bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

The in situ structure of Parkinson’s disease-linked LRRK2

Reika Watanabe1,*, Robert Buschauer1,2,*, Jan Böhning1,3,*, Martina Audagnotto1, Keren Lasker4, Tsan Wen Lu5, Daniela

Boassa6, Susan Taylor5,7, and Elizabeth Villa1,

1Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA 2Present address: Center, University of Munich, 81377, Germany 3Present address: Sir William Dunn School of Pathology, University of Oxford, OX1 2JD, UK 4Department of Developmental Biology, Stanford University, Stanford, 94305, USA 5Department of Chemistry and Biochemistry University of California San Diego, La Jolla, CA 92093, USA. 6National Center for Microscopy and Imaging Research, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA. Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA 7Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, USA *These authors contributed equally.

Mutations in leucine-rich repeat 2 (LRRK2) are the most Pases and modulates their membrane localization and func- frequent cause of familial Parkinson’s disease. LRRK2 is a tion (11). LRRK2 forms filamentous structures surrounding multi-domain containing a kinase and GTPase. Using microtubules (MTs) in cells, and most of the major muta- in situ cryo-electron tomography and subtomogram averaging, tions associated with familial PD enhance filament forma- we reveal a 14-Å structure of LRRK2 bearing a pathogenic mu- tion (12, 13). Pharmacological inhibition of LRRK2’s ki- tation that oligomerizes as a right-handed double-helix around nase also causes reversible recruitment of both wild-type and microtubules, which are left-handed. Using integrative mod- mutant LRRK2 to MTs (13, 14), emphasizing the need for eling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is detailed structural information of LRRK2 bound to MTs to exposed to the cytoplasm. We identify two oligomerization in- design effective PD therapeutics. terfaces mediated by non-catalytic domains. of one To determine the structure of MT-associated LRRK2, we of these abolishes LRRK2 microtubule-association. Our work used cryo-electron tomography (cryo-ET) to image LRRK2 demonstrates the power of cryo-electron tomography to ob- filaments in situ (Fig. 1). We grew HEK-293T human cells tain structures of previously unsolved in their cellu- expressing LRRK2 bearing the pathogenic I2020T muta- lar environment and provides insights into LRRK2 function and tion tagged with YFP (referred to as “LRRK2” here) on pathogenicity. EM grids. After vitrification, cells containing filamentous Correspondence: [email protected] LRRK2 were identified by cryo-fluorescence microscopy. Cryo-focused ion beam (cryo-FIB) milling was then used Parkinson’s disease (PD) is the second most common neu- to prepare thin slices for cryo-ET (Fig. 1A-C). LRRK2- rodegenerative disease. About 10% of patients show a clear decorated MTs were identified by correlative light and elec- family history and in the leucine-rich repeat kinase tron microscopy (CLEM) as regions where MT bundles in the 2 (LRRK2) are the most frequent cause of familial PD (1, 2). transmission electron microscopy (TEM) images overlapped LRRK2 is a 286 kDa protein with an amino-terminal half with the YFP signal of LRRK2 in the fluorescence images containing repeating motifs and a carboxy-terminal half com- (Fig. 1D-F). Subsequently, cryo-ET was performed in these posed of kinase and GTPase domains surrounded by protein- regions (Fig. 1G-I). protein interaction domains (3). Most of the pathogenic mu- We first characterized the MTs in our cryo-ET tomograms. tations are found in these two catalytic domains, but all result MTs are formed from heterodimers of alpha- and beta-tubulin in hyperactivation of the kinase (1, 2). Hyperactivation of and polymerize into polar left-handed hollow tubes, which in LRRK2’s kinase is also reported in non-familial PD cases (4), human cells typically contain 13 protofilaments (15). We an- suggesting that suppression of kinase activity might be bene- notated each MT individually by computationally determin- ficial for a wide range of PD patients. ing its coordinates (Fig. S1A). Within bundles, most of the A major barrier to understanding the mechanism of LRRK2 decorated MTs were aligned parallel to each other (Fig. 1G- is the lack of structural information for the protein, which to I), with a center-to-center distance of 70 ± 18 nm (Fig. S2A). date only includes structures of domains, either single or in This regular distribution of angles and distances suggests that tandem. Many of these are from a distantly related bacte- LRRK2 mediates MT bundling. Most MTs were either fully rial homolog (5–7), two are single domains from the human decorated or not decorated, and these two populations co- protein (8, 9), and two are electron microscopy (EM) struc- existed within the same cellular regions (Fig. 1G). Protofil- tures, whose resolution was too low for molecular models ament number and polarity for each MT was determined to be built (6, 10). LRRK2 is involved in multiple cellular by subtomogram averaging (Fig. S1B). MTs within a given processes, including neurite outgrowth and synaptic morpho- bundle largely had the same polarity (Fig. S2B,C). Among genesis, membrane trafficking, autophagy, and protein syn- LRRK2-decorated MT, we observed significant populations thesis (3). LRRK2 phosphorylates a subgroup of Rab GT- of non-conventional 11- (7.5%) and 12- (69.6%) protofila-

