plunge-frozen in liquid ethane using an Vitrobot (Thermo Fisher Scientific) at 95% humidity and 6°C. The data was acquired using a Titan Krios (Thermo Fisher Scientific) equipped with a spherical aberration (Cs) corrector operating at an acceleration voltage of 300 kV together with a Gatan K3 direct electron detector and a Gatan GIF energy filter. Automated data collection was conducted using SerialEM⁶⁷ with hardware binning in super-resolution mode, with a pixel size of 0.86 Å using a defocus range between -0.8 and -2.5 µm. Six movies per hole were taken leading to a total dataset of 11,549 movies where each had a total exposure dose of 50.26 e-/Å² split over 50 frames.

## Cryo-EM of the tetramer CiSEPT7-9

3 µL of the freshly purified septin tetrameric complex (composed of two copies
each of CiSEPT7 and CiSEPT9) at a concentration of 1.2 mg/mL was applied to a glow-
discharged gold grid (1.2/1.32 300 mesh, Au-Flat). Subsequently, the grid was plunge-
frozen in liquid ethane using an Vitrobot (Thermo Fisher Scientific) at 95% humidity and
6°C. The data was acquired using a Titan Krios G4 (Thermo Fisher Scientific) with an
acceleration voltage of 300 kV, equipped with a Falcon 4i camera operating in electron
event representation (EER) mode and a Selectris X energy filter. Automated data
collection was conducted using SerialEM⁶⁷ in counting mode, with a pixel size of 0.73 Å
and a defocus range between -1.5 and -3.0 µm. Five movies per hole were taken and a
dataset consisting of a total of 13.405 movies was acquired, where each had a total
exposure dose of 49.33 e-/Å².

Cryo-EM data processing

For the dataset corresponding to the octameric septin complex CiSEPT2-6-7-9,
the patch motion- and CTF-correction (CTF: contrast transfer function) were carried out
using the software cryoSPARC.⁶⁸ Micrographs with estimated fit resolutions below 5 Å
and those whose CTF failed to be fitted were excluded from the subsequent analysis.
Preliminary data from the same sample had previously shown the coexistence of
tetramers, hexamers and octamers. Representative 2D classes of octamers and
hexamers from this preliminary data were used as templates for a template picker job,
resulting in 2.36 million selections. Multiple rounds of 2D classification were performed
to distinguish between hexameric and octameric particles. From these, a subset of 22k
particles was selected to calculate 2 *ab initio* reconstructions for the octameric complex.
After applying heterogeneous refinement, the best 3D class, composed of 16k particles,
was used in a homogeneous refinement job, obtaining a map at approximately 10 Å. The
resolution was estimated from the Fourier Shell Correlation (FSC) applying a standard
threshold of 0.143.⁶⁹⁻⁷³

In order to obtain a 3D map of the hexameric complex, a new round of template
 picker was performed using only 2D classes of hexamers. Subsequent rounds of 2D
classification yielded particles for 2 *ab initio* reconstructions. The resulting particles from
the best 3D class were re-extracted using a box of 512 x 512 pixels. Local CTF refinement
was performed and a total of 300k particles were used for a non-uniform refinement,
followed by a local refinement, resulting in a final map with an average resolution of 3.3
Å but reaching 2.5 Å in some regions. The map was subjected to a deep learning
sharpening method using DeepEMhancer⁷⁴ yielding the final map for structural
interpretation.
Regarding the dataset for the tetrameric septin complex of CiSEPT7-9, a similar
series of processing steps was conducted. Following patch motion- and CTF-correction
with the software cryoSPARC, micrographs demonstrating CTF fits with resolutions
better than 5 Å were selected for processing. 297 particles were manually picked for a
first round of 2D classification. The resultant class averages were employed as references
for a subsequent round of template-based particle picking, leading to a selection of 4.86
million particles. These particles underwent three rounds of 2D classification. A total of
977k particles, members of visually assessed good 2D classes, were employed to
generate 3 *ab-initio* reconstructions. Subsequent heterogeneous refinement resulted in
the selection of 529k particles from the most refined class. These particles were re-
extracted using a box size of 480 x 480 pixels. Further refinements were conducted,
including local CTF refinement, non-uniform refinement, and local refinement. The final
resulting map displayed a resolution of 2.7 Å, with specific regions, notably the G-
interface exhibiting local resolutions up to 2.5 Å. The sharpening of the map was
performed using DeepEMhancer<sup>74</sup> prior to structure interpretation.

Model building and refinement

Atomic model building was possible for both tetrameric and hexameric maps due
to their good resolution. Structure predictions derived from AlphaFold<sup>75,76</sup> were used as
initial models. Regions that were visibly incompatible with the map (such as the N- and
C-termini for which no interpretable density was observed) were excised and then the
monomers assembled into preliminary models for the CiSEPT7-9-9-7 tetramer and
CiSEPT2-6-7-7-6-2 hexamer with the software PyMOL.<sup>77</sup> The fit of the model into the
map was initially performed by treating each monomer as a rigid body with the “Dock in
map” function in the software Phenix.<sup>78,79</sup> The nucleotides were subsequently added,
and the model was manually adjusted in the software COOT<sup>80</sup>, followed by rounds of
real-space refinement in PHENIX.<sup>78,79</sup> The geometric quality of the model was evaluated
with MOLPROBITY<sup>81</sup> and EMRinger<sup>82</sup> was used to validate side-chain model-map
correlation. In the case of the CiSEPT2-6-7-9 octamer, a hybrid model assembled using
the hexamer and tetramer was treated as a single rigid body for building into the map
using UCSF Chimera.<sup>83</sup> No subsequent refinement was possible in this case, due to the
limited resolution.
Bioinformatics analysis

**Analysis of the Ramachandran plot and Asx(Sw2).** A database of 11,600 non-redundant proteins from the Protein Data Bank (PDB) with resolutions < 2Å was used and their torsion angles (φ and ψ) were extracted to generate the distribution profile of the classical Ramachandran plot. The plot was divided into 4x4° cells and the number of occurrences per cell was counted. Subsequently, the cell with the highest number of occurrences was identified and used as a reference to compare and quantify the distribution within the plot. A scale was established in which cells were divided into ten ranges and coloured accordingly. For example, all cells in which the number of occurrences fell within the range from 90% to 100% of the maximum value (the cell with the highest number of occurrences) were grouped and coloured dark red. From 80% to 90% coloured scarlet, from 70% to 80% orange, and so on. To generate the Ramachandran plot for "Asx", φ and ψ angles of asparagine (Asn) and aspartate (Asp) were filtered from the database and treated as described previously. The φ and ψ angles of Asn and Asp from the Sw2 of independent chains of septin structures listed in table 3 were extracted and plotted on the classical and Asx-specific Ramachandran plots.

