bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Cryo-EM structure of -SMCR8-WDR41 reveals the role as a GAP for Rab8a and 2 Rab11a 3 Dan Tanga, 1, Jingwen Shenga, 1, Liangting Xua, 1, Xiechao Zhanb, Jiaming Liua, Jiang Huia, Xiaoling 4 Shua, Xiaoyu Liua, Tizhong Zhanga, Lan Jianga, Cuiyan Zhoub, c, Wenqi Lib, c, Wei Chenga, Zhonghan 5 Lia, Kunjie Wanga, Kefeng Lua, Chuangye Yanb, 2, Shiqian Qia, 2 6 aDepartment of Urology, State Key Laboratory of Biotherapy, West China Hospital, College of Life 7 Sciences, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China, 8 610041, bBeijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for 9 Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China, 10084, cState Key 10 Laboratory of Membrane Biology, Tsinghua University, Beijing, China, 10084. 11 1These authors contributed equally to this work. 12 2To whom correspondence may be addressed. Email: [email protected] or 13 [email protected] (ORCID: 0000-0002-7589-8877). 14 15 Classification: Biochemistry 16 17 Conflict of interest: The authors declare that they have no conflicts of interest with the contents of 18 this article. 19 20 Author contributions: Q.S. and Y.C. initiated the project. T.D., J.S., X.L. and Q.S. designed and 21 performed the biochemical analysis. Y.C. and Z.X. performed the cryo-EM analysis. Q.S. and Y.C. 22 analyzed the structure. X. S., X. L., and T. Z. purified Rabs. Z.C. and L.W. performed the AUC 23 analysis. Q.S. and Y.C. wrote the manuscript. All the authors discussed the results and commented 24 on the manuscript. Coordinates and structure factor of the structure reported here have been 25 deposited into the Data Bank with PDB Code: 6LT0, and EMDB code: EMD-0966. 26 27 Keywords: C9ORF72, SMCR8, WDR41, GAP activity, cryo-EM 28 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

29 Abstract 30 A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of 31 amyotrophic lateral sclerosis (ALS) and (FTD). Recently, C9ORF72, 32 together with SMCR8 and WDR41, has been shown to regulate and function as GEF. 33 However, the precise function of C9ORF72 remains unclear. Here, we report the cryo-EM structure of 34 the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 Å. The structure reveals the 35 dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of 36 C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two 37 protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are 38 joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C- 39 terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of 40 the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison 41 and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the 42 arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to 43 the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only 44 illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP 45 activity of the C9ORF72-SMCR8 complex. 46 47 Significance Statement 48 C9ORF72, together with SMCR8 and WDR41, has been shown to form a stable complex that 49 participates in the regulation of membrane trafficking. We report the cryo-EM structure of the 50 C9ORF72-SMCR8-WDR41 complex at atomic resolution. Notably, the stoichiometry of the three 51 subunits in the C9ORF72-SMCR8-WDR41 complex is 2:2:2. Interestingly, the C-termini of C9ORF72 52 and the DENN domain of SMCR8 mediate the dimerization of the two C9ORF72-SMCR8-WDR41 53 protomers in the complex. Moreover, WDR41 binds to the DENN domain of SMCR8 by the C-terminal 54 helix without direct contact with C9ORF72. Most importantly, the C9ORF72-SMCR8 complex works 55 as a GAP for Rab8a and Rab11a in vitro, and the Arg147 of SMCR8 is the arginine finger. 56 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

57 Introduction 58 59 Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are the most common 60 neurodegenerative diseases and show overlapping pathology, genetic abnormalities, and patient 61 symptoms(1-3). Disrupted RNA and protein homeostasis have been indicated to be the general causes 62 of ALS-FTD(4). In 2011, the expanded intronic hexanucleotide repeat (GGGGCC) in the 5’ noncoding 63 region of the C9ORF72 was found to accounted for most cases of familial ALS and FTD as well 64 as some sporadic cases of both diseases, representing a historic discovery(5-7). The two potential 65 mechanisms of disease onset related to repeat expansion identified to date are gain-of-function and 66 loss-of-function(3, 8). Studies suggest that neurotoxic materials, including dipeptide repeat 67 (DRPs) and RNA G-quadruplexes, are generated based on the expanded hexanucleotide repeat, which 68 is described as “gain of function”(9-11). In addition, the expanded hexanucleotide repeat can also inhibit 69 the of C9ORF72 and thereby decrease the production of C9ORF72 protein(5, 6, 12, 13). 70 However, the precise function of C9ORF72 is unclear. 71 72 Recently, C9ORF72, together with SMCR8 (Smith-Magenis syndrome chromosomal region candidate 73 gene 8) and WDR41 (WD40 repeat-containing protein 41), has been shown to form a stable complex 74 that participates in regulating macroautophagy (hereafter referred to as autophagy) by directly 75 interacting with the ULK1 complex(14-16). C9ORF72 knockout causes a defect in starvation-induced 76 autophagy, indicating that C9ORF72 regulates autophagy positvely(14, 16-18). SMCR8 was so named 77 because its gene location on the is close to the gene related to Smith-Magenis syndrome; 78 however, it has no relationship with this syndrome(19). Interestingly, SMCR8 knockout results in an 79 increase in ULK1 , which suggests that SMCR8 is a negative regulator of autophagy(20, 80 21). WDR41, the function of which is unknown, is a WD40 repeat-containing protein located on the ER 81 (Fig. 1a)(22, 23). The subunits of the ULK1 complex, to which the C9ORF72-SMCR8-WDR41 complex 82 (hereafter referred to as CSW) binds, are still controversial. Although studies have suggested that the 83 CSW complex interacts with the ULK1 complex via ATG101 or FIP200(14, 16), the details of the 84 interaction between these complexes have yet to elucidated. 85 86 Both C9ORF72 and SMCR8 are predicted to be members of the DENN (differentially expressed in 87 normal and neoplastic cells) family, although they share low sequence similarity (Fig. 1a)(24-26). DENN 88 domain-containing proteins are well known for functioning as guanine exchange factors 89 (GEFs) for many Rab (27-30). Hence, C9ORF72 and SMCR8 may participate in regulating 90 membrane trafficking by mediating the activity of Rab GTPases(28). Consistent with this hypothesis, 91 studies showed that the CSW complex but not C9ORF72 alone has a significant ability to stimulate the 92 exchange of GDP for GTP of Rab8a and Rab39b in vitro(14, 17).Besides, C9ORF72 has also been 93 suggested to interact weakly with Rab11a(31). However, the physiological targets of the CSW complex 94 need further analysis. Additionally, C9ORF72 and SMCR8 have been predicted to be similar to FLCN 95 and FNIP. Before being shown to function as a GTPase activating protein (GAP) of RAG on , 96 the FLCN-FNIP complex was indicated to be a Rab35 GEF in vitro(32-34). Therefore, understanding 97 the structure and biochemical properties of the CSW complex may shed light on the functions of this 98 neurodegenerative disease-related complex. 99 100 In this manuscript, we determined the cryo-electron microscopy (cryo-EM) structure of the CSW 101 complex, which revealed that the complex is composed of two copies of the three proteins, consistent 102 with the biophysical analysis results. The C-terminal tail of C9ORF72 mediates the dimerization of two 103 protomers of the CSW complex by binding to the DENN domain of SMCR8 in the other protomer, this 104 observation was corroborated by biochemical analysis. In the protomer, C9ORF72 and WDR41 are 105 held together by SMCR8 without direct contact with each other. WDR41 binds to the DENN domain of 106 SMCR8 by the C-terminal helix. The overall structure of C9ORF72-SMCR8 resembles that of the FLNC- 107 FNIP2 complex. Biochemical analysis revealed that C9ORF72-SMCR8 has GAP activity for Rab8a and 108 Rab11a and that Arg147 of SMCR8 is critical to the GAP function. Together, our results reveal the 109 assembly basis of the CSW complex and sheds light on the mechanism of its Rab GAP activity. 110 111 Results 112 The CSW complex is a dimer of trimer 113 The CSW complex was previously reported to form a stable complex at a stoichiometry of 1:1:1(17). 114 After testing different expression systems, the well-behaved CSW complex was expressed and 115 purified using Sf9 cells (Fig. 1b, S1). Curiously, the elution volume of the CSW complex in gel filtration 116 corresponded to a molecular mass of ~400 kD, a mass larger than that of the CSW complex with a bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. 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117 1:1:1 stoichiometry of the three subunits (Fig. 1b, S1). To determine the accurate molecular mass of 118 the CSW complex, an analytical ultracentrifugation (AUC) experiment was carried out. Interestingly, 119 the measured sedimentation coefficient of the CSW complex was ~10S, which corresponded to a 120 molecular mass of ~405 kD (Fig. 1c). The theoretical molecular masses of C9ORF72, SMCR8, and 121 WDR41 are ~54 kD, ~105 kD, and ~48.5 kD, respectively. The intensity of Coomassie blue suggested 122 that the molar ratio of the three subunits was 1:1:1 (Fig. 1b, S1). Collectively, these observations 123 suggested that the CSW complex might contain two copies of each of the three subunits to yield a 124 total molecular mass of 415 kD. 125 126 We carried out single-particle cryo-EM analysis of the CSW complex (Fig. S2, S3, Table S1) and solved 127 the cryo-EM density map at a resolution of 3.2 Å (Fig. 1d, 1e). The resolution of the core domain ranges 128 from 3.4 Å to 3.2Å, which clearly shows the side chains at the core region (Fig. S3), and provides us 129 with the details of the CSW complex at atomic resolution. The map revealed that the CSW complex is 130 a dimer of C9ORF72-SMCR8-WDR41, which is consistent with the data obtained from analytical gel 131 filtration and AUC analysis (Fig. 1b, 1c, 1d). The model of the CSW complex was built by combining 132 homology modelling and de novo model building, which allowed us to visualize the structure of the CSW 133 complex in details. 134 135 Both the C-terminal region of C9OFR72 and the DENN domain of SMCR8 are required for the 136 dimerization of the CSW protomers 137 The CSW complex is a homodimer of C9ORF72-SWCR8-WDR41 with a width of ~ 130 Å and a height 138 of 150 Å (Fig. 2a, S4). The two protomers present 2-fold symmetry and match well with each other after 139 180º rotation (Fig. 2a). The observed buried area between the two protomers is ~1544 Å2. 140 141 The structure model reveals that the dimerization of the two CSW protomers is mainly mediated by the 142 C-terminal region of C9ORF72 and the DENN domain of SMCR8 (Fig. 2a, S4a). The density of the 143 C9ORF72 C-terminal region (461-481, hereafter C9ORF72CTR) is ambiguous in both protomers (Fig. 144 2b). Moreover, C9ORF72CTR is highly conserved cross species (Fig. 2c). To assess the contribution of 145 C9ORF72CTR to dimerization, a mutant with C9ORF72CTR deleted (C9ORF72∆C) was constructed. As 146 expected, the C9ORF72∆C-SMCR8 complex is capable of binding to WDR41 as tightly as the 147 C9ORF72-SMCR8 complex (Fig. 2d, S1). However, the elution volume of the C9ORF72∆C-SMCR8- 148 WDR41 complex in gel filtration was delayed by ~0.9 ml relative to that of the CSW complex, which 149 corresponded to a molecular mass of ~200 kD (Fig. 2d, S1). Consistent with this observation, the AUC 150 experiment showed that the sedimentation coefficient of the C9ORF72∆C-SMCR8-WDR41 complex is 151 9.87S, corresponding to a molecular mass of ~199 kD (Fig. 2e). Together, these observations indicate 152 that the C9ORF72∆C-SMCR8-WDR41 complex is monomeric in solution and that C9ORF72CTR is critical 153 to the dimerization of two CSW protomers. 154 155 The structure of CSW does not show where the C9ORF72CTR packs on the DENN domain of SMCR8 156 (700-937, SMCR8DENN); therefore, the assembly status of the complex of C9ORF72 and SMCR8 Longin 157 domain (1-349, SMCR8Longin) was evaluated. AUC analysis showed that the sedimentation coefficient 158 of C9ORF72-SMCR8Longin was ~4.35S (Fig. 2f), which corresponds to a molecular mass of ~ 85.6 kD 159 and suggests C9ORF72-SMCR8Longin is monomeric in solution. Gel filtration analysis indicated that 160 C9ORF72-SMCR8Longin is a monomer is solution as well (Fig. S5). 161 162 Collectively, the data support that both the C-terminal region of C9OFR72 and the DENN domain of 163 SMCR8 together mediate the dimerization of the CSW protomers together. 164 165 Organization of the CSW protomer 166 The C9ORF72-SMCR8-WDR41 complex in one protomer adopts an elongated rod shape, in which the 167 C9ORF72-SMCR8 complex resembles the FLCN-FNIP2 complex (Fig. 3a)(35, 36). Structural 168 comparison shows that C9ORF72 corresponds to FNIP2, whereas SMCR8 resembles FLCN (Fig. 3b, 169 S6a), which is consistent with the bioinformatic analysis(24, 37). 170 171 Superimposition of the C9ORF72-SMCR8 complex and FLCN-FLIP2 dimer revealed that the position 172 of the Longin domain shifts by approximately 40 Å when the DENN domains are superimposed well 173 (Fig. S6a). Although the C9ORF72-SMCR8 complex and FLCN-FNIP2 dimer show a slight difference 174 in overall conformation, the Longin and DENN dimer of the C9ORF72-SMCR8 complex can be 175 superimposed well on their counterparts in FLCN-FNIP2 (Fig. 3b, S6b, c). Intriguingly, SMCR8Longin and 176 SMCR8DENN are connected by αL5, αL6 and the following loop, whereas the Longin domain and DENN bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