Watanabe et al. | bioRχiv | November 10, 2019 | 1–22 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. 1. Correlative light and electron microscopy of mutant LRRK2-decorated microtubule bundles. (A) Fluorescence micrograph (FM) of a grid with HEK-293T cells expressing YFP-tagged LRRK2(I2020T) (shown in green). (B) Overlay of the fluorescence signal (green) corresponding to YFP-LRRK2(I2020T) and cryo-SEM image (greyscale) of the area marked in A. (C) Overlay of the initial FM image and an SEM image after FIB-milling of the area marked in B. (D) Overlay of the initial FM image (green) and cryo-TEM image of the area marked in B. (E and F) Enlarged TEM image of the area marked in D, with (E) and without (F) the overlaid FM image. The asterisks indicate mitochondria, which appears fragmented. (G) A slice of a tomogram showing non-decorated (white arrows) and LRRK2-decorated (green arrows) MTs. (H) Slice of a tomogram showing LRRK2-decorated MT bundle. (I) Annotated undecorated (green) and decorated (green with LRRK2 in gray) MTs and membranes (purple) in the tomogram in I. (J) Distribution of decorated microtubules with various protofilament numbers observed in LRRK2(I2020T)-expressing cells. (K) Percentage of decorated (green bar) and undecorated (white) MTs of the three different classes. Scale bars: 500 µm (A), 20µm (B), 5 µm (C), 2µm (D), 1µm (E, F), 100 nm (G,H). ment MTs, in addition to the 13-protofilament MTs (22.8%) respectively (Fig. 2A). This suggests some conformational that are more typical in human cells (Fig. 1J) (15). Although flexibility in LRRK2 to accommodate different MT geome- we used Taxol to increase the percentage of cells showing tries. Despite this apparent flexibility, LRRK2 showed a LRRK2 filamentous structures (12), Taxol treatment in the clear preference for the smaller, and less common 11- and absence of over-expression of LRRK2 did not alter protofil- 12-protofilament MTs, which were almost exclusively dec- ament number and showed a significantly different distance orated (90% and 79%, respectively; Fig. 1K). This was in and angle distribution of MTs within bundles (Fig. S3). contrast to 13-protofilament MTs, where only a minority was Next, we used subtomogram averaging to resolve the he- decorated (36%; Fig 1K). Furthermore, LRRK2 bound to lical arrangement of LRRK2 bound to MTs (Fig. 2A and 11-protofilament MTs appeared better resolved than LRRK2 Fig. S1C,D). We found that LRRK2 forms a right-handed bound to 13-protofilament MTs, despite the smaller dataset double helix around MTs with all three protofilament num- for LRRK2 bound to 11-protofilament MTs (Fig. 2A), sug- bers (Fig. 2A). Thus, there is a symmetry mismatch be- gesting that LRRK2 forms a more stable helix around these tween the LRRK2 helix and the MT, which is a left-handed smaller MTs. While the physiological significance of this ob- helix (Fig. 2A,B; helical parameters in table S1). Along servation remains to be determined, unconventional MT ge- the LRRK2 helix, we identified repeating units (protomers) ometries in neurons have been observed (15, 16). Since loss with a C2 symmetry axis perpendicular to the filament axis. of dopaminergic (DA) neurons is the hallmark of PD pathol- The number of LRRK2 protomers per turn matches the MT ogy, DA neurons could have MTs predisposed to be sta- protofilament number, with 11, 12 and 13 LRRK2 pro- bly decorated by the pathogenic filament-forming mutants of tomers per turn around 11-, 12- and 13-protofilament MTs, LRRK2. This stable decoration, even if sparse could enhance

2 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. 2. LRRK2 forms double-stranded right-handed helices around MTs with different protofilament numbers. (A) Structures of LRRK2 bound to MTs composed of 11, 12, and 13 protofilaments. The S-shaped protomer is highlighted in each reconstruction by a black outline. The total length of MTs used for averaging (in µm) in shown below each MT type. The end-on views highlight that the number of protomers matches the number of protofilaments in each MT type. (B) Structure of the MTs shown in A after excluding LRRK2 with a mask. The polarity of microtubules in B is indicated with (-), meaning that the plus end of the microtubule is pointing into the page, and the minus end towards the reader. (C) LRRK2 dimer density determined by subtomogram averaging (Fig. S1E-K). (D) Local resolution of the map in C determined by ResMap. (E) The same subtomogram average shown in C is displayed at a higher threshold to highlight the fit of the known crystal structure of a LRRK2’s WD40 dimer into the density. (F) Close up of a WD40 dimer showing the hole at the center of the beta propeller. (G and H) Location of two different 2-fold symmetry axis in the LRRK2 filament (both perpendicular to the filament’s axis). The first one (G) is at the center of the protomer identified in A, while the second (H) lies at the WD40–WD40 interface. activity of LRRK2 or disturb microtubule integrity and ax- to have resolutions of ∼12 Å. To date, the only in situ struc- onal transport (17) enough to compromise cellular integrity. tures of comparable resolution are of ribosomes (18, 19) and To determine the three-dimensional architecture of LRRK2 proteasomes (20), whose structures had previously been de- in the MT-associated filaments, we performed further termined by other methods. subtomogram-averaging by extracting individual protomer A salient feature in our map was the C-terminal WD40 do- subtomograms from 12-protofilament MT-bound LRRK2 as main of LRRK2, with the hole in the beta propeller being this was the most abundant species in our dataset (Fig. 2A, visible at higher thresholds (Fig. 2E,F). This established that Fig. S1E-M). We obtained a 14-Å resolution map (Fig. 2C, the repeating unit corresponds to a LRRK2 dimer, and can gold-standard FSC in Fig. S1K). Local resolution analysis be defined as two different symmetric protomers (Fig. 2A, (Fig. 2D) showed the region corresponding to the protomer G). Thus, MT-associated LRRK2 filaments are formed by

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 3 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. 3. The in situ architecture of LRRK2 bound to MTs. (A) Schematic representation of the domain organization of LRRK2. ARM: Armadillo repeats; ANK: Ankyrin repeats; LRR: Leucine-rich repeat; ROC: Ras of complex (GTPase); CORN/CORC: C-terminal of ROC; KIN: Kinase. PD mutations are indicated. (B) The subtomogram average of the MT-bound LRRK2 filament (Fig. 2C) is shown with the protomer used for model building displayed at higher threshold (blue) within the density of the filament (semi-transparent lighter blue). (C) Molecular model of MT-bound LRRK2, shown in the same two orientations used in B. Domains are colored for the protomer (a LRRK2 dimer) with neighboring LRRK2 monomers shown in light grey. A cartoon representation of the domain architecture of the protomer is shown on the left side. (D) Two views of the molecular model for a LRRK2 monomer seen along the axis of the filament (and the MT). The second monomer in the protomer is shown as an outline. The ROC and KIN domains are labeled to highlight their location next to, and away from the MT, respectively. (E) Molecular model of the protomer viewed from the MT surface.

ROC and CORC domains are labeled to highlight that dimerization is mediated by the CORC domain. (F) A low-threshold isosurface of a cryo-ET map of LRRK2 bound to 12-pf MT highlights a connection between adjacent strands in the LRRK2 double helix (colored in blue). (G) Subtomogram average obtained using another alignment mask (Fig.S1I,L,M), highlighting hook-like structures (in blue) protruding from the central protomer. (H) The same molecular model for the LRRK2 protomer shown in C with a model for the LRR repeats, which were docked into the blue densities shown in G. (I-L) Architecture of the MT-bound LRRK2 filament. (I) Schematic representation of the LRRK2 filament using the same cartoon of a LRRK2 dimer introduced in C. The filament is viewed in a direction perpendicular to its axis (and that of the MT). The blue lines connecting adjacent strands represent the LRR domains. (J) Close-up of the middle map in Fig. 2A showing a stretch of LRRK2 filament corresponding to the schematic representation in I. (K, L) The two possible LRRK2 dimers – CORC-mediated in blue (K) and WD40-mediated in purple (L) – are shown in solid colors inside the semi-transparent density for the filament, and their location within the cartoon in I is highlighted by contours of the same color. Scale bars: 5 nm (B, G, H); 20 nm (F).