**Elbow analysis with KinkFinder 1.01.** The search for helical kinks (elbows) with angles between 50° and 80° (average angles observed between α5 and α6 in septins) was performed using the KinkFinder 1.01 software<sup>63</sup>, using the database of 11,600 non-redundant proteins from PDB described above. Approximately 1100 proteins were identified which presented at least one helical kink and these were manually inspected using PyMol v2.05<sup>76</sup> to detect glutamines (Gln) pointing towards the Kink and completing the hydrogen bonds of the main chain as observed in septins.<sup>9</sup>
**Accession numbers**

The data underlying this article are available in the GenBank (accession number XM_002125416.2, XM_002123318.1, XM_002131313.2, and XM_002129160.2) and Protein Data Bank (PDB identifiers 9BHT and 9BHW).

**CRediT authorship contribution statement**

**Deborah C. Mendonça:** Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing - review & editing. **Sinara T.B. Morais:** Methodology, Validation, Formal analysis, Investigation, Data curation. **Heloísa Ciol:** Methodology, Validation, Formal analysis, Investigation, Data curation. **Andressa P.A. Pinto:** Methodology, Validation, Formal analysis, Investigation, Data curation. **Diego A. Leonardo:** Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing - review & editing. **Humberto D'Muniz Pereira:** Validation, Formal analysis, Investigation, Data curation. **Napoleão F. Valadares:** Formal analysis, Investigation, Data curation, Writing - review & editing. **Rodrigo V. Portugal:** Validation, Formal analysis, Data curation. **Bruno P. Klaholz:** Supervision, Funding, Validation, Formal analysis, Data curation, Writing - review & editing. **Richard C. Garratt:** Conceptualization, Resources, Data curation, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. **Ana P.U. Araujo:** Conceptualization, Resources, Data curation, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.

**Conflict of interest**

All the authors declare that they have no conflicts of interest in relation to this work.

**Acknowledgements**

We gratefully acknowledge the support of the São Paulo Research Foundation (FAPESP) via thematic grant 2020/02897-1. D.C.M (2018/20209-5, 2021/10247-0 and 2023/06866-1) and D.A.L. (2021/08158-9) received scholarships from FAPESP and S.T.B.M from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES). This work benefited from access to the Electron Microscopy Facility of CBI (Strasbourg) and has been supported by iNEXT-Discovery project number 24895. The
electron microscope facility was supported by the Region Grand Est, FEDER, the French
Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 / France
2030 program, Instruct-ERIC and iNEXT-Discovery. We would like to thank Alexandre
Durand and Nils Marechal for the excellent technical assistance at CBI. We also thank
LNNano/CNPEM for access to the EM facility via project TEM-25932. We are most
grateful to Michael McMurray and Jeremy Thorner for helpful discussions, particularly
concerning structural aspects of the G-interface.
## References

1. Pan, F., Malmberg, R.L., Momany, M., (2007). Analysis of septins across kingdoms reveals orthology and new motifs. BMC Evol Biol 7, 1-17. https://doi.org/10.1186/1471-2148-7-103

2. Shuman, B., Momany, M., (2022). Septins From Protists to People. Front Cell Dev Biol 9. https://doi.org/10.3389/fcell.2021.824850

3. Sirajuddin, M., Farkasovsky, M., Hauer, F., Kühlmann, D., Macara, I.G., Weyand, M., Stark, H., Wittinghofer, A., (2007). Structural insight into filament formation by mammalian septins. Nature 449, 311-317. https://doi.org/10.1038/nature06052

4. Kinoshita, M., (2003). Assembly of Mammalian Septins. J Biochem 134, 491-496. https://doi.org/10.1093/jb/mvg182

5. Sellin, M.E., Sandblad, L., Stenmark, S., Gullberg, M., (2011). Deciphering the rules governing assembly order of mammalian septin complexes. Mol Biol Cell 22, 3152-3164. https://doi.org/10.1091/mbc.e11-03-0253

6. Kim, M.S., Froese, C.D., Estey, M.P., Trimble, W.S., (2011). SEPT9 occupies the terminal positions in septin octamers and mediates polymerization-dependent functions in abscission. Journal of Cell Biology 195, 815-826. https://doi.org/10.1083/jcb.201106131

7. Soroor, F., Kim, M.S., Palander, O., Balachandran, Y., Collins, R.F., Benlekbir, S., Rubinstein, J.L., Trimble, W.S., (2020). Revised subunit order of mammalian septin complexes explains their in vitro polymerization properties. Mol Biol Cell 32, 289-300. https://doi.org/10.1091/mbc.E20-06-0398

8. de Freitas Fernandes, A., Leonardo, D.A., Cavini, I.A., Rosa, H.V.D., Vargas, J.A., D'Muniz Pereira, H., Nascimento, A.S., Garratt, R.C., (2022). Conservation and divergence of the G-interfaces of Drosophila melanogaster septins. Cytoskeleton n/a. https://doi.org/https://doi.org/10.1002/cm.21740

9. Marques da Silva, R., Christe dos Reis Saladino, G., Leonardo, D.A., Pereira, H.M., Sculaccio, S.A., Araujo, A.P.U., Garratt, R.C., (2023). A key piece of the puzzle: The central tetramer of the Saccharomyces cerevisiae septin protofilament and its implications for self-assembly. J. Struct. Biol. 215, 107983. https://doi.org/https://doi.org/10.1016/j.jsb.2023.107983

10. Pinto, A.P.A., Pereira, H.M., Zeraik, A.E., Ciol, H., Ferreira, F.M., Brandão-Neto, J., DeMarco, R., Navarro, M.V.A.S., Risi, C., Galkin, V.E., Garratt, R.C., Araujo, A.P.U., (2017). Filaments and fingers: Novel structural aspects of the single septin from Chlamydomonas reinhardtii. J. Biol. Chem. 292, 10899-10911. https://doi.org/10.1074/jbc.M116.762229

11. Zeraik, A.E., Pereira, H.M., Santos, Y. V., Brandão-Neto, J., Spoerner, M., Santos, M.S., Colnago, L.A., Garratt, R.C., Araújo, A.P.U., DeMarco, R., (2014). Crystal structure of a Schistosoma mansoni septin reveals the phenomenon of strand slippage in septins dependent on the nature of the bound nucleotide. J. Biol. Chem. 289, 7799-7811. https://doi.org/10.1074/jbc.M113.525352

12. Grupp, B., Denkhaus, L., Gerhardt, S., Vögele, M., Johnsson, N., Gronemeyer, T., (2024). The structure of a tetrameric septin complex reveals a hydrophobic element essential for NC-interface integrity. Commun Biol 7, 48. https://doi.org/10.1038/s42003-023-05734-w
13. Hussain, A., Nguyen, V.T., Reigan, P., McMurray, M., (2023). Evolutionary degeneration of septins into pseudoGTPases: impacts on a hetero-oligomeric assembly interface. Front Cell Dev Biol 11. https://doi.org/10.3389/fcell.2023.1296657.