177 domain of FLCN are connected by a flexible loop (Fig. S4b). This observation might explain the 178 structural discrepancy between the two complexes. 179 180 The interaction of C9ORF72 and SMCR8 in one protomer is mediated by the Longin domains and 181 DENN domains, respectively, which is consistent with the observations in the FLCN-FNIP2 structure 182 (Fig. 3b, c, S6a, b, c)(35, 36). Importantly, C9ORF72CTR and SMCR8DENN also mediate the dimerization 183 of the CSW protomers (Fig. 2a, b). Thus, there are two interfaces between the DENN domains of 184 C9ORF72 and SMCR8: the interface consisting of αD10 of C9ORF72 and αD11 of SMCR8, which 185 mediates the intra-protomer interaction, and the interface between C9ORF72CTR and SMCR8DENN 186 mediates the dimerization of the CSW protomer (Fig. 3d). This feature distinguished the DENN domains 187 of the C9ORF72-SMCR8 protomer from other members of the DENN domain-containing proteins. 188 189 The structure of WDR41 reveals an 8-blade β-propeller with the N-terminal first strand and C-terminal 190 last three strands coming together to form the first propeller, generating a “Velcro” closure that stabilizes 191 the architecture of WDR41 (Fig. 3a, S4d)(22). WDR41 binds to the SMCR8 DENN domain via its N- 192 terminal β-strand (βN) and C-terminal helix (CTH) without direct physical contact with C9ORF72 (Fig. 193 3a, S4a, S4d). 194 195 The interface of SMCR8-WDR41 196 As mentioned above, WDR41 binds to SMCR8 mainly via its C-terminal helix (WDR41CTH) and the very 197 N-terminal β-strand (Fig. 3a). WDR41CTH packs against the groove formed by αD11, αD12, and αD13 198 of SMCR8, whereas the N-terminal β-strand of WDR41 forms an antiparallel β-sheet with βD6 of 199 SMCR8 (Fig. 3a, 4a). 200 201 To verify this observation, a mutant of WDR41 with a C-terminal helix deleted (436-459, WDR41∆C) and 202 SMCR8Longin were purified successfully and subjected to the pulldown assay. In the pulldown assay, 203 C9ORF72-SMCR8 was able to interact with WDR41 but not WDR41∆C (Fig. 4b, S7a). In contrast, 204 C9ORF72 was not capable of binding to WDR41 or WDR41∆C (Fig. 4b, S7a). Moreover, SMCR8Longin 205 was not able to bind to either WDR41 or WDR41∆C (Fig. 4b, S7a). Collectively, these observations 206 support that WDR41 binds to the DENN domain of SMCR8 via its C-terminal helix. 207 208 The density map of the interface was clear enough to carry out a detailed alanine mutagenesis analysis. 209 The residues on WDR41CTH facing SMCR8, including S438, R441, S442, L445, F446, and L449, were 210 individually mutated to either alanine or arginine (Fig. S7b). However, none of the mutated residues 211 disrupted the interaction between WDR41 and the C9ORF72-SMCR8 complex (Fig. S7b). Therefore, 212 combinations of these were examined. The mutant (WDR41-mutant) with all six key residues 213 mutated abolished the interaction of WDR41 and SMCR8 (Fig. 4c, S7c). Interestingly, WDR41L445R- 214 F446R-L449R blocked binding to the C9ORF72-SMCR8 complex, whereas WDR41S438A-R441A-S442A had little 215 effect on binding (Fig. 4c, S7c). The double mutant WDR41L445R-L449R had obvious effects on binding, 216 whereas WDR41L445R-F446R and WDR41F446R-L449R had weaker phenotypic effects than WDR41L445R-F446R- 217 L449R (Fig. 4c, S7c). 218 219 On the SMCR8 side, all residues (T862-F863-H865-L867-E907A-K910-Y913-M914) involved in binding 220 to L445, F446, and L449 of WDR41 were mutated to alanine, and the resulting construct was termed 221 SMCR8M1. The C9ORF72-SMCR8M1 complex was purified and subjected to a pulldown assay. 222 Compared with wild-type C9ORF72-SMCR8, C9ORF72-SMCR8M1 reduced the binding ability to 223 WDR41 by ~50% (Fig. 4d, S7d). Two other mutants, SMCR8M2 (T862A-F863A-H865A-L867A) and 224 SMCR8M3 (E907A-K910A-Y913A-M914A), were also tested and showed little effect on the binding to 225 WDR41 (Fig. 4d, S7d). The alanine mutational analysis was probably unable to completely destroy the 226 hydrophobic cavity. Hence, we attempted to mutate these residues to arginine, however, a single 227 of any residues to arginine caused C9ORF72-SMCR8 to precipitate. We also tested the effect 228 of βD6 of SMCR8 on the binding to WDR41 by deleting the C-terminal region of SMCR8 (923-937, 229 SMCR8∆C). The pulldown assay showed that SMCR8∆C had little effect on the interaction of C9ORF72- 230 SMCR8 and WDR41 (Fig. S7e), indicating that the antiparallel β-sheet formed between βD6 of SMCR8 231 and βN of WDR41 is not essential to the binding of SMCR8 and WDR41. 232 233 Collectively, these observations demonstrate that the hydrophobic interaction of the C-terminal helix of 234 WDR41 and the DENN domain of SMCR8 is critical to the binding of WDR41 to SMCR8. 235 236 The C9ORF72-SMCR8 complex is a GAP for Rab8a in vitro bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

237 The CSW complex has been reported to function as a GEF of Rab7a, Rab8a, Rab11a, Rab39a, and 238 Rab39b(14, 17, 31). Hence, a bioluminescence-based GTPase activity assay was carried out to assess 239 whether the CSW complex stimulates these Rabs(38). In this type of assay, unhydrolyzed GTP can be 240 transformed into a bioluminescence signal. A higher the bioluminescence signal indicates less GTP 241 hydrolyzation by Rabs. Stimulated by either the CSW complex or the C9ORF72-SMCR8 complex but 242 not C9ORF72 alone, the amount of GTP consumed by Rab8a and Rab11a increased by approximately 243 100% and 50%, respectively (Fig. 5a). However, C9ORF72, the CSW complex, and the C9ORF72- 244 SMCR8 complex had little effect on Rab7a, Rab39a, and Rab39b (Fig. 5a). Interestingly, the CSW 245 complex and C9ORF72-SMCR8 complex showed similar effects on Rab8a and Rab11a, indicating that 246 WDR41 is not essential for the stimulation of target Rabs (Fig. 5a). Moreover, Rab8a and Rab11a play 247 critical roles in coordinating primary ciliogenesis and growth(39, 40). Therefore, we focused on 248 the relationships between Rab8a/11a and the CSW complex and the C9ORF72-SMCR8 complexes in 249 vitro. 250 251 Since the CSW complex was previously identified as Rab GEF, we investigated whether the CSW 252 complex and C9ORF72-SMCR8 complex promote the nucleotide exchange rate of Rab8a and Rab11a. 253 Hence, a fluorescence-based GEF activity assay was performed using N-methylanthraniloyl (MANT)- 254 GDP(41, 42). Compared with buffer, the CSW complex and the C9ORF72-SMCR8 complex were 255 unable to accelerate mant-GDP release from Rab8a and Rab11a (Fig. 5b, c), suggesting that neither 256 complex functions as a GEF for Rab8a and Rab11a. 257 258 As previously mentioned, the C9ORF72-SMCR8 complex shares a similar overall structure with the 259 FLCN-FNIP2 heterodimer, the GAP of RagC/D(33-36). Arg164 on the loop of β6-β7 of FLCN is critical 260 to the GAP activity of the FLCN-FNIP2 complex(35, 36). Although the density of the β6-β7 loop in the 261 C9ORF72-SMCR8 complex is not clear, sequence alignment shows that Arg147 of SMRC8 is 262 conserved across species and corresponds to Arg164 of FLCN (Fig. 5d, e). Hence, we investigated 263 whether the C9ORF72-SMCR8 complex has GAP activity for Rab8a and Rab11a. The effect of Arg147 264 of SMCR8 was probed by the mutation R147A using a bioluminescence assay. 265 266 Excitingly, compared with wild-type C9ORF72-SMCR8, C9ORF72-SMCR8R147A showed no obvious 267 stimulatory effect on Rab8a and Rab11a (Fig. 5f). Intriguingly, C9ORF72∆C-SMCR8 showed a 268 stimulatory effect on Rab8a and Rab11a similar to that of wild-type C9ORF72-SMCR8, suggesting that 269 the dimerization of CSW protomers is not essential for the stimulatory effect on Rab8a and Rab11a 270 (Fig. 5f). Moreover, C9ORF72∆C-SMCR8R147A eliminated the stimulatory effect on Rab8a and Rab11a 271 (Fig. 5f). 272 273 C9ORF72 showed no stimulatory activity against Rab8a and Rab11a. Hence, we attempted to test 274 whether SMCR8 alone had GAP activity against Rab8a and Rab11a. However, SMCR8 cannot be 275 purified alone. Fortunately, we were able to successfully purify SMCR8Longin and C9ORF72-SMCR8Longin. 276 Interestingly, SMCR8Longin showed no stimulatory effects on Rab8a and Rab11a (Fig. 5f), whereas 277 C9ORF72-SMCR8Longin showed an effect on Rab8a and Rab11a similar to that of C9ORF72-SMCR8 278 (Fig. 5f). As expected, C9ORF72-SMCR8 Longin-R147A showed no GAP stimulatory effects on Rab8a and 279 Rab11a (Fig. 5f). 280 281 Together, the data indicate that CSW and the C9ORF72-SMCR8 complex but not C9ORF72 or SMCR8 282 alone can exhibit GAP activity for Rab8a and Rab11a in vitro. Additionally, the data support that the 283 dimerization of C9ORF72-SMCR8 is not required for the complex’s GAP activity. 284 285 To further assess the responses of Rab8a and Rab11a to stimulation by the C9ORF72-SMCR8 286 complex, a titration experiment was performed at a fixed concentration of Rab. With increasing 287 concentration of the wild-type C9ORF72-SMCR8 complex, Rab8a hydrolyzed more GTP and almost 288 exhausted the GTP when the molar ratio of Rab8a and C9ORF72-SMCR8 complex reached 1:1. In 289 contrast, the C9ORF72-SMCR8R147A complex showed no obvious stimulatory effect on Rab8a (Fig. 5g). 290 291 Similarly, Rab11a consumed more GTP with increasing concentration of the C9ORF72-SMCR8 292 complex but not that of the C9ORF72-SMCR8R147A complex (Fig. 5h). However, the limit of GTP 293 hydrolyzed by stimulated Rab11a was ~ 50% of the total amount of GTP (Fig. 5h). This phenomenon 294 might have been caused by the slow intrinsic nucleotide exchange rate of Rab11a (Fig. 5c), which might 295 have prevented Rab11a from efficiently accessing GTP when the ratio between GTP:GDP in the system 296 was 1:1(43). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