4 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. 4. The WD40:WD40 interface is necessary for LRRK2 filament formation. (A) The subtomogram average of the MT-bound LRRK2 filaments is shown with the MT in grey, the LRRK2 filament in blue, and the WD40-WD40 dimer connecting two protomers in pink/maroon. (B) The crystal structure of the WD40 dimer with monomers colored as in A, and the G2385 residues at the interface shown as yellow spheres. (C) HEK-293T cells transiently expressing Halo-tagged LRRK2 were labeled with TMR-ligand. Representative images are shown for cells expressing wild-type (left panels) and G2385R (right panels) LRRK2, incubated in the absence (top) and presence (bottom) of 100 nM MLi-2, a LRRK2 kinase inhibitor, for 2 hours. The contrast was inverted for better visualization of the LRRK2 signal where black is the highest signal and white is no signal, i.e. background. (D) Percentage of transfected cells showing LRRK2 filament formation, defined by bright rod-like filamentous structures. Data represent three independent experiments. The total number of transfected cells considered in the analysis was 1705 for wild-type/-MLi-2; 1374 for wild-type/+MLi-2; 1464 for G2385R/-MLi-2; 1472 for G2385R/+MLi-2. two homotypic interactions: a yet-to-be-determined interface from a model of an isolated inactive LRRK2 dimer in solu- within the protomer we defined (Fig.2G) and a WD40:WD40 tion built on a negative-stained EM map (6). This suggests interface (Fig.2H). Notably, a recent X-ray structure of a that either LRRK2 uses a different COR:COR dimerization dimeric isolated human LRRK2 WD40 domain (9) fits un- interface from that observed in the reported structures or that equivocally into our cryo-ET map of MT-bound LRRK2 conformational changes are involved in the formation of fila- (Fig.2E, F). We could therefore use the position and orien- ments. tation of the WD40 domain to determine the position of the In our structure, the ROC (GTPase) domain is closest to the neighboring kinase domain, which is connected to the WD40 MT surface (Fig. 3C,D), consistent with observations that domain through a 9 amino-acid linker. The lack of charac- the ROC domain of LRRK2 is sufficient for interaction with teristic arch-shaped densities for the N-terminal Armadillo tubulin heterodimers (23). GTP binding and hydrolysis by (ARM) and Ankyrin (ANK) or Leucine-rich repeat (LRR) the ROC domain have been proposed to be involved in dimer- domains in the best resolved area is consistent with the pre- ization (22), filament formation around MTs (13), and regu- vious observation that the four C-terminal domains, namely lation of kinase activity (24). The kinase domain is distal to the WD40, kinase, ROC and COR domains (Fig. 3A) are suf- the MT and exposed to the cytoplasm (Fig. 3C). Since in- ficient for filament formation in the cell (12). hibitors that capture the kinase in an ATP-bound active-like With the WD40 domain as an anchor point, we used an inte- state (25, 26) are known to induce filament formation (13, 14) grative modeling approach (21) (Fig. S4) to build a model of and all other filament forming LRRK2 mutants (N1437H, LRRK2 into the S-shaped protomer map (Fig. 3) by com- R1441G/C and Y1699C) also results in hyper-kinase activ- bining available crystallographic and homology models of ity in cells (11–13), we expect that our structure captures an the WD40, kinase, ROC and COR domains. Systematic active kinase conformation, which may be facilitated through sampling of all possible combinations of domain placements oligomerization. Taken together, our data suggests that MT- within the cryo-EM density map, subject to polypeptide con- binding plays a scaffolding role in the oligomerization and nectivity and steric clashes restraints, yielded a single con- activation of LRRK2. Figuration of the domains of LRRK2. The structure of hu- Outside the protomer, our tomograms showed densities con- man LRRK2(I2020T) bound to a MT is shown in Fig. 3C-E. necting the two strands of the LRRK2 helices that decorate Our model reveals protein-protein interfaces mediated by the MTs, suggesting inter-helix contacts (Fig. 3F). We generated C-terminal of two COR domains within a protomer (Fig. 3C, a 18-Å resolution map encompassing a larger volume around E). Although our map and model are in agreement with the the protomer that revealed two hook-like structures stem- crystal structure of a WD40 dimer(, they differ from previous ming from the protomer (Fig. 3G, Fig. S1I,L,M, Fig. S5). crystal structures of ROC-COR dimers from distantly-related Given their shape and adjacency to the ROC domain, we pro- bacterial homologs of LRRK2 (5, 22). Our model also differs pose that these densities likely represent the LRR domains,

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 5 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