14. Hu, Q., Milenkovic, L., Jin, H., Scott, M.P., Nachury, M. V, Spiliotis, E.T., Nelson, W.J., (2010). A Septin Diffusion Barrier at the Base of the Primary Cilium Maintains Ciliary Membrane Protein Distribution. Science 329, 436–439. https://doi.org/10.1126/science.1191054.

15. Tada, T., Simonetta, A., Batterton, M., Kinoshita, M., Edbauer, D., Sheng, M., (2007). Role of Septin Cytoskeleton in Spine Morphogenesis and Dendrite Development in Neurons. Current Biology 17, 1752–1758. https://doi.org/10.1016/j.cub.2007.09.039.

16. Kim, S.K., Shindo, A., Park, T.J., Oh, E.C., Ghosh, S., Gray, R.S., Lewis, R.A., Johnson, C.A., Attie-Bittach, T., Katsanis, N., Wallingford, J.B., (2010). Planar Cell Polarity Acts Through Septins to Control Collective Cell Movement and Ciliogenesis. Science 329, 1337–1340. https://doi.org/10.1126/science.1191184.

17. Menon, M.B., Gaestel, M., (2022). Editorial: Emerging Functions of Septins—Volume II. Front Cell Dev Biol 10. https://doi.org/10.3389/fcell.2022.949824.

18. Woods, B.L., Gladfelter, A.S., (2021). The state of the septin cytoskeleton from assembly to function. Curr. Opin. Cell. Biol. 68, 105–112. https://doi.org/https://doi.org/10.1016/j.ceb.2020.10.007.

19. Spiliotis, E.T., McMurray, M.A., (2020). Masters of asymmetry—lessons and perspectives from 50 years of septins. Mol Biol Cell 31, 2289–2297. https://doi.org/10.1091/mbc.E19-11-0648.

20. Mostowy, S., Cossart, P., (2012). Septins: the fourth component of the cytoskeleton. Nat Rev Mol Cell Biol 13, 183–94. https://doi.org/10.1038/nrm3284.

21. Van Ngo, H., Mostowy, S., (2019). Role of septins in microbial infection. J Cell Sci 132, jcs226266. https://doi.org/10.1242/jcs.226266.

22. Robertin, S., Mostowy, S., (2020). The history of septin biology and bacterial infection. Cell Microbiol 22, e13173. https://doi.org/https://doi.org/10.1111/cmi.13173.

23. Finger, F.P., Kopish, K.R., White, J.G., (2003). A role for septins in cellular and axonal migration in C. elegans. Dev Biol 261, 220–234. https://doi.org/10.1016/S0012-1606(03)00296-3.

24. Ihara, M., Kinoshita, A., Yamada, S., Tanaka, H., Tanigaki, A., Kitano, A., Goto, M., Okubo, K., Nishiyama, H., Ogawa, O., Takahashi, C., Itohara, S., Nishimune, Y., Noda, M., Kinoshita, M., (2005). Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa. Dev Cell 8, 343–352. https://doi.org/10.1016/j.devcel.2004.12.005.

25. Takizawa, P.A., DeRisi, J.L., Wilhelm, J.E., Vale, R.D., (2000). Plasma Membrane Compartmentalization in Yeast by Messenger RNA Transport and a Septin Diffusion Barrier. Science 290, 341–344. https://doi.org/10.1126/science.290.5490.341.

26. Ewers, H., Tada, T., Petersen, J.D., Racz, B., Sheng, M., Choquet, D., (2014). A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks. PLoS One 9(12), e113916. https://doi.org/10.1371/journal.pone.0113916.
27. Mavrakis, M., Azou-Gros, Y., Tsai, F.-C., Alvarado, J., Bertin, A., Iv, F., Kress, A., Brasselet, S., Koenderink, G.H., Lecuit, T., (2014). Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles. Nat Cell Biol 16, 322-334. https://doi.org/10.1038/ncb2921.

28. Spiliotis, E.T., Nakos, K., (2021). Cellular functions of actin- and microtubule-associated septins. Current Biology 31, R651-R666. https://doi.org/https://doi.org/10.1016/j.cub.2021.03.064.

29. Vrabioiu, A.M., Mitchison, T.J., (2006). Structural insights into yeast septin organization from polarized fluorescence microscopy. Nature 443, 466-469. https://doi.org/10.1038/nature05109.

30. Bridges, A.A., Jentzsch, M.S., Oakes, P.W., Occhipinti, P., Gladfelter, A.S., (2016). Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton. Journal of Cell Biology 213, 23-32. https://doi.org/10.1083/jcb.201512029.

31. Shi, W., Cannon, K.S., Curtis, B.N., Edelmaier, C., Gladfelter, A.S., Nazockdast, E., (2023). Curvature sensing as an emergent property of multiscale assembly of septins. Proceedings of the National Academy of Sciences 120, e2208253120. https://doi.org/10.1073/pnas.2208253120.

32. Nakazawa, K., Kumar, G., Chauvin, B., Di Cicco, A., Pellegrino, L., Trichet, M., Hajj, B., Cabral, J., Sain, A., Mangenot, S., Bertin, A., (2023). A human septin octamer complex sensitive to membrane curvature drives membrane deformation with a specific mesh-like organization. J Cell Sci 136, jcs260813. https://doi.org/10.1242/jcs.260813.

33. Cannon, K.S., Woods, B.L., Crutchley, J.M., Gladfelter, A.S., (2019). An amphipathic helix enables septins to sense micrometer-scale membrane curvature. Journal of Cell Biology 218, 1128-1137. https://doi.org/10.1083/jcb.201807211.

34. Beber, A., Taveneau, C., Nania, M., Tsai, F.-C., Di Cicco, A., Bassereau, P., Lévy, D., Cabral, J.T., Isambert, H., Mangenot, S., Bertin, A., (2019). Membrane reshaping by micrometric curvature sensitive septin filaments. Nat Commun 10, 420. https://doi.org/10.1038/s41467-019-08344-5.

35. Leonardo, D.A., Cavini, I.A., Sala, F.A., Mendonça, D.C., Rosa, H.V.D., Kumagai, P.S., Crusca Jr, E., Valadares, N.F., Marques, I.A., Brandão-Neto, J., Munte, C.E., Kalbitzer, H.R., Soler, N., Usón, I., André, I., Araujo, A.P.U., D'Muniz Pereira, H., Garratt, R.C., (2021). Orientational Ambiguity in Septin Coiled Coils and its Structural Basis. J Mol Biol 433, 166889. https://doi.org/https://doi.org/10.1016/j.jmb.2021.166889.

36. Mendonça, D.C., Guimarães, S.L., Pereira, H.D., Pinto, A.A., de Farias, M.A., de Godoy, A.S., Araujo, A.P.U., van Heel, M., Portugal, R. V., Garratt, R.C., (2021). An atomic model for the human septin hexamer by cryo-EM. J Mol Biol 433, 167096. https://doi.org/https://doi.org/10.1016/j.jmb.2021.167096.