297 298 In a word, C9ORF72-SMCR8 showed GAP activity against Rab8a and Rab11a in vitro, which was 299 contrary to our expectation. Furthermore, Arg147 of SMCR8 is critical to the GAP activity of C9ORF72- 300 SMCR8. 301 302 Discussion 303 The CSW complex is critical to a variety of cellular processes and is strongly associated with familial 304 ALS and FTD. In addition, the CSW complex is highly conserved in animals (Fig. S9-11). Nevertheless, 305 the exact functions of the CSW complex remain unclear. Here, we investigated this important complex 306 using recombinant proteins, biochemical assays, and cryo-EM analysis. 307 308 First, we determined that the stoichiometry of C9ORF72, SMCR8, and WDR41 in the CSW complex is 309 2:2:2. Both biochemical analysis and the structure of the CSW complex revealed that the CSW complex 310 is a dimer of C9ORF72-SMCR8-WDR41. Although the density map of the dimerization interface is 311 unclear, mutagenesis analysis, AUC analysis and analytical gel filtration indicated that C9ORF72CTR 312 and SMCR8DENN are necessary for the dimerization of C9ORF72-SMCR8-WDR41. Notably, 313 dimerization of C9ORF72-SMCR8-WDR41 or C9ORF72-SMCR8 is not essential to the complex’s GAP 314 activity (Fig. 5f). To obtain detailed information on the dimer interface of C9ORF72-SMCR8-WDR41, a 315 higher-resolution structure is needed, and determining the biological significance of dimerization 316 requires in vivo genetic and functional analyses. 317 318 Additionally, we clarified the relationship among C9ORF72, SMCR8, and WDR41 based on the 319 structure of CSW. WDR41 binds to a groove on SMCR8DENN without direct contact with C9ORF72 (Fig. 320 4). WDR41 and C9ORF72 are joined together by SMCR8. A triad of L445, F446, and L449 from 321 WDR41CTH is critical to the binding of WDR41 to SMCR8. Mutants containing L445R, F446R, and 322 L449R do not have the ability to bind to SMCR8. Eight residues of SMCR8DENN involved in the binding 323 to WDR41CTH were assessed by alanine mutagenesis analysis since arginine mutagenesis crashed 324 SMCR8. All eight residues have to be mutated into alanine simultaneously to cripple the binding of 325 SMCR8 to WDR41. Interestingly, WDR41 is not essential to the GAP activity of the CSW complex. 326 Previous studies showed that WDR41 locates on ER and participates in the autophagosome- 327 pathway(15, 16, 23). Thus, the role of WDR41 in the CSW complex might be localizing the complex on 328 appropriate positions. 329 330 We found that the DENN domain of C9ORF72 can interact with two DENN domains of SMCR8 331 simultaneously, vice versa (Fig. 3d). Not only does such a binding mode enable the DENN dimer of 332 C9ORF72-SMCR8 to mediate intra-protomer interaction as well as the inter-protomer interaction, it 333 distinguishes these DENN domains from others, including the DENN domains of FLCN and FNIP2. 334 These observations enhance our understanding of the functional evolution of the DENN domain. 335 336 Most importantly, the GAP activity of C9ORF72-SMCR8 was identified based on the structure of 337 C9ORF72-SMCR8 and a GTPase activity assay. The structure of C9ORF72-SMCR8 resembles that of 338 FLCN-FNIP2, which is a GAP of RagC/D. The Longin dimers in both structures adopt similar 339 organization patterns (Fig. S4c). Intriguingly, Lst7, the homologue of FLCN in yeast, contains only the 340 Longin domain(44, 45). Moreover, the catalytic arginine of FLCN-FNIP2 is located in the Longin domain 341 of FLCN(35, 36). Together, these data indicate that the Longin dimer might be functionally conserved 342 during evolution. Based on the structure and sequence alignment, Arg147 of SMCR8 was found to 343 correspond to Arg164. However, previous studies indicate that the CSW complex might function as a 344 GEF of many Rabs. Hence, we screened Rabs that can be stimulated by CSW or the C9ORF72- 345 SMCR8 complex in vitro using reconstituted proteins. Serendipitously, CSW and C9ORF72-SMCR8 346 were found able to stimulate Rab8a and Rab11a at similar levels, which indicated that WDR41 is not 347 essential for the stimulating effect of CSW on Rab8a and Rab11a. Curiously, neither CSW nor 348 C9ORF72-SMCR8 accelerated the nucleotide exchange rate of Rab8a and Rab11a in vitro, indicating 349 that CSW and C9ORF72-SMCR8 are not GEFs for Rab8a and Rab11a. Mutagenesis analysis 350 combined with GAP-stimulated GTPase activity assay confirmed that Arg147 of SMCR8 is essential to 351 its GAP activity for Rab8a and Rab11a in vitro. In addition, both C9ORF72 and SMCR8 are required 352 for GAP activity, as evidenced by the finding that neither C9ORF72 nor SMCR8Longin shows GAP activity 353 toward Rab8a and Rab11a. It is of interest to dissect how C9ORF72 and SMCR8 recognize and 354 collaboratively activate Rab8a/11a. Unfortunately, we have not yet been able to obtain a stable complex 355 of C9ORF72-SMCR8-Rab8a/11a, indicating that the interaction of C9ORF72-SMCR8 and Rab8a/11a 356 might be weak or ephemeral. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

357 358 Our results demonstrate that CSW is a dimer of C9ORF72-SMCR8-WDR41 and is able to stimulate the 359 GTPase activity of Rab8a and Rab11a as a GAP. Rab8a and Rab11a function coordinately in many 360 fundamental cellular processes, including receptor recycling, ciliogenesis, and axon growth(39, 40, 46). 361 It is tempting to propose the following model: CSW takes two containing Rab8a/11a via 362 WDR41 simultaneously and then activates the Rabs located on the surface to prompt the fusion of the 363 two endosomes. 364 365 Although the GAP activity of the C9ORF72-SMCR8 complex has been tested in vitro, the specific 366 biological processes in which CSW and the C9ORF72-SMCR8 complex participate as a GAP require 367 further exploration. The findings and the biochemical data presented in our manuscript will definitely 368 facilitate future functional studies of CSW and the C9ORF72-SMCR8 complex. 369 370 371 Materials and methods 372 protein purification 373 Protein were expressed in E. coli BL21 (DE3) cells or Sf9 cells and purified using FPLC 374 chromatography. 375 376 Analytical ultracentrifugation 377 Analytical Ultracentrifugation Sedimentation experiments were carried out at 20 °C in an XL-I 378 analytical ultracentrifuge (Beckman-Coulter) equipped with Rayleigh Interference detection (655 nm). 379 380 Structure determination 381 The structure of CSW complex were determined using cryo-EM. 382 383 GTPase activity assay 384 GTPase activity assays were carried out using GTPase-Glo assay kit (Promega, V7681). 385 386 Nucleotide exchange assay 387 Releasing of MANT-GDP was recorded by monitoring the decrease in fluorescence emission at 388 448 nm (excited at 360 nm) in intervals of 15 s at 25 °C for 3600 seconds. Data were collected using 389 384-well plate (Corning, 3701) in BioTek Synergy2. 390 391 Acknowledgements: We thank the Tsinghua University Branch of China National Center for Protein 392 Sciences (Beijing) for providing the cryo-EM facility support. We thank the computational facility 393 support on the cluster of Bio-Computing Platform (Tsinghua University Branch of China National 394 Center for Protein Sciences Beijing). We thank Jianhua He, Bo Sun, Wenming Qin, Huan Zhou, Feng 395 Yu from BL17U, BL18U, and BL19U at National Facility for Protein Science in Shanghai, Zhangjiang 396 Laboratory (NFPS, ZJLab), China for providing technical support and assistance in crystal testing. We 397 also thank staff from Protein Preparation and Identification Facility at Technology Center for Protein 398 Science, Tsinghua University for the assistance with AUC data collection. This work was supported by 399 National Key R&D Program of China grant 2017YFA0506300 (Q.S.), 2018YFC1004601 (S.Q.), and 400 NSFC grants 81671388 (Q. S.), 31770820 (L. K.). 401 402 References:

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509 Figure Legends 510 511 Figure 1. The CSW complex is a dimer of C9ORF72-SMCR8-WDR41. 512 A. Schematic diagram of the domain arrangement of C9ORF72 (light-blue), SMCR8 (light-green), and 513 WDR41 (orange). The names and boundaries of domains are labeled. β-N: N-terminal first β strand of 514 WDR41. CTR: C-terminal helix of WDR41. The numbers in WDR41 represent WD40 domains: WD1 515 (41-81), WD2 (88-131), WD3 (137-168), WD4 (177-281), WD5 (226-276), WD6 (281-314), WD7 (326- 516 401), and WD8 (411-432, and β-N). The interactions between different domains are shown in lines: 517 light-blue line, C9ORF72-SMCR8 interaction; orange line, WDR41-SMCR8 interaction. B. Gel filtration 518 (superpose 6 10/300 GL) profile of reconstituted C9ORF72-SMCR8-WDR41 and C9ORF72-SMCR8 519 complex. The horizontal axis is elution volume, and the vertical axis is UV absorption. The UV 520 absorbance is shown in red (C9ORF72-SMCR8-WDR41) and blue (C9ORF72-SMCR8) lines. The 521 peaks of proteins are labeled. The Coomassie blue-stained SDS-PAGE shows the peak fraction of the 522 CSW from gel filtration. C. Analysis of peak fraction from (b) by Sedimentation velocity AUC. C(S) 523 functions calculated from sedimentation velocity data are shown in blue curve. The calculated molecular 524 mass is denoted. Horizontal axis: Sedimentation coefficient. Vertical axis: Continuous sedimentation 525 coefficient distribution. (D, E) Cryo-EM density map of the CSW complex. D. The overall map for the 526 dimer. E. The final map of one protomer of the CSW complex. The components of the CSW complex 527 are indicated in different colors. Light-blue: C9ORF72; light-green: SMCR8; Orange: WDR41. 528 529 Figure 2. The dimer interface of the two protomers in the CSW complex. 530 A. The overall structure of the CSW complex is shown in cartoon. The CSW complex shows a two-fold 531 symmetry. The domains are labeled. The width and height are denoted. C9ORF72, SMCR8, and 532 WDR41 are colored in light-blue, light-green, and orange, respectively. B. The density map of the 533 interface between C9ORF72CTR and SMCR8DENN. The unoccupied ambiguous map is highlighted with 534 an oval circle. The last residue presented in the model was denoted with a red dot and labeled. C. The 535 sequence alignment of C9ORF72CTR in different species. HUMAN: Homo sapiens; Mouse: Mus 536 musculus; Zebrafish: Danio rerio; Xenopus: Xenopus tropicalis. D. Gel filtration (superpose 6 10/300 537 GL) profile of reconstituted C9ORF72∆C-SMCR8-WDR41 complex. C9ORF72∆C represents the 538 construct of C9ORF72 with 461-481 deleted. The UV280 absorption curve of C9ORF72∆C-SMCR8- 539 WDR41 complex is shown in blue, whereas wild-type C9ORF72 -SMCR8-WDR41 complex is shown in 540 orange as comparison. The peak fraction of C9ORF72∆C-SMCR8-WDR41 complex is examined by 541 Coomassie blue stained SDS-PAGE. (E, F). Analysis of by Sedimentation velocity AUC of C9ORF72∆C- 542 SMCR8-WDR41 (E) and C9ORF72-SMCR8Longin (F). C(S) functions calculated from sedimentation 543 velocity data are shown in a blue curve. The calculated molecular mass is denoted. Horizontal axis: 544 Sedimentation coefficient. Vertical axis: Continuous sedimentation coefficient distribution. 545 546 Figure 3. Organization of the CSW protomer. 547 A. The overall structure of the CSW protomer. The key secondary structures are labeled. The colors 548 are consistent with the previous figures. B. The structure of the C9ORF72-SMCR8 complex adopts a 549 similar organization to the FLCN-FNIP2 complex. Left: C9ORF72-SMCR8 complex. Right: FLCN-FNIP2 550 complex. C9ORF72, SMCR8, and WDR41 are colored as (A). FLCN and FNIP2 are colored in green- 551 cyan and wheat. The details of the comparison are presented in Fig. S5. (C, D) Two interfaces between 552 C9ORF72 DENN and SMCR8DENN are shown. C. Comparison of DENN pair of C9ORF72-SMCR8 and 553 FLCN-FNIP. The key helixes are labeled. The red dash line indicates the position of the interface. D. 554 The two interfaces between C9ORF72 DENN and SMCR8DENN are shown. The red dash line indicates the 555 positions of the intra-protomer interface, while the blue dash line indicates the positions of the inter- 556 protomer interface. 557 558 Figure 4. The interface of SMCR8 and WDR41. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