(Fig.3H). Thus, there is a third homotypic interaction, me- ously (12). The construct for the N-terminus Halo-tagged diated by the LRR domains, that connects the strands of the full-length LRRK2 was cloned using the Gibson Assembly double helix, which may determine its regular spacing along method. The LRRK2 gene was cloned by using the primer the MT (Fig.3F). In our density maps, the N-terminal ARM sets: 5’-GCGATAACATGGCTAGTGGCAGC-3’ and 5’- and ANK domains were not resolved, presumably due to their GGGGTTATGCTAGTTACTCAACAGATGTTCGTCTC- flexibility. However, we observed undefined density in the 3’ with pENTR221-LRRK2 (Addgene #39529) as periphery of the LRRK2-decorated MTs but not in undeco- the template. The N-terminal Halo-tagged vec- rated MTs (Fig. S6A, B). The density approximately spans tor segment was cloned by using the primer sets: the distance between decorated MTs within a bundle, sug- 5’-GAGTAACTAGCATAACCCCTTGGC-3’ and 5’- gesting that the ARM and ANK domains may form connec- CACTAGCCATGTTATCGCTCTGAAAGTACAGATC-3’ tions between adjacent MTs. with the pHTN HaloTag® CMV-neo Vector (Promega) as Finally, to test our model we set out to determine if disrupt- template. The two segments were assembled using the NEB- ing one of the two dimer interfaces we observed affected uilder® HiFi DNA Assembly Kit following their suggested the formation of MT-associated LRRK2 filaments in cells. protocol. The G2358R mutation was made by using the We focused on the LRRK2 WD40:WD40 interface because NEB Q5-site directed mutagenesis kit, and the primers were a mutation, G2385R, in the WD40 domain is a risk factor designed based on the online tool NEBBaseChanger. The for PD (27, 28) and has been shown to disrupt WD40 dimer constructs were sequenced and expressed in HEK-293T cells formation in vitro (9). G2385 is located at the WD40:WD40 to confirm the expression of full-length LRRK2. interface between protomers (Fig4A, B). We hypothesized that disrupting this interface would lead to defects in filament TEM grid preparation. For expression of LRRK2, the formation. To test this, we expressed Halo-tagged wild-type HEK-293T cells were transfected with a plasmid encod- or G2385R LRRK2 in human HEK-293T cells in the pres- ing full-length LRRK2(I2020T) fused with YFP at the N- ence or absence of the LRRK2-specific kinase inhibitor MLi- terminus using a Lipofectamine 3000 transfection kit (In- 2 (29), which has been shown to induce LRRK2 filament vitrogen) according to the manufacturer’s protocol. After formation (7, 13). We found that MLi-2 induced filament 2 days, 5µM paclitaxel (aka Taxol, form Science Signal- formation in cells expressing wild-type LRRK2, but not in ing) was added. After 16 to 24 hours of Taxol treatment, cells expressing G2385R LRRK2 (Fig. 4C, D) as shown pre- the cells were detached, and counted with a hemocytometer. viously using another LRRK2 kinase inhibitor, LRRK2-IN- Quantifoil 200 mesh holey carbon R4/1 or R2/1 copper grids 1 (30). These data support our model that the WD40:WD40 (Quantifoil Micro Tools) were glow discharged for 60 s at 0.2 protomer interface is important for the formation of LRRK2 mbar with 20 mA using a PELCO easiGlow glow discharge filaments. Given the correlation between breaking the dimer- system (Ted Pella). Just before deposition of cells on TEM ization interface, loss of filament formation, and change in grids, 1.5 µl of poly-lysine (Sigma) was applied and ∼2000- kinase activity seen in the G2385 LRRK2 mutant (9, 11, 30– 6000 cells were deposited onto a grid by pipetting 3-10 µL of 32), supporting that oligomerization plays a role in regulating detached cells onto the grid. Blotting and plunging was per- LRRK2 kinase activity. formed using a custom-made plunger (Max-Planck-Institute Here, we have shown that recent advances in cryo-ET can be for Biochemistry) using 50/50 mixture of liquid ethane and used to determine novel structures of proteins in situ, mak- propane (Airgas) cooled to liquid nitrogen temperature. The ing it possible to generate structural hypotheses in a cellular grids were clipped onto Autogrids (TFS) and samples were context. We determined the first structure of full-length hu- kept at liquid nitrogen temperature throughout the experi- man LRRK2 and its association with MTs. We also show ments. the power of integrative modeling to extract structures from in situ cryo-ET data. Since well-characterized LRRK2 ki- Cryo-fluorescence microscopy. For cryo-fluorescence nase inhibitors enhance the ability of LRRK2 to form fila- microscopy, grids were observed with a CorrSight micro- ments around MTs (13, 14), the structure presented here not scope (TFS) using EC Plan-Neofluar 5x/0.16NA, EC Plan- only provides insight to understanding the pathogenic state Neofluar 20x/0.5NA, and EC Plan-Neofluar 40x/0.9NA air of familial PD, but is also the first step towards designing objectives (Carl Zeiss Microscopy), an Oligochrome light- improved LRRK2 kinase inhibitors. source, which emits in 4 different channels (405/488/561/640 nm) (TFS) and a 1344×1024 px ORCA-Flash 4.0 camera (Hamamatsu). Acquisition and processing of the data was Materials and Methods performed with the MAPS 2.1 software (TFS). After acqui- Cell culture. HEK-293T cells were cultured in Dulbecco’s sition of a grid map at 5X magnification, regions of interest Modified Eagle’s Medium (DMEM) containing GlutaMAX-I were imaged at 20x or 40x magnification, to identify cells [Thermo Fisher Scientific, (TFS)], 10% HyClone bovine calf with regions containing LRRK2 filamentous structures. serum (GE Healthcare) and 100 U/mL HyClone penicillin- streptomycin (GE Healthcare) at 37°C and 5% CO2. Cryo-focused ion beam milling. Micromachining of frozen hydrated cells was performed in a Scios DualBeam Plasmids. Plasmid encoding full-length LRRK2(I2020T) FIB/SEM microscope equipped with a cryo stage (TFS). The fused with YFP at the N-terminus was described previ- sample chamber of the microscope was kept at a pressure

