{"paper_id":"579b1160-90c2-4cd5-ad85-515a74ff5067","body_text":"Pražák et al. 2024 (preprint) 1 \n\t\n\t\nMolecular architecture of glideosome and nuclear F-\nactin in Plasmodium falciparum \n\t\nVojtech Pražák a,b, Daven Vasishtana,b, Kay Grünewalda,b,c, Ross G. Douglasd and Josie L. Ferreirae* \na Leibniz-Institut für Virologie (LIV), Hamburg 20251, Germany, Centre for Structural Systems Biology, Hamburg 22607, Germany \nb Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU \nc Department of Chemistry, Universität Hamburg, Hamburg 20148, Germany  \nd Biochemistry and Molecular Biology, Interdisciplinary Research Centre and Molecular Infection Biology, Biomedical Research \nCentre Seltersberg, Justus Liebig University Giessen, Giessen 35392, Germany \ne Institute for Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, UK \n*Correspondence should be addressed to J.L.F (josie.ferreira@ucl.ac.uk) \n \nActin-based motility is required for the transmission of malaria sporozoites. However, direct structural \ndata for this process is lacking. We used FIB -milling and electron cryo -tomography to study untreated \nactin in situ and dissect the path of actin filaments during parasite gliding. This revealed unexpected \nstructures, including currently unknown  filaments reinforcing the pellicle and actin bundles in the nu-\ncleus. Implications of these structures for motility and transmission are discussed.  \n\t\nThe Plasmodium falciparum parasite causes the most \nsevere form of malaria in humans 1. Infection occurs \nduring a bite from an infected mosquito, where sporo-\nzoites leave the mosquito salivary glands and are de-\nposited into the skin 2. Within the skin, sporozoites \nmove rapidly (1-2 μm.s-1) and persistently (> 1 hour) to \nencounter and traverse peripheral blood capillar-\nies.  This parasite stage utilises an uncommon form of \nmotility, termed gliding motility. It relies on a special-\nised, unconventional actomyosin motor system, situ-\nated below the plasma membrane, where the myosin \npowerstroke results in the rearward translocation of ac-\ntin filaments (F-actin) and associated adhesins3.  Plas-\nmodium requires two highly sequence divergent actin  \nisotypes for its cellular functions, with actin 1 being ex-\npressed throughout the life cycle and directly involved \nin gliding motility. Biochemically, actin 1 monomers as-\nsemble into F-actin at rates similar to vertebrate actin \nisotypes. However, Plasmodium F-actin appears to be \ndynamically unstable  in vitro and through shrinkage \nand fragmentation, ultimately result in only very short \nfilaments of approximately 100 nm length4–7. Within the \nparasite, actin filaments have historically been difficult \nto visualise and the failure of traditional actin  labelling \ntools on this divergent actin,  has limited our under-\nstanding of dynamics within the cellular context. Re-\ncent work in Plasmodium and its related apicomplexan \nToxoplasma, using the filament recognising actin chro-\nmobody, revealed distinct localisations of actin filament \npools primarily at the front (apical), rear (basal) and nu-\nclear region  of motile cell s8–10. However, resolving \nthese enigmatic actin filaments has proven difficult and \nan in vivo understanding of the arrangement, lengths, \njourney and fate of filaments in highly motile Plasmo-\ndium sporozoites remains unknown. \n \nWe used Focussed Ion Beam milling (FIB-milling) and \nelectron cryo tomography (cryo-ET) to image actin fila-\nments and other subcellular structures in Plasmodium \nfalciparum sporozoites (Fig. S1). Subvolume averaging \nwas used to determine the structure and a complete 3D \nmodel of F-actin within sporozoites (Fig. 1). F-actin was \npresent in all major subcellular compartments: the pel-\nlicular space (the intermembrane space between the \nplasma membrane and the inner membrane complex \nand the primary site for gliding machinery), the cytosol, \nand most remarkably in the nucleus.  