559 A. The details of the interface of SMCR8 and WDR41. The key residues on the interface are shown in 560 stick model. Secondary structures are shown as cartoon. Left: front view; right: back view. B. C9ORF72, 561 SMCR8Longin, C9ORF72-SMCR8Longin complex, and C9ORF72-SMCR8 complex were used to pulldown 562 WDR41 and WDR41∆C. (C, D) Mutational analysis of key residues on WDR41CTH (C) and SMCR8 (D) 563 using pulldown assay. The results are visualized by Coomassie blue-stained SDS-PAGE. Protein 564 markers are labeled at left (unit: kD). c. Wild-type C9ORF72-SMCR8 complex with N-terminal His-tag 565 was used to pull down untagged wild-type or indicated mutants of WDR41. WDR41-mutant: WDR41with 566 mutations of S438A-R411A-S442A-L445R-F446R-L449R. Other mutants are shown as the label. D. 567 Indicated mutants of SMCR8 with N-terminal His-tag were used to pull down untagged wild-type 568 WDR41. WT: wild-type C9ORF72-SMCR8; SMCR8M1: C9ORF72-SMCR8M1 (SMCR8 with mutations of 569 T862A, F863A, H865A, L867A, E907A, K910A, Y913A, M914A); SMCR8M2: C9ORF72- 570 SMCR8M2(SMCR8 with mutations of T862A, F863A, H865A, L867A); SMCR8M3: C9ORF72-SMCR8M3 571 (SMCR8 with mutations of E907A, K910A, Y913A, M914A). 572 573 Figure 5. CSW and C9ORF72-SMCR8 complex are GAPs of Rab8a and Rab11a in vitro. 574 A. The screen of Rabs that can be stimulated by CSW or C9ORF72-SMCR8 complex using 575 bioluminescence based GTPase activity assay. In this assay, the final concentration of Rabs and 576 C9/CS/CSW was 1.5 µM and 0.75 µM, respectively. The GTP in the reaction system containing no 577 protein was normalized to “1.0”. The proteins or protein mixtures added in the reaction system are 578 indicated below. Buffer: Buffer control, C9: C9ORF72, CS: C9ORF72-SMCR8, CSW: wild-type 579 C9ORF72-SMCR8-WDR41, 7a: Rab7a, 8a: Rab8a, 11a: Rab11a, 39a: Rab39a, 39b: Rab39b. The 580 error bars represent mean ± SD (N=3). (B, C) mant-GDP based nucleotide exchange assay for Rab8a 581 (B) and Rab11a (C). The abbreviations of protein names are same to the panel (A). 100 µM GDP was 582 used to initiate the exchange reaction. Reactions without GDP were monitored as a control. The error 583 bars represent mean ± SD (N=3). The intrinsic nucleotide exchange rate of Rab11a is negligible. D. 584 Comparison of the Longin domains of SMCR8 and FLCN. The secondary structures are labeled. The 585 Arg164 of FLCN-Longin domain is highlighted in red. The missing loop of SMCR8Longin is indicated as 586 a red dash line. E. Sequence alignment of the βL4-βL5 loop of SMCR8. Arg147 of human SMCR8 is 587 conserved cross species and corresponds to Arg164 of FLCN. HUMAN: Homo sapiens; Mouse: Mus 588 musculus; Zebrafish: Danio rerio; Worm: Caenorhabditis elegans; Xenopus: Xenopus tropicalis; The 589 secondary structures are shown on top of the sequence in light-green, and the βL4-βL5 is shown in red 590 dash line. F. Test the effect of Arg147 of SMCR8 on the stimulation of Rab8a and Rab11a. The 591 experiment was carried out as panel (A), and the labels are same to the panel (A).C∆S: C9ORF72∆C- 592 SMCR8, CSLongin: C9ORF72-SMCR8Longin, CSR147A: C9ORF72-SMCR8R147A, C∆SR147A: C9ORF72∆C- 593 SMCR8R147A, CSLongin+R147A: C9ORF72-SMCR8Longin+R147A, SLongin: SMCR8Longin. The error bars represent 594 mean ± SD (N=3). (G, H) Measurement of GAP activity to Rab8a (G) and Rab11a (H) using different 595 concentrations of C9ORF72-SMCR8 or C9ORF72-SMCR8Arg147. The final concentration of Rabs in this 596 assay was 2 µM. The concentrations of C9ORF72-SMCR8 or C9ORF72-SMCR8Arg147 are indicated in 597 the horizontal axis. The relative amount of non-hydrolyzed GTP in the system is shown in the vertical 598 axis. Each concentration of GAP was measured in triplicate. The value of each measurement is shown 599 in as "○" (C9ORF72-SMCR8) and "▽" (C9ORF72-SMCR8Arg147) since the error bars of several 600 measurements are too small to be shown clearly in the figure. The curve was fitted to using the 601 Stimulation Model in Graphpad. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Materials and methods Cloning and protein purification The DNAs of human C9ORF72 and SMCR8 were subcloned into pFastBac dual under control of the polyhedron promoter and the p10 promoter, respectively. The DNA of WDR41 was subcloned into pFastBac dual under control of the polyhedron promoter with a N-terminal His8-tag that can be removed by Tobacco Etch Virus (TEV) protease. Both C9ORF72 and SMCR8 were expressed with N-terminal His8-tag that can be removed by TEV. The baculoviruses were generated in Sf9 cells with the bac-to-bac system (Life Technologies). The cells were infected and harvested after 60 hours. Cells were pelleted by centrifugation at 2000 x g for 15 minutes. The pellets were lysed in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM TCEP-HCl, 1 mM PMSF, 0.8 µM Aprotinin, 1 µM Pepstatin, and 10 µM Leupeptin by French Press. The lysate was then centrifuged at 25,000 x g for 30 min at 4 °C. The supernatants of C9ORF72-SMCR8 complex and WDR41 were loaded onto Ni-NTA resin at 4 °C, respectively. C9ORF72-SMCR8 complex and WDR41 were eluted with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM TCEP-HCl, and 250 mM Imidazole pH 8.0, respectively. The eluate of target proteins was diluted 5-fold with 25 mM Tris-HCl pH 8.0, 2 mM DTT and applied to a Hi-Trap Q HP column. Peak fractions were pooled and digested with TEV protease at 4 °C overnight. TEV and His-tag were removed by loading the solution onto Ni-NTA resin. Target proteins were further purified on a Superose 6 10/300 GL column equilibrated with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, and 2 mM DTT. The peak fractions were pooled and flash-frozen in liquid N2 for storage.

To reconstitute the CSW complex, purified C9ORF72-SMCR8 complex and WDR41 were mixed at a molar ratio of 1:2 and incubated at 4 °C for 45 minutes. Then the mixture was applied to Superose 6 10/300 column equilibrated with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, and 2 mM DTT (or 0.5mM TCEP). Peak fractions were identified using SDS-PAGE and flash-frozen in liquid N2 for storage.

Longin SMCR8 was expressed in E. coli BL21 (DE3) cells with N-terminal His6-tag that can be removed by TEV. After induction with 0.2 mM IPTG overnight at 16 °C, the bacteria were pelleted by centrifugation at 4000 x g for 10 minutes. Target proteins were purified as described above.

C9ORF72 and the C9ORF72-SMCR8Longin complex was expressed in Sf9 cells and purified as the C9ORF72-SMCR8 complex.

The primers for point mutations were designed facilitated by a python script(1). All the mutant constructs were verified by DNA sequencing, and all the mutants were purified as the wild type proteins.

Analytical ultracentrifugation Analytical Ultracentrifugation Sedimentation experiments were carried out at 20 °C in an XL-I analytical ultracentrifuge (Beckman-Coulter) equipped with Rayleigh Interference detection (655 nm). BTN3A1 B30.2 (400 mL, 27 mM, 54 mM, 160 mM, 240 mM respectively) were centrifuged at 50,000 or 30,000 rpm for 8 hours, in an An50Ti rotorusing12 mm double-sector aluminum center pieces. All samples were prepared in the interaction buffer (25 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP). Interference profiles were recorded every 6 min. Data analysis was conducted with the software Sedfit 11.

EM sample preparation and data collection The purified CSW complex was concentrated to approximately 1 mg/mL. Aliquots (4 μL) of the protein complex were placed on glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh). Then, the grids were blotted for 3.0 s or 3.5 s with 100% humidity at 4 °C, and flash-frozen in liquid ethane cooled by liquid nitrogen with Vitrobot (Mark IV, Thermo Fisher Scientific).

Micrographs were collected using a Gatan K2 Summit detector (Gatan Company) mounted on a Titan Krios electron microscope (FEI Company) operating at 300-kV and equipped with a GIF Quantum energy filter (slit width 20 eV). Micrographs were recorded in the super-resolution mode with a normal magnification of 130,000x, resulting in a calibrated pixel size of 0.53 Å. Each stack of 32 frames was exposed for 8 seconds, with an exposing time of 0.25 second per frame. The

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total dose rate was about 5.3 counts/sec/physical-pixel (~4.7 e-/sec/Å2) for each stack. AutoEMation was used for the fully automated data collection(2). All 32 frames in each stack were aligned and summed using the whole-image motion correction program MotionCor2 (3) and binned to a pixel size of 1.06 Å. The defocus value of each image was set from -1.5 to -2.0 μm and was determined by Gctf (4).