6 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

below ∼1×10−6 mbar. A low-magnification SEM image en- e/Å2, respectively. compassing the entire grid using an acceleration voltage of 5 kV, a beam current of 25 pA, and a dwell time of 200 ns Tomogram reconstruction and annotation. The tilt se- was correlated with the grid map from the cryo-light micro- ries were and aligned and dose-weighted according to the scope using MAPS 2.1 (TFS) to identify regions of interest cumulative dose using MotionCor2 (39). Alignment of the on TEM grids. A thick platinum layer (typically ∼2µm) was tilt series and tomographic reconstructions were performed deposited on the sample to improve its conductivity and to using Etomo, part of the IMOD package (40). Since no fidu- reduce streaking on the lamella caused by local variations cial markers were present on the lamellas, tilt-series align- in mass density that result in variations in lamella thick- ment was performed using patch tracking. Contrast trans- ness, known as curtaining (33). An integrated gas injec- fer function (CTF) correction was performed in IMOD (40) tion system (GIS) is used to deposit the precursor compound using defocus values measured by CTFFIND4 (41) prior to trimethyl(methylcyclopentadienyl)platinum(IV) (34, 35). To dose-weighting via Fourier filtering. Tomograms were recon- this end, the grid was placed 11.5 mm away from the GIS structed using weighted back-projection. Tomograms were with a nominal stage tilt of 7°, and the GIS was opened for 13 4x-binned (without CTF correction) and used for segmenta- seconds. Cells were targeted for FIB-milling that satisfied the tion of microtubules using the filament tracing function in following conditions: (1) showed a filamentous LRRK2 phe- Amira (TFS). After filament tracing, the resulting center co- notype; (2) were located within the center of a grid square, ordinates were re-sampled equidistantly using a script written and (3) were located on a grid square that was at most five in MATLAB. For each point along the filaments, initial Eu- squares from the center of the grid (the central ∼1mm2 area ler angles were assigned for subtomogram averaging. Within of the grid). The stage was positioned at a nominal tilt of 11- a filament, angles were assigned such as the direction along 18°, corresponding to a milling angle of 4-11°. FIB milling the filament axis was the same for all particles. The in-plane was performed in three steps with decreasing ion beam cur- Euler angle (orthogonal to the axis of the filament) was ran- rents and a fixed acceleration voltage of 30 kV. By decreasing domized to minimize the effect of the missing wedge. the current in three steps, the beam spread was reduced to a minimum at the final stages of milling, while accelerating the Subtomogram averaging. To determine the protofilament milling process at initial stages. For the rough milling step number of single microtubules, 4x binned tomograms were an ion beam current of 0.3 nA was used. The current was re- used for extraction of subtomograms with a box size of 38 duced to 0.1 nA for the intermediate step and to 30 pA for fine nm along the microtubule at 4 nm spacing using the Dynamo milling. The target lamella thickness was ∼100 nm. During software, and an initial average for each microtubule was the milling process, lamella thickness was estimated utilizing created using the pre-assigned Euler angles described above. thickness-dependent charging effects observed in the SEM Each filament was separately aligned and averaged using Dy- images at different acceleration voltages (35). At the end of namo (42), using the initial average as a reference. Three the session, the grids were transferred out under vacuum and iterations of translational and orientational alignment of the stored in liquid nitrogen. first two Euler angles followed by five iterations of transla- tional alignment and orientational alignment of the third Eu- Cryo-electron tomography. Tomographic tilt series were ler angle, with the latter restricted to 18-36 degrees. Parti- recorded in a Tecnai G2 Polara (TFS) equipped with a field cles were low-pass filtered to 22-30 Å for alignment. 2D emission gun operated at 300 kV, a GIF Quantum LS post- projections along the MT axis were calculated in MATLAB, column energy filter (Gatan) and a K2 Summit 4k×4k pixel and the 2D projections as well as the 3D averages were visu- direct electron detector (Gatan). The FIB-milled grids were ally inspected in MATLAB and IMOD to determine protofil- loaded into the TEM using modified Polara cartridges, which ament number and plus/minus end polarity of each micro- securely accommodate Autogrids (36). The milling slot of tubule. Assignment of MT polarity can be achieved by in- each grid was aligned perpendicular to the tilt axis of the specting a cross section of the averages from the plus end microscope. This way the inherent tilt axis of the slanted (anti-clockwise slew; Fig. S1B, left column) or from the mi- lamella was parallel with the tilt axis of the microscope stage nus end (clockwise slew; Fig. S1B, right column) respec- allowing separation of focus and record areas according to tively (43). Microtubules with unclear or ambiguous protofil- low dose practice along the tilt axis at equal heights. Tilt se- ament number or polarity were discarded. Then, new parti- ries were acquired at a target defocus of 5µm and a pixel cles with a box length of ∼70 nm were extracted from 4x size of 2.2 or 3.5Å using the SerialEM software (37) in binned tomograms, and for each microtubule class (11-, 12- low-dose mode. The dose-symmetric tomography acquisi- or 13-protofilament MTs) all particles representing the re- tion scheme (38), was modified to account for the pre-tilt of spective class were submitted to alignment and averaging in the lamella, and to allow for the acquisition to stop when the Dynamo, using an initial average as a reference. In order image does not yield enough counts with a target dose due to to separate decorated MTs from non-decorated MTs, multi- thickness, which occurs on the side of the pre-tilt first. The reference alignment in Dynamo was conducted using two K2 detector was operated in counting mode and the images templates: (1) a LRRK2-decorated MT template (being the were divided into frames of 0.075 to 0.1 s. The tilt range was average from the previous step) and (2) a non-decorated MT typically between ±50°and ±70°with increments of 2 and template (being the average from the previous step with the 3°and the total electron dose was around 180 e/Å2 and ∼120 density of LRRK2 masked out) using a hollow cylindrical

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 7 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