Surprisingly, and \nunlike some previous in vitro  reports, we consistently \nobserved actin filaments longer than 100 nm, some up \nto 850 nm long (Fig. S2b). The mean length of 200 ± \n140 nm (standard deviation) is likely an underestimate \ndue to filament s being truncated by FIB -milling. The \nglobal F-actin concentration was measured to be 40 ± \n7 μM. \n \nWe first analysed F-actin in the pellicular space with a \nview to understand the types of F -actin dynamics \nneeded for rapid motility. At the apical pole, we ob-\nserved several filaments in close proximity to the pre-\nconoidal rings (Fig. 2). Recent observations in related \napicomplexans Cryptosporidium parvum and Toxo-\nplasma gondii suggest that pellicular actin is nucleated \nat the preconoidal rings, implying that this is a con-\nsistent apicomplexan feature11. However, in C. parvum \nand T. gondii, the channelling of filaments into the pel-\nlicular space is dependent on extrusion of the conoid –  \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.22.590301doi: bioRxiv preprint \n\nMolecular architecture of glideosome and nuclear F-actin in Plasmodium falciparum \n\t\nPražák et al. 2024 (preprint)   2 \n \n \n \n \n \n \n \n \n \nFig. 1: Discrete F-actin populations are found in the sporozoite. a, Slice through a tomogram showing a bundle of actin filaments \nin the nucleus. Insert shows a 3D representation of the actin bundle derived from subvolume averaging. b, Slice through the basal \nends of two sporozoites (see h for positioning of the slicing planes relative to the cell).   c,  Same as b, but overlaid with a 3D \nrepresentation of actin from the whole tomogram. d, Volume in c seen from the side. e, Slice through the average volume of \nsporozoite F-actin. f, Isosurface representation of the volume in e fitted with the molecular model of PDB 6TU415. g, Size distribution \nof pellicular F -actin at different subcellular regions. Filaments that were fully contained within tomograms are shown in black, \nwhereas those that were cut off by FIB milling are shown in grey. Bars represent medians. h, Cartoon representation of a sporozoite \ncell with colours corresponding to structures labelled in a and dotted lines showing lamella orientations for cells 1 and 2 in b and c. \n\t\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.22.590301doi: bioRxiv preprint \n\nMolecular architecture of glideosome and nuclear F-actin in Plasmodium falciparum \n\t\nPražák et al. 2024 (preprint)   3 \na structural feature that is missing in Plasmodium spo-\nrozoites.  \n  \nAnalysis of the length distribution of pellicular F-actin \nshows two distinct length regulatory steps.  (i) As fila-\nments are transported down the cell towards the basal \npole, they increase in length (Fig. 1g) and (ii) at the ba-\nsal pole, actin filaments are disassembled into shorter \nfilaments ( Fig. 1b-d,g).  Short filaments at the basal \npole supports a recently proposed model of severing \nmediated by coronin, an actin binding protein located \nat the basal end of gliding P. berghei  sporozoites12, \nlikely through recruitment of actin depolymerizing fac-\ntors13. The shorter filaments accumulate at the basal \npole, and in some cases form a 1-filament deep shell, \nsuggesting that actin disassembly may be rate-limiting \n(Fig. 1b,c). As actin disassembly occurs within the re-\nstricted pellicular space, a local actin monomer gradi-\nent likely forms. Therefore the path of glideosome actin \nfilaments towards the basal pole, up an actin monomer \ngradient, could account for the elongation we observe \nas filaments move down the cell, prior to their disas-\nsembly at the basal pole. While inspecting individual \ninstances of lateral pellicular actin, we observed two lo-\ncations where filaments were bound by densities con-\nsistent with myosin heads with tails leading to the IMC \n(Fig. 2f).  \n \nThe IMC at the basal pole was dotted with ~25 nm di-\nameter pores (Fig. S 3), a consistent feature with C. \nparvum11, but not T. gondii, which has a large opening \nin the IMC at the basal pore. On two occasions we ob-\nserved filamentous actin protruding through a basal \npore, suggesting that filamentous actin can pass \nthrough. It is possible that these pores could facilitate \nmore efficient exchange of G- and F-actin between the \npellicular space and the rest of the cell. This would re-\ncycle actin and lower the concentration of actin at the \nrear of the sporozoite (up to 60 mM local F -actin con-\ncentration 100 nm from the basal pole end ) and thus \ncould allow for more efficient gliding. Strikingly, we ob-\nserved few pores of the same size in other subcellular \nlocations, indicating