Cryo-EM Image processing and calculation 1,338,452 particles were automatically picked by Gautomatch (developed by Kai Zhang, http:/www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/) from 5,332 micrographs. Two rounds of 2D classification was performed using RELION 3.0(5, 6), resulting 923,886 particles. A small subset of those selected particles was used to generate the initial model of CSW complex with C2 symmetry by RELION 3.0. Two rounds of guided multi- reference classification procedure were applied to these selected particles with C2 symmetry using the program RELION 3.0 (SI Appendix, Fig. S2). Details of this modified procedure were previously described in the manuscript reporting the cryo-EM structure of the human spliceosomal C* complex(7). The 3D volumes of the CSW complex and five bad classes were used as initial references (SI Appendix, Fig. S2). These six references were low-pass filtered to 40 Å. After one round of global classification and one round local classification, 347,925 particles that belong CSW complex were separated from the bad ones. With the coordinates of these 347,925 good particles as additional input for Gautomatch, one round of exclusive particle picking was performed. With this strategy, 1,313,534 particles with different coordinates compared with the 347,925 good particles were picked. After the same procedure, these 1,313,534 particles contribute 259,603 additional particles, all together, 607,528 particles were generated for further calculation. After additional round of multi-reference classification on the same 607,528 particles with pixel size: 2.12 Å, 397,055 particles were classified out and these particles were re-centered and re-extract with pixel size 1.06 Å (SI Appendix, Fig. S2).

For these 397,055 particles with pixel size 1.06 Å, one round multi-reference local 3D classification with C2 symmetry was performed using two references low-pass filtered to different resolution (SI Appendix, Fig. S2). One class with 212,774 particles was generated. The C2 symmetry of the CSW complex is not perfect, these particles were expanded to 425,548 particles by applying a C2 operator in RELION, and further focused on one protomer of the CSW complex with C1 symmetry. With additional round of 3D classification and auto-refinement by applying soft mask, the focused protomer of the CSW complex yielded a reconstruction at an average resolution of 3.2 Å. The WDR41 and C9ORF72 region of this protomer were also further calculated to a relatively higher resolution by applying soft mask for these regions, the resolution was improved to 3.4 and 3.3 Å, respectively (SI Appendix, Fig. S2, S3A-B; Table S1).

The angular distributions of the particles used for the final reconstruction of the CSW complex are reasonable (SI Appendix, Fig. S3C), and the refinement of the atomic coordinates did not suffer from severe over-fitting (SI Appendix, Fig. S3D). The resulting density maps display clear features for the secondary structural elements and side chains of the CSW complex in the core region.

Reported resolutions were calculated on the basis of the FSC 0.143 criterion, and the FSC curves were corrected with high-resolution noise substitution methods(8). Prior to visualization, all density maps were corrected for the modulation transfer function (MTF) of the detector, and then sharpened by applying a negative B-factor that was estimated using automated procedures(9). Local resolution variations were estimated using ResMap(10).

Model Building and refinement The atomic coordinates of the CSW complex was generated by combining homology modelling and de novo model building. The structure of Longin and DENN domains of C9ORF72 and SMCR8 were generated according to the structure of DENND1b(11). For WDR41, the atomic coordinates of crystal structure of WD40 protein FBXW7 (PDB code: 5V4B(12)) was served as the initial template. These structures were docked into the density map and manually adjusted and built by by COOT(13). Sequence assignment was guided mainly by

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bulky residues such as Phe, Tyr, Trp and Arg. Unique patterns of sequences were exploited for validation of residue assignment.

The final overall models of the CSW complex were refined against the 3.2-Å map, using PHENIX(14) in real-space. Overfitting of the model of the CSW complex was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach, and testing the refined model against the other map (15) (SI Appendix, Fig. S3D). The structures of the human CSW complex were validated through examination of the Molprobity scores and statistics of the Ramachandran plots (SI Appendix, Table S1). Molprobity scores were calculated as described (16) .

Pulldown assay For the pull-down assay, indicated versions of His8-tagged SMCR8-C9ORF72 (12.5 μM) and indicated versions of untagged-WDR41(20 μM) were incubated with Ni-NTA agarose beads in incubation buffer (150mM NaCl, 25mM Tris pH 8.0, 0.05% NP40, and 5% glycerol) at 4 ℃ for 45 min. After rinsed with incubation buffer for three times, the resin was resuspended in 1x SDS loading buffer and boiled at 95 ℃ for 5 mins before applied to SDS- PAGE analysis.

GTPase activity assay GTPase activity assays were carried out using GTPase-Glo assay kit (Promega, V7681). Briefly, preparing 2X GTPase solution containing 1mM DTT and Rabs in buffer A (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 20 mM EDTA, and 5 mM MgCl2). The concentration of Rab in this solution is 2-fold of the final concentration. Then, diluting GAP to desired concentration in buffer A containing 10 μM GTP, and dispense 5 μl into each well of a 384-well plate (Corning, 3570). To initiate the GTPase reaction, 5 μl 2X GTPase solution was added into each well. The total GTPase reaction volume is 10 μl. The plate was incubated at 25 °C for 120 min. Then, 10 μl of reconstituted GTPase-Glo reagent was added to the wells to terminate the GTP hydrolysis reactions and the plate was incubated at 25°C for 30 min with shaking. Then, 20 μl of the Detection Reagent (from the kit) was added to all the wells. The luminescence signal was measured using BioTek Synergy2. Noteworthily, the higher the luminescence is, the less the GTP is consumed by GTPases.

Nucleotide exchange assay Purified Rabs were loaded with MANT-GDP (Thermo Fisher, M12414) in the presence of 10 mM EDTA pH 8.0 and 2-fold molar excess of MANT–GDP at 4 °C overnight. Loading reaction was terminated by adding MgCl2 to 50 mM and the Rab8a–MANT– GDP complex was further purified using Superdex75 10/300 (GE Healthcare) in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2 and 2 mM DTT. For the nucleotide exchange assay, 1.6 μM Rab GTPase-MANT-GDP complex were pre-incubated with 0 and 300 nM of indicated C9ORF72- SMCR8 subcomplex for 30 minutes. After baseline stabilization, the nucleotide exchange reaction was triggered by the addition of GDP to a final concentration of 0.1 mM. Releasing of MANT-GDP was recorded by monitoring the decrease in fluorescence emission at 448 nm (excited at 360 nm) in intervals of 15 s at 25 °C for 3600-second. Data were collected using 384-well plate (Corning, 3701) in BioTek Synergy2.

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A B

-SMCR8-WDR41 -SMCR8 mAU ∆C ∆C CSW 100 C9ORF72∆C-SMCR8-WDR41

C9ORF72-SMCR8 CSW C9ORF72C9ORF72-SMCR8C9ORF72 80 C9ORF72∆C-SMCR8 115 kD SMCR8 60 UV280 70 kD C9ORF72 40 55 kD C9ORF72∆C 44 kD 20 WDR41 34 kD

0 28 kD

17 kD 0 5 10 15 20 25 ml

Fig. S1. Gel filtration profiles of different complexes.

A. The elution profiles of different complexes in Superose 6 10/300 GL column are shown here. Red: CSW, blue: C9ORF72∆C-SMCR8-WDR41, orange: C9ORF72 -SMCR8, cyan: C9ORF72∆C- SMCR8. C9ORF72∆C-SMCR8-WDR41 was eluted earlier than C9ORF72 -SMCR8 due to C9ORF72∆C-SMCR8-WDR41 is more elongated than C9ORF72 -SMCR8. B. The peak fractions from gel filtrations are visualized with the Coomassie blue-stained SDS-PAGE.

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ARaw cryo-EM image B Selected 2D averages

Initial 3D model

20 nm Top view Side view C 1,338,452 particles auto-picked by Gautomatch 1,313,534 particles exclusive-picked by Gautomatch ( from 5,332 micrographs ) ( from 5,332 micrographs )

Pixel size: 4.24 Å 2 rounds of 2D classification 2 rounds of 2D classification 923,886 particles 755,898 particles

2 rounds of multi-reference classification 2 rounds of multi-reference classification Supplied as input of Gautomatch 347,925 particles 259,603 particles

Pixel size: 2.12 Å 1 rounds of multi-reference classification

38.0% 12.1% 4.2% 27.4% 14.3% 3.9% of input particles

Pixel size: 1.06 Å Re-extract and recenter the good particles, pixel size 1.06 Å

397,055 particles

Multi-reference classification Particle C2 symmetry expand by RELION 425,548 particles 212,774 particles Local 3D classification 46.4 % 53.6 % and autorefinement Autorefinement C2 symmetry

Local 3D classification Autorefinement and autorefinement 3.3 Å 4.0 Å 3.4 Å 3.2 Å 212,774 particles 347,925 particles 347,925 particles 194,157 particles

Fig. S2. A flow chart for the cryo-EM data processing and structure determination of the human C9ORF72-SMCR8-WDR41 complex.

A. A representative cryo-EM micrograph of the final CSW complex. Scale bar, 20 nm. B. Selected 2D classification averages and an initial model. C. Based on the FSC value of 0.143, the final reconstruction has an average resolution of 3.2 Å for one protomer of the CSW complex. Please refer to the Method for the detailed description.

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A 1 B Dimer One protomer n

o 0.8 i t a l CSW complex 4Å e r r 0.6 (dimer ) o C

l CSW complex 3.2 Å l

e 0.4 (one protomer) h S C9ORF72 region 3.3Å r

e C9ORF72 i

r 0.2 WDR41 region 3.4 Å u

o

F FSC=0.143 WDR41 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 3.0 3.3 3.6 3.9 C Resolution (1/Å) 3.8 4.5 5.0 D 1 Angular distribution FSC between model and cryo-EM maps

0.8 n o i t 0.6 a l 3.8 Å e r r

o FSC=0.5 C

0.4 l l e h S

r 0.2 e i r u o F 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Resolution (1/Å)

Fig. S3. Cryo-EM analysis of the human C9ORF72-SMCR8-WDR41 complex.

A. The average resolutions of the map for CSW complex are estimated to be 3.2 Å for one protomer based on the FSC criterion of 0.143. The average resolutions for WDR41 region and C9ORF72 region are estimated to be 3.4 and 3.3 Å, respectively. B. The local resolutions are color-coded for different regions of the CSW complex, including two sub-regions of WDR41 and C9ORF72. C. Angular distribution of the particles used for the reconstruction of the human CSW complex. Each cylinder represents one view, and the height of the cylinder is proportional to the number of particles for that view. D. The FSC curves of the final refined model of the CSW protomer versus the overall map that it was refined against (black); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (green). The generally similar appearances between the red and green curves indicate that the refinement of the atomic coordinates did not suffer from severe over-fitting. 3.8 Å indicates the resolution of the black curve at FSC=0.5.

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A B

WDR41 C9ORF72-Longin SMCR8-DENN SMCR8 αD12 SMCR8-Longin αD11 αD13 SMCR8-DENN αL6 SMCR8-Longin αL2 αL5 C9ORF72-DENN αD10 βD6 βL3 αD8 C9ORF72-DENN βL4 βD7 SMCR8-DENN βD4 WDR41 βL5 βD1 βL1 βD5 αL4 αD7 βD2 βD3 αD9 SMCR8-Longin βL2 αL3

C9ORF72-Longin

C D αD11 C9ORF72-DENN βD Blade4 αD6 βC αD10 αD12 Blade5 βD βD βC Blade3 βB αD5 βD5 αD13 βB βC Blade6 βA βA βB αD7 βD βC βA βB βD1 βB βA βD αD8 αD14 βC βA βD4 βA βA βD3 Blade2 βC βB βD2 βB βD βB βC αL2 αD9 Blade7 βC Blade1 βD

βD αL3 Blade8 βL4 WDR41 βL6

βL5 WDR41CTH βL2 αL1 αL4 βL1 βL3 C9ORF72-Longin C9ORF72

Figure S4. The architecture of each subunit of CSW.

A. The overall structure of CSW. B. Structure of SMCR8. C. Structure of C9ORF72. d. Structure of WDR41. The secondary structures are labeled. The blue and red dots indicated the N-termini and C-termini of proteins.