classification mask corresponding to the LRRK2 density. The ROC domain (COR), kinase (KIN) and a WD40 domains same alignment parameters as described above were used. In (Fig. 3A). To determine the architecture of LRRK2/I2020T order to further improve the structure of LRRK2 bound to bound to MTs, we applied an integrative approach imple- MTs, particles sorted to class 1 (decorated MTs) were further mented in the open source Integrative Modeling Platform aligned by using a hollow cylindrical alignment mask that in- (IMP) package (48). The integrative modeling process con- cluded LRRK2 but excluded the MT. For obtaining MT aver- sists of four stages: (1) gathering data, (2) choosing how to ages (Fig.2B), all particles (decorated and undecorated) with represent the system and translating the information into spa- the same protofilament count and polarity were aligned by tial restraints, (3) determining an ensemble of structures that using a cylindrical alignment mask containing just the MT re- satisfy these restraints and, (4) validating the model. This ap- gion. The resulting averages were used to estimate the helical proach has been applied to determine the structure of numer- parameters of the LRRK2 double-stranded helix and MTs as ous biological complexes including the 26S proteasome (49), detailed below. Coordinates of particles along the helical path the mediator complex (50) as well as the nuclear pore com- were used to extract particles from unbinned, dose-weighted plex (51). To our knowledge, this work represents the first and non-CTF-corrected tomograms, and used to create an ini- application of integrative modeling to solve a novel structure tial average using pre-assigned orientations. 3D refinement determined in situ using cryo-ET. Given the resolution of the of extracted particles was performed in RELION (44, 45) cryo-ET map, the modeling was limited to the location and to create a template for subsequent classification. This den- orientation of the domains within the density, treated as rigid sity was used as a reference for 3D classification into three bodies. The clear identification into the cryo-ET density map classes, employing a soft mask covering the LRRK2 pro- of the characteristic donut-shape of the WD40 provided the tomer and a regularization parameter of 2-4. Class averages starting point for the LRRK2 domains allocation and allowed were inspected in UCSF Chimera (46), and classes with high us to assess that the assignment of the LRRK2 is possible up levels of noise were excluded. A density corresponding to the to the last four domains (WD40, KIN, COR, ROC) while the ring-shaped WD40 domain was clearly resolved in one class density related to the LRR, ANK and ARM is visible only at (class 1 in Fig. S1G). Particles belonging to this class were higher threshold, presumably due to their intrinsically flexi- subjected to gold-standard refinement using two different bility. masks (masks A and B; Fig. S1H,I) and post-processing with- out B-factor sharpening in RELION. Local-resolution maps Stage 1: Gathering Information. Three different types of data were calculated using ResMap (44, 45). Maps and structures were used for structure determination: A. Cryo-ET map of were visualized using UCSF Chimera (46) and VMD (47). LRRK2(I2020T) protomer: a 14-Å in situ cryo-ET map was determined as detailed above. The protomer corre- Measuring distances/angles between MTs. To calculate sponds to a dimer LRRK2 bound to the MTs (Fig 3B). B. the angles and distances between filaments (microtubules), Atomic models of the domains composing LRRK2: X-ray the distances between all points sampled every 4 nm along crystallography structure of the human LRRK2 WD40 (9) each filament and all points on all other filaments within the (PDB:6DLO) Homology model of the kinase domain of hu- tomogram were measured. The resulting distance matrix was man LRRK2 (Uniprot ID Q5S007, residue 1883-2135) in its then reduced to point pairs with distances of 100 nm or less. active-like conformation was generated by homology mod- For each point along a filament, we calculated (1) a tangent eling with SWISS-MODEL (52, 53). The crystal structure vector along the filament at that point, considering the nearest of the Roco4 Kinase domain bound to AppCp from D. dis- points along the same filament, (2) the distance to the nearest coideum was used as a template (54) (PDB:4F0F, homol- point in each neighboring filament, and (3) the angle between ogy 43%). Alignment was generated by ClustalOmega (55) the vectors at these two points, where an angle of zero corre- (Fig. S7 and S8). A model of the monomeric ROCCOR sponds to parallel filaments. A 2-D histogram of the distance domain of human LRRK2 protein (Uniprot ID Q5S007, between filaments vs angle between filaments was calculated residue 1332-1838 was obtained by homology modeling with in MATLAB. SWISS-MODEL (52, 53). The structure of C.tepidum Roco protein (5) (PDB:3DPU) was used as template and aligned Determining helical parameters. Helical parameters of to the human LRRK2 sequence of domains ROC and COR non-symmetrized MTs and LRRK2 helices were obtained us- by using ClustalOmega (55) (Fig. S7A). Normal model anal- ing an autocorrelation function as implemented in Dynamo. ysis (NMA) with Bio3D package in R (56) was performed To obtain the helical parameters for MT-bound LRRK2, av- on the ROCCOR template revealing a hinge in the COR do- erages of each were obtained by masking out the volume main (L1669-I1689) which corresponds to a missing loop corresponding to the other, i.e., MT helical parameters were area in the template PDB structure. Therefore, we split the obtained from an average obtained with LRRK2 excluded ROCCOR domain into three separate rigid bodies namely, through a mask, and vice versa. CORC, CORN, and ROC. (PDB:3DPU, homology 37%, 37% and 38% respectively; Fig. S7 and S8). C. Since all the do- Integrative Modeling of LRRK2 bound to MTs. LRRK2 mains are part of a single polypeptide chain of LRRK2, the is a 286kDa protein formed of seven domains: armadillo amino acid stretches between the domains were considered as (ARM), Ankyrin (ANK) and Leucin-rich repeat (LRR) do- linkers connecting the N- and C- termini of consecutive do- mains, a Ras of complex proteins (ROC), and C-terminal of mains. A worm-like-chain model (57) was used to model the

8 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

connecting√ linkers, with an average end-to-end distance of scored. From that, we filtered the solutions based on respect- 2 N ∗l , where N is the number of amino acids and l = 3.1Å ing the N-C and having not major compenetration between is the length of one amino acid measured as the typical aver- domains, (corresponding to an EV score of <100,000). This age distance between alpha carbons of adjacent amino acids yielded 786,211 models. Third, the ensemble was clustered (Fig. S8). based on RMSD of 8 Å, leading to 256 clusters, that could be further classified into models with different architectures, Stage 2: System Representation and Translation data into e.g., swaps between ROC, CORN and CORC domains. Next, Spatial Restraints. Each domain was represented as a rigid the models were filtered to allow only clashes of up to 10% of body at atomic resolution. Five types of spatial restraints the alpha carbons within domains as measured in VMD (47). were used during monomer conFiguration and dimer refine- From here, only two clusters remained, one with 31,140 mod- ment in Stage 3. Cryo-ET density restraint: (a) an IMP scor- els, and the other with 394 models. Fourth, dimers were built ing term that considers the percentage of atoms that are in- from each monomer by applying a symmetry transformation. cluded in the map, used both in monomer conFiguration and All models in the second cluster had major clashes at the refinement. (b) Cross correlation of model to map (imple- dimer interface. Thus, only one ensemble of 31,140 mod- mented in MDFF), used to select models in monomer con- els remained corresponding to the same domain architecture. Figuration. Location of WD40: based on the characteristic The ensemble was filtered to satisfy restraints i, iii, iv, and donut shape in the density, the WD40 could be unequivo- v(a), resulting in 1,137 models. B. Dimer refinement. We cally located in the cryo-ET density map and thus its loca- generated an ensemble of LRRK2 dimers by rigid-body fit- tion was constrained to that region in the map (Fig. S8), used ting each monomer model into the dimer density map, as de- in monomer conFiguration. Chain connectivity restraints: tailed above. We then clustered the dimers ensemble into 48 distance restraints between the C-term and N-term of con- clusters based on an RMSD of 8 Å. From each cluster, we secutive domains as described above (Fig. S8), used both refined a representative dimer using a simulated-annealing in monomer conFiguration and dimer refinement. Excluded Monte-Carlo (MC) protocol implemented in IMP. During the volume restraints: Overlap between domains is assessed as refinement, local perturbations in positions and orientations an excluded volume restraint (steric clashes of less than 10% of the ten rigid bodies were sampled and assessed using a and 5% of alpha carbons), used both in monomer conFigu- scoring function including terms i-v. Minima from the refine- ration and dimer refinement respectively. Dimer symmetry ment protocol were gathered and used in the refined ensemble restraints: (a) Overlap between monomers was assessed us- assessment (stage 4). ing excluded volume (steric clashes of less that 10% of alpha carbon atoms between LRRK2 monomers), used in the final Stage 4: Assessment of Data and Structure. Our extensive stage of monomer conFiguration. (b) Symmetry restraints sampling rendered a single architecture of LRR2 dimers. In implemented in IMP used during dimer refinement. order to validate this structure, we first looked at the overall conformation and its congruency with other available data. Stage 3: Ensemble Sampling. To create an ensemble of We found that our model agrees with the following inde- LRRK dimers that incorporates the data available and satis- pendent observations: The LRR domain fits into the den- fies the restraints, we applied a sampling protocol consisting sity and is in the proximity of the ROC domain (Fig. 3H). of two steps: monomer conFiguration and dimer refinement. The LRR model corresponds to residues 129-444 of the LRR A. Monomer conFiguration. To determine the monomer con- in a crystal structure of a homologous Roco protein (PDB Figuration we used the MultiFit module of IMP (58). First, ID: 6HLU), and was fitted using the rigid-body fitting func- rigid-body fitting of each of the individual five domains to a tion of Chimera (46). The ROC domain is proximal to the dimer protomer map were sampled at 5°resolution using col- microtubules, consistent with observations that the ROC do- ores (59). The best 10,000 fits for WD40, ROC, CORN and main of LRRK2 is sufficient for interaction with tubulin het- CORC and 5,000 fits for KIN were considered. WD40 fits erodimers (23). The dimerization of the WD40 observed in were filtered to be placed within the donut-shaped density to a recent crystal structure (9) matches the architecture in our satisfy constraint ii. From these fits, we selected a single po- model, and is consistent with our data showing no filament sition and orientation that matched that of the dimer in the formation (Fig. 4). Although we used the crystal structure to dimer crystal structure (9). Second, we looked at all 5000 assign the WD40 conformation, a preliminary global align- fits of the kinase and selected those that satisfied the distance ment using MultiFit (data not shown) confirmed this to be the linker of 9 residues (restraint iii). Only six fits satisfied this best location of the WD40 independently of the crystal struc- restraint, with an RMSD of 4.7Å between them, and thus we ture. The monomer ensemble obtained was analyzed to as- used a single kinase fit for the subsequent steps. Next, the sess the uncertainty on the positions and orientations of each fits for ROC, CORN and CORC were filtered to include only domain (Fig. S9). The analysis on the domains variability as- those fitted to the same monomer within the protomer map, sessed a narrow conformational space visited by COR, CORC and not overlapping with the placed WD40 and kinase, yield- and a broader conformational space visited by ROC (Fig. S9). ing 1188, 1446 and 1148 models respectively. MultiFit was During refinement, each model improved its conformation in then used to sample all possible combinations of the ROC, terms of the restraints, without major rearrangements. Thus, CORN and CORC domain fits and scored for spatial restraints the ensemble reflects the variability of the structure given the i(a), iii and iv. A total of three million models were initially available experimental data. Altogether, we determined the