37. Szuba, A., Bano, F., Castro-Linares, G., Iv, F., Mavrakis, M., Richter, R.P., Bertin, A., Koenderink, G.H., (2021). Membrane binding controls ordered self-assembly of animal septins. Elife 10, e63349. https://doi.org/10.7554/eLife.63349.

38. Bertin, A., McMurray, M.A., Thai, L., Garcia, G., Votin, V., Grob, P., Allyn, T., Thorner, J., Nogales, E., (2010). Phosphatidylinositol-4,5-bisphosphate Promotes Budding Yeast Septin Filament Assembly and Organization. J Mol Biol 404, 711-731. https://doi.org/10.1016/j.jmb.2010.10.002.
39. Ong, K., Wloka, C., Okada, S., Svitkina, T., Bi, E., (2014). Architecture and dynamic remodelling of the septin cytoskeleton during the cell cycle. Nat Commun 5, 5698. https://doi.org/10.1038/ncomms6698.

40. Mendonça, D.C., Macedo, J.N., Guimarães, S.L., Barroso da Silva, F.L., Cassago, A., Garratt, R.C., Portugal, R. v, Araujo, A.P.U., (2019). A revised order of subunits in mammalian septin complexes. Cytoskeleton 76, 457-466. https://doi.org/10.1002/cm.21569.

41. Cavini, I.A., Leonardo, D.A., Rosa, H. V.D., Castro, D.K.S.V., D'Muniz Pereira, H., Valadares, N.F., Araujo, A.P.U., Garratt, R.C., (2021). The Structural Biology of Septins and Their Filaments: An Update. Front Cell Dev Biol. https://doi.org/10.3389/fcell.2021.765085.

42. Hall, P. A, Jung, K., Hillan, K.J., Russell, S.E.H., (2005). Expression profiling the human septin gene family. J Pathol 206, 269-278. https://doi.org/10.1002/path.1789.

43. Cao, L., Ding, X., Yu, W., Yang, X., Shen, S., Yu, L., (2007). Phylogenetic and evolutionary analysis of the septin protein family in metazoan. FEBS Lett 581, 5526-5532. https://doi.org/10.1016/j.febslet.2007.10.032.

44. Rosa, H.V.D., Leonardo, D.A., Brognara, G., Brandão-Neto, J., D'Muniz Pereira, H., Araújo, A.P.U., Garratt, R.C., (2020). Molecular Recognition at Septin Interfaces: The Switches Hold the Key. J Mol Biol 432, 5784-5801. https://doi.org/10.1016/j.jmb.2020.09.001.

45. Satoh, N., Levine, M., (2005). Surfing with the tunicates into the post-genome era. Genes Dev 19, 2407-2411. https://doi.org/10.1101/gad.1365805.

46. Dehal, P., Satou, Y., Campbell, R.K., Chapman, J., Degnan, B., De Tomaso, A., Davidson, B., Di Gregorio, A., Gelpke, M., Goodstein, D.M., Harafuji, N., Hastings, K.E.M., Ho, I., Hotta, K., Huang, W., Kawashima, T., Lemaire, P., Martinez, D., Meinertzhagen, I.A., Necula, S., Nonaka, M., Putnam, N., Rash, S., Saiga, H., Satake, M., Terry, A., Yamada, L., Wang, H.-G., Awazu, S., Azumi, K., Boore, J., Branno, M., Chin-bow, S., DeSantis, R., Doyle, S., Francino, P., Keys, D.N., Haga, S., Hayashi, H., Hino, K., Imai, K.S., Inaba, K., Kano, S., Kobayashi, K., Kobayashi, M., Lee, B.-I., Makabe, K.W., Manohar, C., Matassi, G., Medina, M., Mochizuki, Y., Mount, S., Morishita, T., Miura, S., Nakayama, A., Nishizaka, S., Nomoto, H., Ohta, F., Oishi, K., Rigoutsos, I., Sano, M., Sasaki, A., Sasakura, Y., Shoguchi, E., Shin-i, T., Spagnuolo, A., Stainier, D., Suzuki, M.M., Tassy, O., Takatori, N., Tokuoka, M., Yagi, K., Yoshizaki, F., Wada, S., Zhang, C., Hyatt, P.D., Larimer, F., Detter, C., Doggett, N., Glavina, T., Hawkins, T., Richardson, P., Lucas, S., Kohara, Y., Levine, M., Satoh, N., Rokhsar, D.S., (2002). The Draft Genome of Ciona intestinalis: Insights into Chordate and Vertebrate Origins. Science 298, 2157-2167. https://doi.org/10.1126/science.1080049.

47. Zeraik, A.E., Rinaldi, G., Mann, V.H., Popratiloff, A., Araujo, A.P.U., DeMarco, R., Brindley, P.J., (2013). Septins of Platyhelminths: Identification, Phylogeny, Expression and Localization among Developmental Stages of Schistosoma mansoni. PLoS Negl Trop Dis 7, 1-14. https://doi.org/10.1371/journal.pntd.0002602.

48. Sirajuddin, M., Farkasovsky, M., Zent, E., Wittinghofer, A., (2009). GTP-induced conformational changes in septins and implications for function. Proc Natl Acad Sci U S A 106, 16592-16597. https://doi.org/10.1073/pnas.0902858106.

49. Weems, A., McMurray, M., (2017). The step-wise pathway of septin hetero-octamer assembly in budding yeast. Elife 6, e23689. https://doi.org/10.7554/eLife.23689.
50. Zent, E., Wittinghofer, A., (2014). Human septin isoforms and the GDP-GTP cycle. *Biol Chem* 395, 169-180. https://doi.org/10.1515/hsz-2013-0268.

51. Kumagai, P.S., Martins, C.S., Sales, E.M., Rosa, H. V.D., Mendonça, D.C., Damalio, J.C.P., Spinozzi, F., Itri, R., Araujo, A.P.U., (2019). Correct partner makes the difference: Septin G-interface plays a critical role in amyloid formation. *Int J Biol Macromol* 133, 428-435. https://doi.org/10.1016/j.ijbiomac.2019.04.105.

52. Iv, F., Martins, C.S., Castro-Linares, G., Taveneau, C., Barbier, P., Verdier-Pinard, P., Camoin, L., Audebert, S., Tsai, F.-C., Ramond, L., Llewellyn, A., Belhabib, M., Nakazawa, K., Di Cicco, A., Vincentelli, R., Wenger, J., Cabantous, S., Koenderink, G.H., Bertin, A., Mavrakis, M., (2021). Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms. *J Cell Sci* 134, jcs258484. https://doi.org/10.1242/jcs.258484.

53. Hovmöller, S., Zhou, T., Ohlson, T., (2002). Conformations of amino acids in proteins. *Acta Crystallographica Section D* 58, 768-776. https://doi.org/10.1107/S0907444902003359.

54. Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., (1993). PROCHECK: a program to check the stereochemical quality of protein structures. *J Appl Crystallogr* 26, 283-291. https://doi.org/10.1107/S0021889892009944.

55. Wilmot, C.M., Thornton, J.M., (1990). β-Turns and their distortions: a proposed new nomenclature. *Protein Engineering, Design and Selection* 3, 479-493. https://doi.org/10.1093/protein/3.6.479.

56. Efimov, A. V, (1993). Standard structures in proteins. *Prog Biophys Mol Biol* 60, 201-239. https://doi.org/10.1016/0079-6107(93)90015-C.

57. Hollingsworth, S.A., Karplus, P.A., (2010). A fresh look at the Ramachandran plot and the occurrence of standard structures in proteins. *Biomolecular Concepts* 1, 271-283. https://doi.org/doi:10.1515/bmc.2010.022.

58. Perskie, L.L., Street, T.O., Rose, G.D., (2008). Structures, basins, and energies: A deconstruction of the Protein Coil Library. *Protein Science* 17, 1151-1161. https://doi.org/10.1110/ps.035055.108.

59. Grupp, B., Lemkul, J.A., Gronemeyer, T., (2023). An *in silico* approach to determine inter-subunit affinities in human septin complexes. *Cytoskeleton* n/a. https://doi.org/10.1002/cm.21749.

60. Macedo, J.N. a, Valadares, N.F., Marques, I. a, Ferreira, F.M., Damalio, J.C.P., Pereira, H.M., Garratt, R.C., Araujo, A.P.U., (2013). The structure and properties of septin 3: a possible missing link in septin filament formation. *Biochem J* 450, 95-105. https://doi.org/10.1042/BJ20120851.

61. Castro, D.K.S. do V., da Silva, S.M. de O., Pereira, H.D., Macedo, J.N.A., Leonardo, D.A., Valadares, N.F., Kumagai, P.S., Brandão-Neto, J., Araújo, A.P.U., Garratt, R.C., (2020). A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface. *IUCR J* 7, 462-479. https://doi.org/10.1107/S2052252520002973.

62. Chelli, R., Gervasio, F.L., Procacci, P., Schettino, V., (2002). Stacking and T-shape Competition in Aromatic-Aromatic Amino Acid Interactions. *J Am Chem Soc* 124, 6133-6143. https://doi.org/10.1021/ja0121639.
63. Wilman, H.R., Shi, J., Deane, C.M., (2014). Helix kinks are equally prevalent in soluble and membrane proteins. Proteins: Structure, Function, and Bioinformatics 82, 1960–1970. https://doi.org/https://doi.org/10.1002/prot.24550.

64. Valadares, N.F., d' Muniz Pereira, H., Ulian Araujo, A.P., Garratt, R.C., (2017). Septin structure and filament assembly. Biophys Rev 9, 481–500. https://doi.org/10.1007/s12551-017-0320-4.

65. Schulz, G.E., Schirmer, R.H., (1979). Principles of protein structure / G. E. Schulz, R. H. Schirmer., Springer advanced texts in chemistry. Springer-Verlag, New York.

66. Seckler, R., Wu, G.M., Timasheff, S.N., (1990). Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex. J Biol Chem 265, 7655–61. http://www.jbc.org/content/265/13/7655.

67. Mastronarde, D.N., (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36–51. https://doi.org/https://doi.org/10.1016/j.jsb.2005.07.007.

68. Punjani, A., Rubinstein, J.L., Fleet, D.J., Brubaker, M.A., (2017). cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290–296. https://doi.org/10.1038/nmeth.4169.

69. Saxton, W.O., Baumeister, W., (1982). The correlation averaging of a regularly arranged bacterial cell envelope protein. J Microsc 127, 127–138. https://doi.org/https://doi.org/10.1111/j.1365-2818.1982.tb00405.x.

70. Rosenthal, P.B., Henderson, R., (2003). Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy. J Mol Biol 333, 721–745. https://doi.org/https://doi.org/10.1016/j.jmb.2003.07.013.

71. van Heel, M., Schatz, M., (2005). Fourier shell correlation threshold criteria. J Struct Biol 151, 250–262. https://doi.org/https://doi.org/10.1016/j.jsb.2005.05.009.

72. van Heel, M., Keegstra, W., Schutter, W.G., van Bruggen, E.F.J., (1982). Arthropod hemocyanin studied by image analysis. Life Chem Rep Suppl 69–73.

73. Vuillemot, R., Mirzaei, A., Harastani, M., Hamitouche, I., Fréchin, L., Klaholz, B.P., Miyashita, O., Tama, F., Rouiller, I., Jonic, S., (2023). MDSPACE: Extracting Continuous Conformational Landscapes from Cryo-EM Single Particle Datasets Using 3D-to-2D Flexible Fitting based on Molecular Dynamics Simulation. J Mol Biol 435, 167951. https://doi.org/https://doi.org/10.1016/j.jmb.2023.167951.

74. Sanchez-Garcia, R., Gomez-Blanco, J., Cuervo, A., Carazo, J.M., Sorzano, C.O.S., Vargas, J., (2021). DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun Biol 4, 874. https://doi.org/10.1038/s42003-021-02399-1.

75. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., Steinegger, M., (2022). ColabFold: making protein folding accessible to all. Nat Methods 19, 679–682. https://doi.org/10.1038/s41592-022-01488-1.

76. Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S.A.A., Ballard, A.J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., Back, T., Petersen, S., Reiman, D., Clancy, E., Zielinski, M., Steinegger, M., Pacholska, M., Berghammer, T., Bodenstein, S., Silver, D., Vinyals, O.,
Senior, A.W., Kavukcuoglu, K., Kohli, P., Hassabis, D., (2021). Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589. https://doi.org/10.1038/s41586-021-03819-2

77. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC., (n.d.).

78. Adams, P.D., Afonine, P. V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.-W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H., (2010). {\it PHENIX}: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D 66, 213–221. https://doi.org/10.1107/S0907444909052925.

79. Klaholz, B.P., (2019). Deriving and refining atomic models in crystallography and cryo-EM: the latest Phenix tools to facilitate structure analysis. Acta Crystallographica Section D 75, 878–881. https://doi.org/10.1107/S2059798319013391.

80. Emsley, P., Cowtan, K., (2004). {\it Coot}: model-building tools for molecular graphics. Acta Crystallographica Section D 60, 2126–2132. https://doi.org/10.1107/S0907444904019158.

81. Chen, V.B., Arendall, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., Richardson, D.C., (2010). MolProbity : all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66, 12–21. https://doi.org/10.1107/S0907444909042073.

82. Barad, B.A., Echols, N., Wang, R.Y.-R., Cheng, Y., DiMaio, F., Adams, P.D., Fraser, J.S., (2015). EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat Methods 12, 943–946. https://doi.org/10.1038/nmeth.3541.

83. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., (2004). UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612. https://doi.org/10.1002/jcc.20084.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Electron micrograph of the recombinant *C. intestinalis* complexes at high salt (500 mM NaCl) and low salt (40 mM NaCl) concentrations. (A) CiSEPT2-6-7, (B) CiSEPT6-7-9, (C) CiSEPT2-6-7-9. The complexes comprising CiSEPT6-7-9 are incapable of polymerization at low salt concentration buffer, attributable to the absence of CiSEPT2 at the terminal position of the oligomer.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Cryo-EM of *Ciona intestinalis* septin complexes. The left column shows representative 2D classes derived from the Cryo-EM data, illustrating the coexistence of tetramers, hexamers and octamers within the same sample. The arrows indicate NC-interfaces. In the centre, the three Coulomb potential density maps corresponding to each complex are shown, specifically highlighting the G and NC interfaces. On the right, the atomic models of the octamer (top) hexamer (centre) and tetramer (bottom) have been colour-coded according to each septin within the complex (CISEPT2 in red, CISEPT6 in blue, CISEPT7 in yellow and CISEPT9 in green).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3.** The β-bridge in *C. intestinalis* septins. (A) The β-bridge at the physiological G-interfaces *CiSEPT2-CiSEPT6*, *CiSEPT7-CiSEPT7*, and *CiSEPT7-CiSEPT9*. Asx, asparagine or aspartic acid, occupies the central position of the β-bridge, with the main chain of amino acids in the Asx-1 and Asx+1 positions forming the hydrogen bonds. (B) A classical but updated version of the Ramachandran Plot showing the 4 favored regions (above) and the position of septin Asx(Sw2) residues superimposed on it (below). (C) Ramachandran plot filtered for only Asx residues (above), showing the position of septin Asx(Sw2) residues superimposed on it (below).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Interactions within the β-meander at the G interfaces. (A-C) the β-meander in *C. intestinalis* septins. Tyrosine of the βb strand guides the tryptophan to interact with His of the βc-α5 loop. In *CiSEPT9*, this tyrosine is replaced by alanine (C), however the indole ring of the tryptophan maintains its orientation towards the histidine. (D) The same region of the G-interface as formed between the yeast septins Cdc3 and Cdc10. In Cdc10, the tyrosine is replaced by a threonine that allows the flip of the tryptophan, changing its orientation away from the histidine (arrow). (E) Alignment showing the β-meander regions in the septin subgroups, highlighting in bold the amino acids involved in the interaction network. Cdc3-Cdc10 PDB code: 8FWP.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** The Hook-loop fitting into the hydrophobic cavity of the neighboring subunit, formed by β1, β2, α1 and α5. (A) α0 and the hook-loop from CiSEPT7 are shown in yellow. Val19 and Phe21 (the PheHL position which is largely conserved in septins) fit into the hydrophobic cavity of CiSEPT6 (blue). The remainder of the CiSEPT6 surface is shown in grey. (B) In CiSEPT9, Phe21 is replaced by Ile11 (conserved for the SEPT3 subgroup) which is shown in red. α0 and the hook-loop of one subunit (including Leu5 and Val9) are shown in green. Together with Ile11 these fit into the hydrophobic cavity formed by the other CiSEPT9 subunit, shown as a green surface. (C) is similar to (B) but shows details of the hook-loop binding cavity composed of residues Phe49, Ile77, Leu84 and Ile271 coming from the neighbouring subunit. (D) Alignment between human septins and *C. intestinalis* septins, showing the regions which form the hook-loop and its binding cavity (α1, β2 and β3). Residues of particular importance in forming the hook-loop binding cavity are shown in bold. The PheHL is indicated with an arrow. Vertical dotted lines indicate where the sequence has been interrupted.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Cross-talk between the G- and NC- interfaces. (A) shows the region of the hook-loop binding cavity (red circle), together with α2/Switch I. SEPT3 when complexed to GMppNp (4Z51) is shown in grey and CiSEPT9/GDP in green. α0 and the hook-loop from the CiSEPT9 NC-interface partner are shown in purple, with the Ile11 occupying the “PheHL position” highlighted in red. (B) A schematic representation of the two states, following the same colour scheme. The arrows on the inset indicate the movements which accompany GTP hydrolysis.</figcaption></figure>
Supplementary Material

<figure><img src="image_9.png" /><figcaption>**Figure S1:** Molecular weight determination of the Septins and complexes using SEC-MALS. The curve corresponds to the differential refractive index. The short bold lines correspond to the estimated experimentally determined molecular masses for each peak. The dotted lines correspond to the expected masses for the oligomer indicated.</figcaption></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

**Figure S2:** Ion exchange chromatography of the released nucleotide from individual septins and complexes thereof after denaturation. (a) CiSEPT7, CiSEPT7-9 and CiSEPT6-7-9; and (b) CiSEPT2-6, CiSEPT2-6-7 and CiSEPT2-6-7-9. The positions of the elution peaks corresponding to the GDP and GTP standards are shown as vertical dotted lines. In the case of CiSEPT2 and CiSEPT6, when expressed in isolation the chromatogram followed the baseline indicating the absence of bound nucleotide.
<caption>(a) CiSEPT2-6</caption>

<caption>(b) CiSEPT7-9</caption>

<caption>(c) CiSEPT2-6-7-9</caption>

**Figure S3:** Electron micrographs of the recombinant purified complexes at high salt concentration (300 mM NaCl). (a) CiSEPT2-6, (b) CiSEPT7-9, and (c) two images of CiSEPT2-6-7-9. Red arrows indicate tetramers and yellow arrows indicate octamers.