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Longin

A 440 158 75 44 29 13.7 B mAU C9ORF72-SMCR8Longin 800 C9ORF72

C9ORF72-SMCR8C9ORF72 600 115 kD

70 kD UV280 C9ORF72 400 55 kD 44 kD SMCR8Longin 34 kD 200 28 kD

0 17 kD ml 0 5 10 15 20 25 Elution Volume

Figure S5. Gel filtration profiles of C9ORF72 and C9ORF72-SMCRLongin.

A. The elution profiles of different complexes in the Superdex 200 10/300 GL column are shown here. Red: C9ORF72-SMCRLongin; blue: C9ORF72. The molecular weight standers are indicated on top. B. The peak fractions from panel (A) were visualized with the Coomassie blue-stained SDS- PAGE.

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A FLCN-DENN FLCN-DENN SMCR8-DENN SMCR8-DENN FLCN-DENN SMCR8-DENN 90° 90°

C9ORF72-DENN FNIP2-DENN C9ORF72-DENN SMCR8-Longin FNIP2-DENN SMCR8-Longin

40 Å SMCR8-Longin FNIP2-Longin FNIP2-Longin C9ORF72-Longin C9ORF72-Longin 40 Å

FNIP2-Longin C9ORF72-Longin FLCN-Longin FLCN-Longin FLCN-Longin FNIP2-Longin FLCN-Longin B FLCN-Longin FNIP2-Longin FLCN-Longin FNIP2-Longin 90° 90°

C9ORF72-Longin

SMCR8-Longin SMCR8-Longin SMCR8-Longin C9ORF72-Longin C9ORF72-Longin αD10 αD10 C FLCN-DENN αD11 FNIP2-DENN FNIP2-DENN FLCN-DENN FNIP2-DENN αD6

FLCN-DENN 90° 90°

αD11 αD6 αD10 SMCR8-DENN αD10 C9ORF72-DENN αD11 C9ORF72-DENN SMCR8-DENN αD6 C9ORF72-DENN SMCR8-DENN αD10 αD10

Figure S6. The architecture of C9ORF72 -SMCR8 complex protomer.

A. Different views of the superimposition of the C9ORF72-SMCR8 complex protomer and the FLCN-FNIP2 complex. In the middle and right panels, the red arrow indicates the distance between SMCR8Longin and FLCNLongin when the DENN dimers of two complexes are overlapped. (B, C) Comparison of Longin dimers (B) and DENN dimers (C) of C9ORF72-SMCR8 complex with those of FLCN-FNIP2 dimer. Three views are presented here. The helixes mediated the formation of DENN dimer are labeled in panel (C).

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+ - - - + C9ORF72-SMCR8 A - + - - - C9ORF72-S8Longin

Longin Longin - - + - - C9ORF72 - - - + - SMCR8Longin Longin ∆C Longin ∆C + + + + - WDR41 WDR41∆C Marker - - - - + MarkerC9ORF72-SMCR8C9ORF72-S8C9ORF72SMCR8WDR41WDR41 MarkerC9ORF72-SMCR8C9ORF72-S8C9ORF72SMCR8WDR41WDR41 115 kD His8-SMCR8 His -C9ORF72 70 kD 8 55 kD His -S8-Longin 44 kD 8 34 kD 28 kD WDR41 17 kD input Protein+Resin His-pulldown B

MarkerC9ORF72-SMCR8WDR41-L445R-F446R-L449RWDR41-S438AWDR41-R441AWDR41-S442AWDR41-L445RWDR41-F446RWDR41-L449RMarkerWDR41-S438AWDR41-R441AWDR41-S442AWDR41-L445RWDR41-F446RWDR41-L449RMarkerWT-WDR41WDR41-L445R-F446R-L449RWDR41-S438AWDR41-R441AWDR41-S442AWDR41-L445RWDR41-F446RWDR41-L449R 115 kD His -SMCR8 70 kD 8 55 kD 44 kD His8-C9ORF72 34 kD 28 kD WDR41 17 kD input WDR41-mutants+Resin His-pulldown C

MarkerC9ORF72-SMCR8WT-WDR41WDR41-mutantWDR41-S438A-R441A-S442AWDR41-L445R-F446R-L449R WDR41-L445R-F446RWDR41-L445R-L449RWDR41-F446R-L449RC9ORF72-SMCR8WT-WDR41WDR41-mutantWDR41-S438A-R441A-S442AWDR41-L445R-F446R-R449R WDR41-L445R-F446RWDR41-L445R-L449RWDR41-F446R-L449RMarkerWT-WDR41WDR41-mutantWDR41-S438A-R441A-S442AWDR41-L445R-F446R-L449RWDR41-L445R-F446RWDR41-L445R-L449RWDR41-F446R-L449R 115 kD His -SMCR8 70 kD 8 55 kD His -C9ORF72 44 kD 8 34 kD 28 kD WDR41 17 kD

input Protein+Resin His-pulldown D

Marker C9ORF72-SMCR8C9ORF72-SMCR8-M1C9ORF72-SMCR8-M2C9ORF72-SMCR8-M3 WT-WDR41MarkerC9ORF72-SMCR8 MarkerWT-SMCR8 C9ORF72-SMCR8-M1C9ORF72-SMCR8-M2C9ORF72-SMCR8-M3WT-WDR41 SMCR8-M1SMCR8-M2SMCR8-M3 E 115 kD His8-SMCR8 70 kD 55 kD His8-C9ORF72 44 kD 34 kD WDR41 28 kD

17 kD

input Protein+Resin His-pulldown MarkerC9ORF72-SMCR8C9ORF72-SMCR8∆CWT-WDR41C9ORF72-SMCR8 C9ORF72-SMCR8∆CWT-WDR41C9ORF72-SMCR8 C9ORF72-SMCR8∆C

115 kD His8-SMCR8 70 kD 55 kD His8-C9ORF72 44 kD 34 kD 28 kD WDR41 17 kD input Protein His +Resin pulldown

Figure S7. Contributions of key residues to the interface of SMCR8 and WDR41.

A. Full profile of pulldown assay of Fig. 4B. The labels are same to Figure 4B. Protein markers are labeled at left (unit: kD). B. Effect of single mutations of WDR41 on the bind to SMCR8 was assessed by pull down assay. The mutated residues are labeled. C. Full profile of pulldown assay of Fig. 4C. The labels are same to Figure 4C. D. Full profile of pulldown assay of Fig. 4D. The labels are same to Figure 4D. E. Effect of βD6 of SMCR8 on the binding to WDR41was assessed by pull- down assay. The labels are same to Figure 4. C9ORF72-SMCR8∆C: C9ORF72-SMCR8 (SMCR8 with 923-937 deleted). WT-WDR41: wild-type WDR41. 11 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

αD12 αD13 WDR41-Blade1 A R437 B C Y55

R440 F887

W69 F883

R444 H904 F446 SMCR8-DENN WDR41CTH Y913 H912

F49 K449 R48

D F85 E F Y126SMCR8-Longin C9ORF72-Longin C9ORF72-Longin SMCR8-Longin beta-sheet F123 beta-sheet R346 F345 M189 Y124 R129 F176 W45 F84 M184 Y157 αL3 R134 F30 F124 αL4 F117 αL4 Y110 R140 F108 F331 G F188 R143 SMCR8-DENN Y330 W767 W145 H147 beta-sheet F191 αL3

R787 I SMCR8-DENN J C9ORF72-DENN αD11 αD11 H766 Y843 Y844

Y415 H C9ORF72-DENN beta-sheet

F859 F401 Y861 F863 F397

H867 H865 F394 H866 H307

Figure S8. EM map for the indicated region.

The core regions of the model fit well with the EM density. (A-J) Different regions of the CSW protomer are shown as indicated. The bulky residues are labeled.

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βL1 βL2 βL3 C9ORF72_HUMAN MSTLCPPPSPAVAKTEIALSGKSPLLAATFAYWDNILGPRVRHIWAPKTEQV-LLSDGEI 59 C9ORF72_MOUSE MSTICPPPSPAVAKTEIALSGESPLLAATFAYWDNILGPRVRHIWAPKTDQV-LLSDGEI 59 C9ORF72_Zebrafish MSSACPPQSPAVAKTEVLVDDCCPVLAATFAYWDNILGPRVRHIWAPKSQGLLLLSDGEV 60 C9ORF72_Xenopus ------0

αL1 βL4 βL5 βL6 C9ORF72_HUMAN TFLANHTLNGEILRNAESGAIDVKFFVLSEKGVIIVSLIFDGNWNGDRSTYGLSIILPQT 119 C9ORF72_MOUSE TFLANHTLNGEILRNAESGAIDVKFFVLSEKGVIIVSLIFDGNWNGDRSTYGLSIILPQT 119 C9ORF72_Zebrafish TFLANHTLNGEILRSAESGAVDVKFFVLAEKGVIIVSLIFDGELKGDKNTCALSIILPQS 120 C9ORF72_Xenopus ------0

αL2 αL3 αL4 C9ORF72_HUMAN ELSFYLPLHRVCVDRLTHIIRKGRIWMHKERQENVQKIILEGTERMEDQGQSIIPMLTGE 179 C9ORF72_MOUSE ELSFYLPLHRVCVDRLTHIIRKGRIWMHKERQENVQKIVLEGTERMEDQGQSIIPMLTGE 179 C9ORF72_Zebrafish ELSFYLPLHAVCVERLKHVIRKGRICMQK------GYNIISMLSSE 160 C9ORF72_Xenopus ------MLTGE 5

αD5 βD1 C9ORF72_HUMAN VIPVMELLSSMKSHSVPEEIDIADTVLNDDDIGDSCHEGFLLNAISSHLQTCGCSVVVGS 239 C9ORF72_MOUSE VIPVMELLASMKSHSVPEDIDIADTVLNDDDIGDSCHEGFLLNAISSHLQTCGCSVVVGS 239 C9ORF72_Zebrafish IVPIMELLTSMKKHSVPEEVDLKDTVLNDDDIGDSCHEDFLHKAISSHLQTCGCSMVVGS 220 C9ORF72_Xenopus VIPIMELLSSIKSHGVPEEIDITSTVLNDDDIGDSCHGDFLINTICNLLKNVVCRMVVSE 65

αD6 αD7 βD2 βD3 αD8 C9ORF72_HUMAN SAEKVNKIVRTLCLFLTPAERKCSRLCEAESSFKYESGLFVQGLLKDSTGSFVLPFRQVM 299 C9ORF72_MOUSE SAEKVNKIVRTLCLFLTPAERKCSRLCEAESSFKYESGLFVQGLLKDATGSFVLPFRQVM 299 C9ORF72_Zebrafish NPEKVNKIVLTLCLFLTPAERKCSRLCHPDGSFKYDTGLFVQGLLKDSTGSFVFPYRQVL 280 C9ORF72_Xenopus DCSQFSNMVRTLCLFLSPTERKCSRLCRSESSFQYESGLFVQGLLKDATGSFVLPFRQVM 125

βD4 βD5 αD9 C9ORF72_HUMAN YAPYPTTHIDVDVNTVKQMPPCHEHIYNQRRYMRSELTAFWRATSEEDMAQDTIIYTDES 359 C9ORF72_MOUSE YAPYPTTHIDVDVNTVKQMPPCHEHIYNQRRYMRSELTAFWRATSEEDMAQDTIIYTDES 359 C9ORF72_Zebrafish YSPYPTTHIDVDINTVKQMPPCHEHTYHQRRYMRAELSALWKAASEDDFSSDNLINAQDS 340 C9ORF72_Xenopus YAPYPTTHIDVDIFTVKQMPPCHEHTYNQRRYMRAELGALWKATSDEEIGMDSVIHSDET 185