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 9 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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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A cryo-FIB lift-out technique enables molecular- assessed for the presence of clear filamentous structures and resolution cryo-ET within native Caenorhabditis elegans tissue. Nature methods, 16(8): 757–762, August 2019. quantified in three independent experiments. 20. Q Guo, C Lehmer, A Martinez-Sanchez, T Rudack, F Beck, H Hartmann, M Perez-Berlanga, F Frottin, M S Hipp, F U Hartl, D Edbauer, W Baumeister, and R Fernandez-Busnadiego. In ACKNOWLEDGEMENTS Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment. We thank Daniel Castano Diez and Paula Perez Navarro for assistance with Dy- Cell, 172(4):696–705 e12, February 2018. namo; Guillaume Castillon, Alexis Rohou, Junru Hu, and members of the Villa lab 21. K Lasker, M Topf, A Sali, and H J Wolfson. Inferential optimization for simultaneous fitting of for stimulating discussions and technical support. This work was supported by an multiple components into a CryoEM map of their assembly. Journal of Molecular Biology, NIH Director’s New Innovator Award 1DP2GM123494-01 (to E.V.), a Michael J. Fox 388(1):180–194, April 2009. Foundation grant (to S.T., E.V and D.B.), and a Branfman Family Foundation award 22. E Deyaert, M Leemans, R K Singh, R Gallardo, J Steyaert, A Kortholt, J Lauer, and (to D.B). M.A. is supported by a postdoctoral fellowship from the Visible Molecular W Versees. Structure and nucleotide-induced conformational dynamics of the Chlorobium Cell Consortium at UC San Diego. T.L. is supported by a Taiwan MOE fellowship. tepidum Roco protein. Biochem J, 476(1):51–66, January 2019. We acknowledge the use of the UC San Diego cryo-Electron Microscopy Facility 23. P N Gandhi, X Wang, X Zhu, S G Chen, and A L Wilson-Delfosse. The Roc domain of (partially supported by a gift from the Agouron Institute to UC San Diego), the NIH leucine-rich repeat kinase 2 is sufficient for interaction with microtubules. 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Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 11 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Supplementary Data Effects of Taxol. To examine the effect of Taxol incubation (5 µM) performed to increase the percentage of cells showing LRRK2 filamentous structure (12), MTs in the HEK-293T cells incubated with Taxol, in the absence of LRRK2 overexpression, were similarly analyzed (Fig. S3). In Taxol-treated HEK-293T cells, MTs were less clustered compared to LRRK2 decorated MTs (Fig. S3A, B). The center-to-center distance between MT was the average distance of 47 ± 32 nm (peak 31 nm) (Fig. S3C). The wider distribution compared to LRRK2 expressing Taxol-treated cells (Fig. S2) indicates more random MT arrangements in the Taxol-treated cell suggesting that LRRK2 itself mediates MT bundle formation observed in Fig.1H, I and Fig. S2. The 31 nm peak distance likely corresponds to the closest possible packing (non-decorated 13-protofilament MT diameter is 25 nm; Fig. S3A-C). In these cells, 8% of the total MTs belonged to 12-protofilament MTs, and no 11-protofilament MTs were found, while 92% of MTs observed were 13-protofilament MTs suggesting that Taxol treatment used in our experiments did not drastically affect MTs population in HEK-293T cells (Fig. S3D).