that their location is regulated. It \nwould be reasonable to speculate that the posterior/ba-\nsal polar ring complex14 could be involved in the locali-\nsation of the pores. Notably, we have not observed a \nring-like structure at the basal po le, but rather a thick \namorphous layer, which we refer to as basal polar ma-\ntrix. \n \nIn C. parvum , IMC surface filaments (IMCFs) form \ntracks to guide actin filaments down the cell11. We did \nnot observe these in P. falciparum. Rather, the whole \nouter IMC surface is reinforced by a network of thin fil-\naments that bear no ultrastructural similarity to IMCFs \n(Fig. 2c-f). We refer to these filaments as pellicular \nintermediate filaments (PIFs). PIFs were always ori-\nented parallel to the long parasite axis and each other \nwith a spacing of approximately 20 nm (~5 - 40 nm) \nbetween them with few clear cross-links. The observed \nfeatures prompted us to hypothesise that PIFs are \nstructural elements that allow the force generated by \nthe glideosome to be distributed along the entire IMC. \nAlthough it has been suggested that the gliding ma-\nchinery is directly connected to subpellicular microtu-\nbules (SPMTs)16, with our current approach, we see no \ndirect connection between PIFs, actin and SPMTs \nthrough the IMC membranes (Fig. S 4). We observe \nglobular densities between the IMC membranes, but \nthese are not statistically associated with PIFs ( Fig. \nS4d).  \n \nApart from motility, F-actin is implicated in multiple cel-\nlular roles including intracellular transport, transcrip-\ntional regulation and cell structural support. We there-\nfore analysed the populations of F -actin that we ob-\nserved in compartments other than th e pellicular \nspace. In the cytosol, F -actin was found primarily as \nindividual filaments, with some bundles (10 filaments, \n4 bundles consisting of 2-8 filaments). It was not asso-\nciated in any obvious pattern with any subcellular ele-\nments, but it was typically oriented along the apico-ba-\nsal axis. The presence of F-actin in the cytosol was not \nunexpected as it has been suggested by other meth-\nods9. However, what was surprising, was the large \namount of F-actin that we observed within the nucleus. \nIn fact, the nucleus contains the majority of observed \nF-actin in sporozoites: approximately 60%. It was \nfound predominantly in bundles of ~3 -8 filaments with \nat least one filament less than 10 nm from the nuclear \nmembrane (global median distance of all filaments was \n21 ± 11 nm, Fig. 1a). Whether this is due to specific \nbinding to a component of the membrane or due to \nmarginalisation is not clear. While ther e have been \nmany reports of the presence of nuclear actin in mam-\nmalian cells, this is the first direct evidence of nuclear \nF-actin in the absence of staining or stabilising agents. \n \nIndeed, actin chromobody signals have indicated F-ac-\ntin accumulation at the nuclear position (in ~20% of \nsporozoites) during motility and invasion of apicom-\nplexan parasites, suggesting that a nuclear actin cage \nfacilitates efficient invasion and/or protects the nucleus \nfrom damage when the parasite undergoes con-\nstriction8,9. However, here we have observed extensive \nbundles within the nucleus itself  (Fig. 1a and 2d) . \nWhether this simply serves as an intra-nuclear protec-\ntive structure or fulfils additional molecular roles, such \nas mechanosensing, in the nucleus requires further in-\nvestigation.  \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.22.590301doi: bioRxiv preprint \n\nMolecular architecture of glideosome and nuclear F-actin in Plasmodium falciparum \n\t\nPražák et al. 2024 (preprint)   4 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nFig. 2: Visualising discrete actin populations within the 3D cellular context of motile sporozoites. Central cartoon repre-\nsents the approximate position of volumes a-c within a cell. a, Apical end of showing a single actin filament being nucleated at \nthe pre-conoidal rings (grey). b, Basal end of a sporozoite, showing cytoplasmic actin bundles as well as a build-up of pellicular \nactin. White arrowhead indicates an actin filament going through a basal pore. Shown is also an invagination of the IMC (ob-\nserved in three cells). c, A lateral section showing nuclear, cytosolic and pellicular