αD10 αD11 C9ORF72_HUMAN FTPDLNIFQDVLHRDTLVKAFLDQVFQLKPGLSLRSTFLAQFLLVLHRKALTLIKYIEDD 419 C9ORF72_MOUSE FTPDLNIFQDVLHRDTLVKAFLDQVFHLKPGLSLRSTFLAQFLLILHRKALTLIKYIEDD 419 C9ORF72_Zebrafish YTPDLNIFQDVMHKDTLVKSFIDEVFLLKPGLSLRSVYLSHFLLLLHRKALTLLRYIEDE 400 C9ORF72_Xenopus FTPNLNLFLSNFHV---FFFFTFQVFQLKPNLSLRSTFLAQFLLVLHRKALTLIKYIEDD 242

αD12 αD13 αD14 C9ORF72_HUMAN TQKGKKPFKSLRNLKIDLDLTAEGDLNIIMALAEKIKPGLHSFIFGRPFYTSVQERDVLM 479 C9ORF72_MOUSE TQKGKKPFKSLRNLKIDLDLTAEGDLNIIMALAEKIKPGLHSFIFGRPFYTSVQERDVLM 479 C9ORF72_Zebrafish TQKGKKPFRSLRNLKTDLDLTVEGDLNIIMAMAEKLRAGLHSFVFGKSFLTSVQERDLLI 460 C9ORF72_Xenopus TQKGKKPFKSLRSLKTDLDLAAEGDLYIIMALAEKIKPGLHSFIFGSSFHTSMQERDVLM 302

C9ORF72_HUMAN TF 481 C9ORF72_MOUSE TF 481 C9ORF72_Zebrafish NF 462 C9ORF72_Xenopus TF 304

Figure S9. Sequence alignment of C9ORF72

HUMAN: Homo sapiens; Mouse: Mus musculus; Zebrafish: Danio rerio; Xenopus: Xenopus tropicalis; The deleted residues are indicated as red. The secondary structures are labeled on top of the sequences.

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SMCR8_HUMAN ------MISAPDVVAFTKEEE---YEEEPYNEPALPEEYSVPLFPFA 38 SMCR8_MOUSE ------MISAPDVVAFTKEDE---YEEEPYNEPALPEEYSVPLFPYA 38 SMCR8B_Zebrafish ------MIGSPDLVAFTKETD---FSEITTDSSVLPEDLSVPMYPYT 38 SMCR8_Worm MQAVIALCHFCENHGPRVVMTCQPMRDVDDKGASCTASTSLGPSTSSSPPGIIVPLIKFD 60 SMCR8_XENLA ------MIGSPDLMPFTMEEE---YEDPFQGSSGLPEEYSVPLFPFS 38

SMCR8_HUMAN SQGA------42 SMCR8_MOUSE SQGA------42 SMCR8B_Zebrafish G-DA------41 SMCR8_Worm GECVVKEKTNTEDGTSYPHYGNCTKFTIDTEDRCSACSSFRNGPCLLSNDHQTKTSYISS 120 SMCR8_XENLA QANR------42

βL1 βL2 SMCR8_HUMAN ------NPWSKLSGAKFSRDFILISEFS----EQVGPQPLLTIPNDTKVFGTFD-----L 87 SMCR8_MOUSE ------NPWSKLSGAKFSRDFILISEFS----EQVGPQPLLTIPNDTKVFGTFD-----L 87 SMCR8B_Zebrafish ------TPWSKISSAKLKKDFILISEFS----EQVGPQPLLTVPLETKACGTFD-----L 86 SMCR8_Worm EVALQDRVYERVKNACLRSLSCEVSAASKKNPVQPPPPPLSR-STTPRKIHPYVIHEEPL 179 SMCR8_XENLA ------SPWSRLSAAKFSRDFILISEFS----EQVGPQPLLTIPDDQRICGDFD-----L 87

αL1 βL3 βL4 SMCR8_HUMAN NYFS-LRIMSVDYQASFVGHPPGSAYPKL--NFVEDSKVVLGDSKEGAFAYVHHLTLYDL 144 SMCR8_MOUSE NYFS-LRIMSVDYQASFVGHPPGSAYPKL--NFVEDSKVVLGDSKEGAFAYVHHLTLYDL 144 SMCR8B_Zebrafish NYFS-LRIMSVDYQTSLAGSP-GYGSFKL--NFVEDSKVVLADSREGVFAYVHHLTLYDL 142 SMCR8_Worm HDFSGTRIP---PSSTVLQEPIEDGAFEQPAAEEADGYVIFGDTDN-GFCFAYIFRLADA 235 SMCR8_XENLA NYFS-LRIMSVDYQASFVGHPPGSSYPKL--NFVEDSKVVLGDSKEGAFAYVHHLTLYDL 144

βL5 αL2 αL3 SMCR8_HUMAN EARGFVRPFCMAYISADQHKIMQQFQELSAEFSRASECLKTGNRKAFAGELEKK--LKDL 202 SMCR8_MOUSE EARGFVRPFCMAYISADQHKIMQQFQELSAEFSKASECLKMGNRKAFAGELEKK--LKDL 202 SMCR8B_Zebrafish EARGFVRPLCLAYVSSDENKIIQQFQRISTEFNKVSECLKTGNRKNFANELEVK--LRDL 200 SMCR8_Worm KARGFYRLFSLIVVSNDLTFLTNNFEYFKTTLGGIKNDLQKLAQDVFSREIDTGEKLSED 295 SMCR8_XENLA EARGFVRPFCMAYISMDEEKIMRQFMELSSDFSKASECLKTGNRKAFANELEKK--LRDL 202

SMCR8_HUMAN DYTRTVLHT------ETEIQKK------218 SMCR8_MOUSE DYTRTVLHT------ETEIQKK------218 SMCR8B_Zebrafish EYTRVVLQK------ELNTVSVKCS------SEREPILNGVH 230 SMCR8_Worm DIHKPEFQKLVGKMPSWYRRKIAIDTDRNLTVVTANNDIWVQLHRHMMWTLRSPTLACLD 355 SMCR8_XENLA DYTRTVLQT------QMEEQMK------218

SMCR8_HUMAN ------218 SMCR8_MOUSE ------218 SMCR8B_Zebrafish SFERNADEVKLNEKSSH---TDEISPQEKDGCGNSRKVEVKLENENRSHFEHEQYGKQRK 287 SMCR8_Worm QV------MEGKPTQDMMVSALGTGGIPNCGTRTEASKPT-PEN------L 393 SMCR8_XENLA ------218

SMCR8_HUMAN -ANDKGFYSSQAIEKANELASV------EKSIIEHQDLLKQIRSYPHRK-LKGHDLCP 268 SMCR8_MOUSE -ANDKGFYSSQAIEKANELANV------EKSIIEHQDLLRQIRSYPRQK-TKIPDLQP 268 SMCR8B_Zebrafish DKPDKTSCPMPLANKNDELASV------EKLIQDYKSLLKQVTCYPTRK-LRDSEYSP 338 SMCR8_Worm HAVDRFNIPVQYQLKSMCIASVSSSVAIQNEKILREEMPLIIEIQNIPPSFMMRVHEKN- 452 SMCR8_XENLA -ADDKGFYTTQTIDKANELANV------EKSIIEHQDLLKQIQLYPYRK-IKETDFQP 268

SMCR8_HUMAN GEMEHIQDQAS-----QASTTSNPDES---ADTDLYTCRP----AYTPKLIKAKSTKCFD 316 SMCR8_MOUSE GDTEHTQDQAD-----QVSTTSNPEES---ANADLYTCRP----AYTPKLIKAKSTKCFD 316 SMCR8B_Zebrafish YEPDDLPQSFDLDLDSQ------FAGPMLECSVFTYTNTP----SQTLQQINSTSSSRFD 388 SMCR8_Worm ------ESALATEPDDFEG----CENETIKGKVSMAQLANLKIIAT------488 SMCR8_XENLA YEPETLQDSDVPNESEQPLAISDPEESLVEEENSGYLPRS----SYTPKFIKAKSSKCFD 324

αL4αL4 αL5αL5 αL6 SMCR8_HUMAN KKLKTLEELCDTEYFTQTLAQLSHIEHMFRGDLCYLLTSQIDRALLKQQHITNFLFEDFV 376 SMCR8_MOUSE KKLKTLEELCDTEYFTQTLAQLSHIEHMFRGDLCYLLTSQIDRVLRKQQPITNFLFEDFV 376 SMCR8B_Zebrafish KRLKTLEELCDDYFYQQALQQLYSIERTFRGDACYLYTQQLCRNLLRNLKSTNFLFEDPC 448 SMCR8_Worm -QLNTCSEP------GDLDLLI------503 SMCR8_XENLA KKLKTLEELVDIYFFTQTIDLLTVVEKRYRGDLSYLLMKQLEMGLLEQQMVSNFLFEDIA 384

SMCR8_HUMAN EVDDRMVEKQESIPSKPSQDRPPSSSLEECPIPKVLISVGSYKSSVESVLIKMEQELGDE 436 SMCR8_MOUSE EVDDRM-EKQENVPSQPSQDRLPPKPVEECPIPKVLISVGSYKSSVESVLIKMEQELGDE 435 SMCR8B_Zebrafish DLDDDVGLQIGQST------IQQPSFLPAPSFLSGPVSLESYASCVEMVPIKLELGGSSQ 502 SMCR8_Worm ------503 SMCR8_XENLA TWDTKQSSIHFSNTLALAQALNSPKFVEEPGNLKNSASLESYKSSVESVPIKIEQEMFEE 444

SMCR8_HUMAN EYKE------VEVTELSSFDPQENLDYLDMDMKGSISSGESI 472 SMCR8_MOUSE EYTG------VEATEARSFDPQENLDYLDMDMKGSISSGESI 471 SMCR8B_Zebrafish SQVQHSTLNTPSKDNRPQVADKSPAEVEMKGEIISAPDCQGNVESVSNLMKTSISSGDSI 562 SMCR8_Worm ------503 SMCR8_XENLA EIHQ------ETGELSVSDTLDNCEYNDLDIKGSVSSSESI 479

Figure S10-1

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

SMCR8_HUMAN EVLGTEKSTSVLSKSDSQAS---LTVPLS-----PQVVRSKAVSHRTISEDSIEVLSTCP 524 SMCR8_MOUSE EVLGTEKSASVLSKSDSQAS---LTVPLS-----PHVVRSKAVSHRTISEDSIEVLSTCP 523 SMCR8B_Zebrafish EVLGTERSFRSQGANTLVETAMHRPPPLSSATALEGLKQGRVPTRRTCSEDSIEVL--CI 620 SMCR8_Worm ------503 SMCR8_XENLA EVLRTEKSVALFTHSESQPC---L--RLS-----PQAPRRKVGSRRTISGDSIEVLSMCP 529

SMCR8_HUMAN SEALIPDDFKASYPSAINEEESYPDGNEGAIR-FQASISPPELGETEEGSIENTPSQIDS 583 SMCR8_MOUSE SEALIPDDFKASYPSAINEEEAYAD-NEGAIH-FQASAGSPEPDETQEGNLENIPSQIDS 581 SMCR8B_Zebrafish TESISPDELRASYPCAIDEESPEQETDEKNSSQYQEDNN--EKSIYVQGKI----SADHE 674 SMCR8_Worm ------503 SMCR8_XENLA SESVLPEDFRPAYQVAIHEEEPDGVDVDGEQP-CNSTIN------EENEPSANDLSGDP 581

SMCR8_HUMAN SCCIGKESDGQLVLPST-----PAHTHSDEDGVVSSPPQRHRQKDQGFRVDFSVENANPS 638 SMCR8_MOUSE SCCIGKESEGHLVPLPT-----PAYTLSDEDSVVSIPPQRYIQKDQGLHVDFGVENTDPS 636 SMCR8B_Zebrafish NACLKKLH------PSVTVTPPDCPLTLE------ETS------700 SMCR8_Worm ------503 SMCR8_XENLA ACCIGMETSGLLEQTGNVTPVPDDDDDDDNSTVVSVPPQSYRHTDHAFQVKFSVDGTEIH 641