12 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 13 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S1. Subtomogram averaging. (A) Individual microtubules are automatically annotated using the filament tracing function of Amira (TFS). The annotated MTs (grey) are indicated in a slice of a tomogram showing LRRK2-decorated MT. (B) Single microtubule averaging sampled at every 4 nm was performed using Dynamo to determine the protofilament number and polarity of individual MTs using 4 binned tomograms. Examples of a cross-section of averages from the plus end (anti-clockwise slew, left column) and from the minus end (clockwise slew, right column). The total length of each class of MTs unambiguously determined from LRRK2 tomograms is shown. (C) After sorting of MTs according to the protofilament number, larger subtomograms (70 nm) were re-extracted and averaged to obtain initial LRRK2/MT averages. (D) Dynamo multi-reference alignment using two different templates: (1) LRRK2/MT and (2) MT only, using a hollow cylindrical classification mask corresponding to LRRK2 density, resulting in averages for LRRK2/13-protofilament MT (class 1) and non-decorated 13-protofilament MT (class 2). The same protocol was followed for 11-pf and 12-pf MTs. (E) Helical parameters of the LRRK2 helix on 12-protofilament MT were estimated and used for extraction of subtomograms containing the LRRK2 protomer from un-binned and non CTF-corrected tomograms and used to create an initial average using pre-assigned orientations. (F) 3D refinement of extracted particles was performed in RELION to create a template for subsequent classification. (G) The refined average from (F) was used as a reference for 3D classification into three classes in RELION. Classes with high levels of noise (classes 2 and 3) were excluded. (H, I) Particles belonging to the good class (class 1) were subjected to gold-standard refinement and post-processing in RELION using two different masks shown in grey (A and B). (J, L) The final subtomogram averages from LRRK2/12-protofilament MT. (K,M) FSC curves for the two maps shown in J and L, respectively.

Fig. S2. LRRK2-decorated MTs are found in bundles with consistent polarity. (A) Relative angle and distance between MT segments revealing parallel bundles with a regular center-to-center distance of 70 ±18 nm (peak ∼67 nm) between microtubules. (B) Slice of a tomogram showing bundles of LRRK2-decorated MT. Sale bar, 200 nm. (C) Individual MT analysis with Dynamo shows that MTs found in bundles have consistent MT polarity.

14 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S3. MTs in Taxol-treated HEK-293T cells (A) A slice of a tomogram showing MTs in HEK-293T cells treated with 5 µM Taxol. Scale bars, 100 nm. (B) The annotated MTs (in green) in the corresponding tomogram. (C) Relative angle and center-to-center distance between MT from 5 Taxol-treated cellular tomograms. The average distance is 47 ± 32 nm (peak ∼31 nm). (D) Graph showing the percentage of different MT classes found in 5 tomograms of Taxol-treated cells from two biological duplicates.

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 15 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S4. Integrative structure determination of the domain architecture of LRRK2. First, we gather data generated by various experiments and computational methods. Second, the five C-terminal domains of LRRK2 are represented as rigid bodies and the data are translated into spatial restraints. Third, an ensemble of structures that satisfy the restraints is sampled using Multifit, an IMP module that allows simultaneously fitting the rigid bodies respecting the a priori restraints imposed. The ensemble is clustered into distinct domain arrangements and representative structures are further refined. Finally, the refined models are analyzed for accuracy and certainty (See Materials and Methods for details).

16 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S5. Local resolution of subtomogram-average 3-D reconstructions. Local resolution for the map shown in Fig.3G.

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 17 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S6. Putative location of LRRK2’s N-terminal domains. (A) The cross-section density view of LRRK2/13-pf MT at lower threshold. The weak outermost density highlighted with a pair of blue dotted circles is observed. (B) The cross-section density view of an undecorated 13-pf MT at lower threshold. No outer density is observed in this cross-section. Two pairs of dotted blue circles shown in A and B correspond to 50 nm and 67 nm in diameter, respectively. Cf. the peak of center to center distance between LRRK2-decorated MT bundles is ∼67 nm (Fig. S2A). Scale bar: 25 nm.

18 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S7. Sequence alignment used for homology modeling of LRRK2. (A) Alignment of the LRRK2 kinase domain to the D. discoideum Roco4 kinase domain template structure PDB ID code 4F0F (bound to AppCp). (B) Alignment of the LRRK2 CORC (C-terminal end of ROCCOR) domain to the C. tepidum Roco ROCCOR template structure PDB ID 3DPU. (C) Alignment of the LRRK2 CORN and ROC to the C.tepidum Roco ROCCOR template structure PDB ID 3DPU.

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 19 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S8. Experimental data, statistical inference and physical principles considered in the integrative modeling procedure. (A) Comparative modeling templates for WD40,

KIN, CORC, CORN, and ROC. The PDB template, the organism, the homology, and the relative modeled region are reported for each domain. (B) Distance restraints and (C) exclude volumes pairs considered in the integrative modeling approach.

20 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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.

Fig. S9. Ensemble Variability Analysis Analysis of the ensemble variability for WD40 (red), KIN (pink), CORC (orange), CORN (yellow) and ROC (green). (A) Representative structure from the ensemble, with the position and orientation indicated by an arrow. (B) Probability density function of the localization of the centroid of each domain within the density. A blue asterisk indicates the potential location of the C-terminus of the LRR domain. (C) Orientation variability analysis of each domain within the ensemble; each arrow indicates the orientation of the domain with respect to their center of mass colored accordingly to the domain analyzed. Scale bar: 5 nm.

Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2 bioRχiv | 21 bioRxiv preprint doi: https://doi.org/10.1101/837203; this version posted November 10, 2019. 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. Parameters for the LRRK2 helices and MTs found in cells.

Structural Type Rotation (°) Z-rise (Å) LRRK2/11-pt 33.3 31.5 LRRK2/12-pt 30.3 28.8 LRRK2/13-pt 27.8 26.9 MT 11-3 -32.4 11.7 MT 12-3 -30.1 10.5 MT 13-3 -27.7 9.4

Table S2. Cryo-ET data collection

LRRK2-expressing cells Taxol-treated cells data set 1 data set 2 data set 1 data set 2 Voltage 300 Detector Gatan K2 Energy filter slit width (eV) 20 Target Defocus (µm) -5.0 Electron exposure (e/Å) ∼180 ∼120 Tilt range (min/max, step) -60°/+60°, 2° -60°/+60°, 3° Tilt scheme bi-directional dose symmetrical bi-directional dose symmetrical Tomograms used 6 12 3 2 Pixel size (Å) 3.5 2.2 2.7 2.2

22 | bioRχiv Watanabe et al. | The in situ structure of Parkinson’s disease-linked LRRK2