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

**Figure S4.** CiSeptin octamer production. (a) Final purification step using size exclusion chromatography (Superose 6 increase 10/300 GL column), with the highlighted blue peak corresponding to the octameric complex. (b) Coomassie-stained 12% SDS-PAGE including a molecular mass standard (MW in kDa) and the eluted fraction from the size exclusion column. All four septins are identified.
<figure><img src="image_18.png" /><figcaption>**Figure S5:** Average and local resolution analyses performed in cryoSPARC and visualized in Chimera for the Coulomb potential maps. (a) Hexameric septin complex, CiSEPT2-6-7. (b) Tetrameric septin complex, CiSEPT7-9. Colour keys indicate the local resolution in Å.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**Figure S6.** In an attempt to confirm the position of CISEPT2 at the ends of the oligomer, we produced a fusion protein including MBP at the N-terminus (CISEPT2-MBP) and used it for negative stain TEM analyzes. (a) The complex eluted from the amylose affinity chromatography shown on 12% SDS-PAGE (MW in kDa); (b) Electron micrograph of the purified complex at high salt concentration (500 mM); (c) Eight representative class averages (~30 particles each) derived from processing a total of 173,000 particles from the micrographs. It is clear from the distribution of the addition density (which does not form part of the linear hexamer) that the MBP is associated with the terminal subunit, thereby identifying the position of CISEPT2.</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure S7.** Middle section of the physiological G interfaces of *C. intestinalis*. The formation of the bifurcated salt-bridge is observed in *CiSEPT2-CiSEPT6* and *CiSEPT7-CiSEPT7*, however, in *CiSEPT7-CiSEPT9* it is lost due to the substitution of glutamic acid by serine in *CiSEPT9*.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Figure S8.** Elbow in physiological NC interfaces of *C. intestinalis* septins. In all cases, the glutamine of the α0 helix completes the hydrogen bond network in the kink of α5-6.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Figure S9.** Examples of kinks with angles between 50° and 80°. We identified a small molecule (1UZA) and residues different from glutamine (4GEK and 4J3H) that form polar interactions with kinks. We also identified examples where glutamine forms only a single hydrogen bond with the kink (3VT7), but only two examples where glutamine completes the hydrogen bonding network as observed in septins (3UPL and 5LKY).</figcaption></figure>
<table><caption>**Table S1:** Constructs used in this work</caption><thead><tr><th>Construct<br>number (#)/description</th><th>Expression vector</th><th>cloning sites</th><th>Residues<sup>§</sup></th><th>molecular mass<br>(including tag) kDa</th></tr></thead><tbody><tr><td>1 CiSEPT2-MBP</td><td>pRSFDuetTM-1*</td><td>BamHI/NotI</td><td>1 – 413</td><td>92.3</td></tr><tr><td>2 CiSEPT2-SUMO</td><td>pETSUMO**</td><td>BamHI/NotI</td><td>1 – 413</td><td>59.1</td></tr><tr><td>3 CiSEPT2-His</td><td>pET28a (Merck)</td><td>BamHI/NotI</td><td>1 – 413</td><td>47.1</td></tr><tr><td>4 CiSEPT6-SUMO</td><td>pETSUMO**</td><td>BamHI/XhoI</td><td>1 – 431</td><td>61.6</td></tr><tr><td>5 CiSEPT6</td><td>pETDuet (Merck)</td><td>BamHI/XhoI</td><td>1 – 431</td><td>49.6</td></tr><tr><td>6 CiSEPT7-His</td><td>pETDuet (Merck)</td><td>BamHI/HindIII</td><td>1 – 423</td><td>48.9</td></tr><tr><td>7 CiSEPT7-SUMO</td><td>pETSUMO**</td><td>BamHI/HindIII</td><td>1 – 423</td><td>60.9</td></tr><tr><td>8 CiSEPT7-His</td><td>pETDuet(Merck)</td><td>BamHI/HindIII</td><td>1 – 423</td><td>48.9</td></tr><tr><td>+</td><td></td><td></td><td></td><td></td></tr><tr><td>CiSEPT9</td><td></td><td>Ndel/XhoI</td><td>1 – 358</td><td>40.4</td></tr><tr><td>9 CiSEPT9-strep</td><td>pETDuet</td><td>Ndel/XhoI</td><td>1 – 356</td><td>41.5</td></tr></tbody></table>

* Mendonça et al. 2019.