SMCR8_HUMAN SRDNSCEGFPAYELDPSHLLASRDISKTSLDNYSDTTSYVSSVASTSSDRI-PSAYP--- 694 SMCR8_MOUSE PRDNSCEMFPAYELDPSCLLASRDVSKMSLDNYSDTTSYMGSAASTSSDRI-PSAPP--- 692 SMCR8B_Zebrafish -FQDSCQ-----ATESATMLLLDEPSRMVPDDLSDCFSYRSTTASTTSECTFPACLPKDK 754 SMCR8_Worm ------503 SMCR8_XENLA SNKVSPLGLSKPEQDSTYLMSSGESVRLINEEYSDSTSYRSSTST-CSDRT-PSPAP--C 697

αD7 αD8 βD1 αD9 SMCR8_HUMAN AGLSSDRHKKRAGQNALKFIRQYPFAHPAIYSLLSGRTLVVLGEDEAIVRKLVTALAIFV 754 SMCR8_MOUSE AGLSSERHKKRAGQNALKFIRQYPFAHPAIYSLLSGRTLVVLGEDETIVRKLVTALSIFV 752 SMCR8B_Zebrafish REGGTRRRRGRVGRAALQFMRQFPFAVHAVFSLLSGRTLVVLGSEEAAVRRLVTALSVYL 814 SMCR8_Worm ------RHIVTGGQVVVESREKEYCRQFLLAISNLL 533 SMCR8_XENLA VTGLSERHKRKAGQNALMFIRQYPFSHPAIYSLLCGRTLIVLGEEEAFVRRLVTALSIFV 757

βD2 βD3 SMCR8_HUMAN P------SYGCYAKPV--KHWASSPLHIMDFQKWKLIG------784 SMCR8_MOUSE P------NYGCYAKPV--KHWISSPLHIMDFQKWKLIG------782 SMCR8B_Zebrafish P------HLTKYKDSI--QPWTSTPLQLTDLLNWKLIG------844 SMCR8_Worm PIGCIKLATYKEQYLPEYSSRFNLIGGPHNMDIPLEVSDVMVFRVSPRDVSIVDIEQMSL 593 SMCR8_XENLA P------NHGHVAKPV--NTWMTSPLHIIDFQKWKLIG------787

βD3 αD10 βD4 βD5 SMCR8_HUMAN ------LQRVAS--PAGAGTLHALSRYSRYTSILDLDNKTLRCPLYRGTLVPRLADHRTQ 836 SMCR8_MOUSE ------LQRVAS--PANVGTLHTLSRYSRYTSILDLDSKTLRCPLYRGTLVPRLADHRTQ 834 SMCR8B_Zebrafish ------FDRMCS--FNPSSLPHCLDHYSRYLSILDVDQKTLHCPTYSGSLINLLVEPKSH 896 SMCR8_Worm VNCVIEVRRRPENDGSNGPLPRIVNRYRDLLLDSDVTDTILETA------637 SMCR8_XENLA ------LQRVGS--PSSTNILHSLNRYSRYASVLDADYKTLRCPVYKGAFLQRLADHRTQ 839

αD11 αD12 SMCR8_HUMAN IKRGSTYYLHVQSMLTQLCSKAFLYTFCHHLHLPTHDKETEELVASRQMSFLKLTLGLVN 896 SMCR8_MOUSE IKRGSTYYLHVQSMLTQLCSKAFLYTFCHHLHLPAHSEETQEAVASRQTSFLKLNLGLVN 894 SMCR8B_Zebrafish FKRGNTYFTFAQSVQSKLVTKAFLLTFSHGHPSPSRPQGSSGT-----EC---FLSELHT 948 SMCR8_Worm LRTTREGWMLK----AKI-----CYQMTKQLSHP------QVFEHITGCGV 673 SMCR8_XENLA IRRGSTYYMHVQSMLTQLSSKAFLFTFCHHVHLPIREKESEESIAQRRAAFLHQLLGLSE 899

αD13 βD6 βD7 SMCR8_HUMAN EDVRVVQYLAELL----KLHYMQESPGTSHPMLRFDYVPSFLYKI 937 SMCR8_MOUSE EDIRVVQYLAELL----KLHYMQESPGTTHPLLRFDYVPSFLYKI 935 SMCR8B_Zebrafish DDKKILRYLSELI----KLHFMEVTPNV----LLFSYTTTSIFKL 985 SMCR8_Worm EDQQVVSYWTAGLSNAYKLHVLTSIQQSQTSTAR------707 SMCR8_XENLA EDSKIVEYLSELL----KKHYLKESGTQLLPMLRFDYMSNFLYKI 940 Figure S10-2

Figure S10. Sequence alignment of SMCR8.

HUMAN: Homo sapiens; Mouse: Mus musculus; Zebrafish: Danio rerio; Worm: Caenorhabditis elegans; Xenopus: Xenopus tropicalis; The mutated residues are indicated as red. The secondary structures are labeled on top of the sequences.

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

β8D β1A β1B WDR41_HUMAN MLRWLIGGGREPQGLAEKSPLQTIGEEQTQNPYTELLVLKAHHDIVRFLVQLDDYRFASA 60 WDR41_MOUSE MLRWLIGGGREPQGLAEKAALQTIGEDQGQNPYTELLVLEAHRDIVRFLVRLDDFRFASA 60 WDR41_Zebrafish MLRWILGGR-EAQGAVEKSAALFIGEEQPKNCFTEMQVLKGHFDIVRFLVQIDDLRFASA 59 WDR41_XENTR MLRWLLGSREAHNG-VEKSAVVDIGEEQTQNPYTELSVLQGHNDIVRFLLQIDESRFASA 59

β1C β1D β2A β2B β2C WDR41_HUMAN GDDGIVVVWNAQTGEKLLELNGHTQKITAIITFPSLESCEEKNQLILTASADRTVIVWDG 120 WDR41_MOUSE GDDGIIVVWNAQTGEKLLELRGHTQKITAVIAFPPLDSCEASSQLLLTASADRTVGVWDC 120 WDR41_Zebrafish GDDGLVLIWNVETGDCLLELRGHTQQITAMTSYTFIRGGNT-HTALITASSDRTLSLWDP 118 WDR41_XENTR CDDGSVFIWDVQTGDILCELRGHTQKITAIVAWGASDILQGKTDIILTASSDKTVIAWDC 119

β2D β3A β3B β3C β3D β4A WDR41_HUMAN DTTRQVQRISCFQSTVKCLTVLQRLDVWLSGGNDLCVWNRKLDLLCKTSHLSDTGISALV 180 WDR41_MOUSE DTGRQIQRVTCFQSTVKCLTVLQRLDIWLSGGSDLGVWNRKLDLLCKTSHLSDTGISALV 180 WDR41_Zebrafish DSGNRVQNISDLQSSVKCLLVLNLLDMWLSGGNELCVWNQDFELQCKTVHHSDTGISAMV 178 WDR41_XENTR DSVQQVQKATDFHSTVKTLLVLQSLDVWLSGGNELRVWNREFNLLCETGYFVDGGISSLI 179

β4B β4C β4D β5A β5B WDR41_HUMAN EIPKNCVVAAVGKELIIFRLVAPTEGSLEWDILEVKRLLDHQDNILSLINVNDLSFVTGS 240 WDR41_MOUSE EIPGNCVAAAVGRELIIFRLVTPTEELPEWDIIEVKRLLDHQDNILSLANINDTGFVTGS 240 WDR41_Zebrafish ELPKNFIAAAMDKEIVIFKLNVSGSD---VAVMAIRHLADHQDTIHSLISINDGLFASGS 235 WDR41_XENTR ELPKNCVAAAIGKDLIIFKLSVASRESDQWDVSEVRCLTAHQDIIRSLITVNDLTFVSGS 239

β5C β5D β6A β6B WDR41_HUMAN HVGELIIWDALDWTMQAYERNFWDPSPQLDTQQEIKLCQKSNDISIHHFTCDEENVFAAV 300 WDR41_MOUSE HVGELLIWDALDWTVQACERTFWSPTAQLDAQQEIKLFQKQNDISINHFTCDEENIFAAV 300 WDR41_Zebrafish HIGELIIWDSVDWTIQGYELILWEESKGDGHSEVKVTQQKQIERSIQHMSSDGELLVAAV 295 WDR41_XENTR HAGELIVWDALDWTFKAVERNFSEILSPQDAPQEIKLLQTPEEISVQHLASDEESVFAAV 299

β6C β6D β7A β7B β7C WDR41_HUMAN GRGLYVYSLQMKRVIACQKTAHDSNVLHVARLPNRQLISCSEDGSVRIWELREKQQLAAE 360 WDR41_MOUSE GRGLYVYNLQLKRVIACQKTAHDSNILHIDKLPNRQLISCSEDGAVRMWEVREKQQLAAE 360 WDR41_Zebrafish GSGLYVYNVLTKTVVAYRKMAHDSNVLHTMI-QTDGLMSCSEDGSVRMWELQDL-PLPAE 353 WDR41_XENTR GRGLYVYNLQVKRVIAYQKRAHDSSIQHIAKLPNGQFVSCSEDGSVRIWELRSK-PLHAE 358

β7D β8A β8B WDR41_HUMAN PVPTGFFNMWG-FGRVSKQASQPVKKQQENATSCSLELIGDLIGHSSSVEMFLYFEDHGL 419 WDR41_MOUSE PVPTGFFNMWG-FGRVNKQASQPVKKQEENVTTCSLELIGDLIGHSSSVEMFLYFEDHGL 419 WDR41_Zebrafish PASSGFFGMFGTFGRSSKHAGPPAKKLPEVPDLRSLELIGDLIGHSGAVQMFVSFEQKAL 413 WDR41_XENTR SVPAGFFSMWG-FGKAAKQSSHAVKKPHDTGIVATLELIGNLIGHSGSVQMFLYFKDHGL 417

β8C α WDR41_HUMAN VTCSADHLIILWKNGERESGLRSLRLFQKLEENGDLYLAV--- 459 WDR41_MOUSE VTCSADHLIILWKNGERESGVRSLKLFQKLEENGDLYPESP-- 460 WDR41_Zebrafish VTCSTDHLLIVWKDGELQSQLRSLAVFQKLEENQQL------449 WDR41_XENTR VTCSADHLIIIWKEGNRESRLRSLMLFQKLEQNGDLNPRFSLN 460

Figure S11. Sequence alignment of WDR41.

HUMAN: Homo sapiens; Mouse: Mus musculus; Zebrafish: Danio rerio; Xenopus: Xenopus tropicalis; The mutated residues are indicated as red. The secondary structures are labeled on top of the sequences.

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S1. Cryo-EM data collection and refinement statistics.

Data collection EM equipment FEI Titan Krios Voltage (kV) 300 Detector Gatan K2 Pixel size (Å) 1.061 Electron dose (e-/Å2) 45.6 Defocus range (µm) -1.5 ~ -2.0 Reconstruction Software RELION 3.0 Number of used Particles 347,925 Accuracy of rotation (˚) 1.496 Accuracy of translation (Å) 0.708 Final Resolution (Å) 3.2 Model building Software Coot Refinement Software Phenix Map sharpening B-factor (Å2) -119.5 Average Fourier shell correlation 0.76 R-factor 0.35 Model composition Protein residues 1,133 Model Validation R.m.s. deviations Bonds length (Å) 0.010 Bonds Angle (˚) 1.060 Ramachandran plot statistics () Preferred 86.38 Allowed 13.34 Outlier 0.28 Molprobity score 1.91

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.045708; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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