F-actin. Dark lines on the outer surface of the \nIMC represent pellicular intermediate filaments (PIFs). d, A slice along the surface of the outer leaflet of the outer IMC membrane. \nSome filaments have been highlighted in green. e, A slice through the average volume of PIFs showing their position relative to \nthe IMC membranes. f, A slice through the tomogram (position shown in c) showing a pellicular actin filament (blue) connected \nto a PIF by two densities (red) consistent with myosin dimensions (observed twice). \n\t\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.22.590301doi: bioRxiv preprint \n\nMolecular architecture of glideosome and nuclear F-actin in Plasmodium falciparum \n\t\nPražák et al. 2024 (preprint)   5 \nMaterials and Methods \nObtaining sporozoites \nP. falciparum sporozoites (strain: NF54-ΔPf47-5’csp-GFP-Luc: \nexpressing a GFP-Luciferase fusion protein under the control of \nthe csp  promoter, genomic integration, no selection marker) \nwere prepared at TropIQ (Nijmegen, Netherlands). Gameto-\ncytes were fed to 2 day old female Anopheles stephensi mos-\nquitoes. Mosquito infection was confirmed 7 days post feeding \nby midgut dissection. At 7 days post infection, mosquitoes re-\nceived an extra non -infectious blood meal to boost sporozoite \nproduction. Two weeks post infection, sporozoites were isolated \nusing salivary gland dissection and shipped at room temperature \nin Leibovitz medium with 10% heat inactivated human serum. \n \nCryo-grid preparation \nP. falciparum sporozoites were checked under the fluorescent \nmicroscope and then diluted 1:4 into RPMI medium (without \nphenol red). 3 μl of parasites were applied onto a freshly \nplasma-cleaned UltrAufoil R1.2/1.3 300 mesh EM grid (Quanti-\nfoil) in a humidity controlled facility. Excess liquid was manually \nback-blotted and grids were plunged into a reservoir of \nethane/propane using a manual plunger. Grids were stored un-\nder liquid nitrogen until imaging. \n \nCryo FIB milling  \nGrids were clipped into autogrids modified for FIB preparation17 \nand loaded into either an Aquilos or an upgraded Aquilos2 cryo-\nFIB/SEM dual -beam microscope (Thermofisher Scientific). \nOverview tile sets were recorded using MAPS software (Ther-\nmofisher Scientific) before being sputter coated with a thin layer \nof platinum. Good sites with parasites were identified for lamella \npreparation before the coincident point between the electron \nbeam and the ion beam was determined for each point by stage \ntilt. Prior to milling, an organometallic platinum layer was depos-\nited onto the grids using a GIS (gas-injection-system). Lamellae \nwere milled manually until under 300 nm in a stepwise series of \ndecreasing currents. Milling was performed at the lowest possi-\nble angles to increase lamella length in thin cells. Finally, polish-\ning of all lamella was done at the end of the session as quickly \nas possible but always within 1.5 h to limit ice contamination \nfrom water deposition on the surface of the lamellae. Before re-\nmoving the samples, the grids were sputter coated with a final \nthin layer of platinum. Grids were stored in liquid nitrogen for a \nmaximum of 2 weeks before imaging in the TEM. \n \nTilt-series collection \nCryo-EM FIB-milled grids were rotated by 90° and loaded into a \nTitan Krios microscope (Thermofisher) equipped with a K3 direct \nelectron detector and (Bio -) Quantum energy filter (Gatan). \nTomographic data was collected with SerialEM with the energy-\nselecting slit set to 20 eV. Datasets were collected using the \ndose-symmetric acquisition scheme at a ± 65° tilt range with 3° \ntilt increments. For all datasets, 5 - 10 frames were collected and \naligned on the fly using SerialEM and the total fluence was kept \nto less than 120 e −Å2. Defoci between 3 and 8 μm underfocus \nwere used to record the tilt series’. \n \nTomogram reconstruction  \nFrames were aligned on the fly in SerialEM 18; CTF estimation, \nphase flipping and dose -weighting was performed in IMOD 19. \nTilt-series’ were aligned in IMOD either using patch-tracking or \nby using nanoparticles (likely gold or platinum) on lamella sur-\nfaces as fiducial markers. Tomograms were binned 4x and fil-\ntered in IMOD or by using Bsoft20. \n \nSubvolume averaging  \nSubvolume averaging was performed using