** Pinto et al., 2017.

§ Initial and final aa of construct, based on the full-length protein.
<table><caption>**Table S2:** Cryo electron microscopy data collection, structure determination, refinement and validation statistics.</caption><thead><tr><th></th><th>EMDB: 44552<br>PDB: 9BHT</th><th>EMDB: 44555<br>PDB: 9BHW</th></tr></thead><tbody><tr><td><strong>C. intestinalis Septin Complex</strong></td><td>Hexamer<br>C/SEPT2-6-7</td><td>Tetramer<br>C/SEPT7-9</td></tr><tr><td><strong>Data collection</strong></td><td></td><td></td></tr><tr><td>Microscope</td><td>FEI Titan Krios with Cs corrector</td><td>FEI Titan Krios G4</td></tr><tr><td>Magnification</td><td>81,000</td><td>165,000</td></tr><tr><td>Voltage (kV)</td><td>300</td><td>300</td></tr><tr><td>Electron exposure (e<sup>-</sup>/Å<sup>2</sup>)</td><td>50.26</td><td>41.83</td></tr><tr><td>Detector</td><td>Gatan K3 with GIF energy filter</td><td>Falcon 4i with Selectris X energy filter</td></tr><tr><td>Physical pixel size (Å)</td><td>0.86</td><td>0.729</td></tr><tr><td>Defocus range (μm)</td><td>-0.8 to -2.5</td><td>-1.5 to -3.0</td></tr><tr><td>Final number of particles</td><td>304274</td><td>529597</td></tr><tr><td>Symmetry imposed</td><td>C1</td><td>C1</td></tr><tr><td>Box size (pixels)</td><td>512 x 512</td><td>480 x 480</td></tr><tr><td>Average map resolution (Å)</td><td>3.26</td><td>2.72</td></tr><tr><td>FSC threshold</td><td>0.143</td><td>0.143</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td></tr><tr><td>Initial model used</td><td>AlphaFold prediction</td><td>AlphaFold prediction</td></tr><tr><td>Chains</td><td>6</td><td>4</td></tr><tr><td>Atoms</td><td>12715</td><td>10044</td></tr><tr><td>Protein residues</td><td>1563</td><td>1248</td></tr><tr><td>Model-to-map fit, CC_mask</td><td>0.75</td><td>0.80</td></tr><tr><td>Bonds (r.m.s.d)</td><td></td><td></td></tr><tr><td>Length (Å) (# > 4σ)</td><td>0.004 (0)</td><td>0.003 (0)</td></tr><tr><td>Angles (°) (# > 4σ)</td><td>0.979 (13)</td><td>0.599 (0)</td></tr><tr><td>B-factors (Å<sup>2</sup>) min/max/mean</td><td></td><td></td></tr><tr><td>Protein</td><td>17.09/160.66/77.54</td><td>23.22/168.32/65.28</td></tr><tr><td><strong>Validation</strong></td><td></td><td></td></tr><tr><td>MolProbity score</td><td>2.19</td><td>1.29</td></tr><tr><td>Clash score</td><td>18.55</td><td>5.34</td></tr><tr><td>EMRinger score</td><td>2.46</td><td>5.20</td></tr><tr><td>Ramachandran Plot (%)</td><td></td><td></td></tr><tr><td>Outliers</td><td>0.46</td><td>0.00</td></tr><tr><td>Allowed</td><td>5.89</td><td>1.45</td></tr><tr><td>Favored</td><td>93.65</td><td>98.55</td></tr><tr><td>Rotamer outliers (%)</td><td>1.00</td><td>0.00</td></tr><tr><td>Cβ outliers (%)</td><td>0.00</td><td>0.00</td></tr><tr><td>Peptide plane (%)</td><td></td><td></td></tr><tr><td>Cis proline/general</td><td>10.5/0.0</td><td>12.5/0.0</td></tr><tr><td>Twisted proline/general</td><td>0.0/0.0</td><td>0.0/0.0</td></tr></tbody></table>
<table><caption>**Table S3:** Estimated molecular masses from SEC-MALS analysis and their theoretical values.</caption><thead><tr><th>Construct description</th><th>SEC-MALS Molar Mass (kDa)</th><th>Oligomeric State by SEC-MALS</th><th>Theoretical Molar Mass of the Monomer (kDa)</th><th>Theoretical Molar Mass of the Oligomer (kDa)</th></tr></thead><tbody><tr><td>CiSEPT2</td><td>51.14</td><td>monomer</td><td>47.1</td><td></td></tr><tr><td>CiSEPT6</td><td>55.19</td><td>monomer</td><td>49.6</td><td></td></tr><tr><td>CiSEPT7</td><td>44.11/115.1</td><td>monomer/dimer</td><td>48.9</td><td>97.8 (dimer)</td></tr><tr><td>CiSEPT9</td><td>not determined</td><td></td><td>40.4</td><td></td></tr><tr><td>CiSEPT2-6</td><td>199.00</td><td>tetramer</td><td></td><td>193.4</td></tr><tr><td>CiSEPT7-9</td><td>188.40</td><td>tetramer</td><td></td><td>178.6</td></tr><tr><td>CiSEPT2-6-7</td><td>324.80</td><td>hexamer</td><td></td><td>291.2</td></tr><tr><td>CiSEPT6-7-9</td><td>258.70</td><td>hexamer</td><td></td><td>277.8</td></tr><tr><td>CiSEPT2-6-7-9</td><td>337.10</td><td>octamer</td><td></td><td>372.0</td></tr></tbody></table>
<table><caption>Table S4: φ and ψ values.</caption><thead><tr><th>Septin</th><th>PDB code</th><th>Amino acid</th><th>Chain</th><th>φ</th><th>ψ</th></tr></thead><tbody><tr><td>SEPT3</td><td>4Z51</td><td>Asn</td><td>A,B</td><td>-111.3</td><td>112.4</td></tr><tr><td rowspan="2">SEPT9</td><td rowspan="2">5CYP</td><td rowspan="2">Asn</td><td>A</td><td>-124.3</td><td>127.9</td></tr><tr><td>B</td><td>-123.7</td><td>128.2</td></tr><tr><td rowspan="2">SEPT9</td><td rowspan="2">5CYO</td><td rowspan="2">Asn</td><td>A</td><td>-112.5</td><td>114.2</td></tr><tr><td>B</td><td>-112.1</td><td>114.0</td></tr><tr><td rowspan="2">SEPT3</td><td rowspan="2">4Z54</td><td rowspan="2">Asn</td><td>A</td><td>-122.9</td><td>109.0</td></tr><tr><td>B</td><td>-109.9</td><td>132.1</td></tr><tr><td rowspan="2">SEPT12</td><td rowspan="2">6MQ9</td><td>Asn</td><td>A</td><td>-119.3</td><td>115.9</td></tr><tr><td>Asn</td><td>D</td><td>-116.7</td><td>112.9</td></tr><tr><td rowspan="2">SEPT12</td><td rowspan="2">6MQK</td><td>Asn</td><td>A</td><td>-108.2</td><td>113.2</td></tr><tr><td>Asn</td><td>D</td><td>-104.4</td><td>121.9</td></tr><tr><td rowspan="2">SEPT7-SEPT3<sub>T282Y</sub></td><td rowspan="2">6UQQ</td><td>Asp</td><td>SEPT7 (B)</td><td>-98.8</td><td>121.4</td></tr><tr><td>Asn</td><td>SEPT3 (D)</td><td>-107.0</td><td>98.8</td></tr><tr><td rowspan="2">SEPT2-SEPT6</td><td rowspan="2">6UPA</td><td>Asn</td><td>SEPT2 (A)</td><td>-117.4</td><td>101.2</td></tr><tr><td>Asn</td><td>SEPT6 (B)</td><td>-101.4</td><td>118.8</td></tr><tr><td rowspan="2">SEPT2-SEPT11</td><td rowspan="2">6UPQ</td><td>Asn</td><td>SEPT2 (A)</td><td>-111.8</td><td>101.4</td></tr><tr><td>Asn</td><td>SEPT11 (B)</td><td>-97.5</td><td>107.5</td></tr><tr><td rowspan="2">SEPT2-SEPT8</td><td rowspan="2">6UPR</td><td>Asn</td><td>SEPT2 (A)</td><td>-99.8</td><td>141.8/150.0</td></tr><tr><td>Asn</td><td>SEPT8 (B)</td><td>-108.6</td><td>120.1</td></tr><tr><td rowspan="2">DmSep1-DmSep2</td><td rowspan="2">8DKT</td><td>Asp</td><td>DmSEPT1 (A)</td><td>-98.9</td><td>104.1</td></tr><tr><td>Asn</td><td>DmSEPT2 (B)</td><td>-109.3</td><td>99.7</td></tr><tr><td rowspan="2">SEPT7</td><td rowspan="2">6N0B</td><td>Asp</td><td>B</td><td>-113.0</td><td>101.5</td></tr><tr><td>Asp</td><td>C</td><td>-111.9</td><td>100.6</td></tr><tr><td rowspan="2">ScCdc3-ScCdc10</td><td rowspan="2"></td><td>Asp</td><td>ScCdc 10 (A)</td><td>-105.9</td><td>104.9</td></tr><tr><td>Asn</td><td>ScCdc 3(B)</td><td>-114.4</td><td>103.9</td></tr><tr><td rowspan="2">CiSEPT2-CiSEPT6-CiSEPT7</td><td rowspan="2"></td><td>Asn</td><td>CiSEPT2 (A)</td><td>-129.3</td><td>93.6</td></tr><tr><td>Asn</td><td>CiSEPT6 (B)</td><td>-86.1</td><td>84.3</td></tr><tr><td rowspan="5">CiSEPT7-CiSEPT9</td><td rowspan="5"></td><td>Asp</td><td>CiSEPT7 (C)</td><td>-93.5</td><td>90.9</td></tr><tr><td>Asp</td><td>CiSEPT7 (D)</td><td>-111.5</td><td>126.8</td></tr><tr><td>Asn</td><td>CiSEPT6 (E)</td><td>-90.1</td><td>117.5</td></tr><tr><td>Asn</td><td>CiSEPT2 (F)</td><td>-102.0</td><td>133.6</td></tr><tr><td>Asp</td><td>SEPT7 (A)</td><td>-107.2</td><td>121.4</td></tr><tr><td rowspan="3"></td><td rowspan="3"></td><td>Asp</td><td>SEPT9 (B)</td><td>-111.7</td><td>108.7</td></tr><tr><td>Asp</td><td>SEPT9 (C)</td><td>-107.9</td><td>119.3</td></tr><tr><td>Asp</td><td>SEPT7 (D)</td><td>-107.7</td><td>113.4</td></tr></tbody></table>