PEET 21 as de-\nscribed previously 22. Model processing was done using \nTEMPy23, Scipy 24, Scikit -learn25, Matplotlib 26 and Numpy 27 in \nPython 3. Initial models were generated manually   by picking \nline segments using pairs of IMOD model points and then inter-\npolating particles at 1 voxel (1.3 nm) increments. The initial Y \naxes were aligned with the line segments and initially Y axis ro-\ntation angles were randomised. The initial reference was gener-\nated by averaging particles with the starting orientations, thus \ngenerating a featureless cylinder. A small subset of particles \n(~700) were refined to create a reference with F -actin features \nwhich was then used for alignment of ~ 70k initial positions. Du-\nplicate and low scoring particles were removed. In order to im-\nprove model completeness and allow separation of particles into \ntwo independent halves, the subvolume positions were then fit-\nted to a spline-smoothed helical model allowing for small varia-\ntion in helical pitch (Figure S2). Subvolume positions were then \ngenerated based on the best fitting model parameters. These \nwere split into two halves and aligned independently. Overlap-\nping particles between the two half-maps were removed before \ngenerating final half-maps. Fourier Shell Correlation was meas-\nured using Bsoft, suggesting 27 Å resolution at the 0.143 cutoff. \nParticles from the two half -datasets (11487 total) were then \ncombined and aligned together. The final volume was sharp-\nened using Bsoft with an arbitrarily chosen B-factor of -3000 for \nfitting and visualisation.  \n \nSegmentation and visualisation  \nMembrane segmentation was performed in IMOD, using draw-\ning tools followed by linear interpolation. These were then \nresampled using open3d to achieve an isotropic coordinate dis-\ntribution, which were then used to generate a volume using \nIMOD imodmop. F-actin, microtubules, apical polar ring and pre-\nconoidal rings were backplotted: average volumes were placed \ninto 3D volumes using coordinates determined by SVA. Actin \nand microtubule models were smoothed for backplotting. Sur-\nface visualisation was performed usin g UCSF ChimeraX or \nopen3d. Volume sections were visualised using IMOD 3dmod. \nPlots were generated using Matplotlib. \n \nLength measurements \nFilament lengths for comparison of nuclear, cytosolic and pellic-\nular filament lengths were derived from helical models based on \nsubvolume averaging positions (see above). Filament lengths \nfor comparison of apical, lateral and basal pellicular filament \nlengths were measured manually using 3dmod.  \n \nF-actin concentration \nThe number of actin subunits in observed F-actin was estimated \nfrom subvolume averaging (15,058) and manual length meas-\nurements (17165, assuming 38 nm per 13 subunits). The sub-\nvolume averaging-derived value is likely an underestimate due \nto cross-correlation based particle cleaning; it is the number of \nparticles after the first alignment step of the two independent da-\ntasets. The two estimates were used to calculate the experi-\nmental error, expressed as standard deviation. The total ob-\nserved volume of 29 tomo grams with an average thickness of \n244 nm was 4.2 x 10 -17 m3, of which cells made up approxi-\nmately 7/12. 9.7 x 10-19 mol in 2.4 x 10-14 L corresponds to 4.0 x \n10-5molL-1    \n\t\n\t\n\t\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.22.590301doi: bioRxiv preprint \n\nMolecular architecture of glideosome and nuclear F-actin in Plasmodium falciparum \n\t\nPražák et al. 2024 (preprint)   6 \nReferences \n1.\t Cowman,\tA.\tF.,\tHealer,\tJ.,\tMarapana,\tD.\t&\tMarsh,\tK.\tMalaria:\tBiology\t\nand\tDisease.\tCell\t167,\t610–624\t(2016).\t\n2.\t Amino,\t R.\t et\t al.\t Quantitative\t imaging\t of\t Plasmodium\t transmission\t\nfrom\tmosquito\tto\tmammal.\t Nat.\tMed.\t12,\t220–224\t(2006).\t\n3.\t Heintzelman,\tM.\tB.\tGliding\tmotility\tin\tapicomplexan\tparasites.\t Semin.\t\nCell\tDev.\tBiol.\t46,\t135–142\t(2015).\t\n4.\t Schmitz,\t S.\tet\t al.\t Malaria\t Parasite\t Actin\t Filaments\t are\t Very\t Short.\tJ.\t\nMol.\tBiol.\t349,\t113–125\t(2005).\t\n5.\t Vahokoski,\tJ.\tet\tal.\tStructural\tDifferences\tExplain\tDiverse\tFunctions\tof\t\nPlasmodium\tActins.\tPLoS\tPathog.\t10,\te1004091\t(2014).\t\n6.\t Lu,\tH.,\tFagnant,\tP.\tM.\t&\tTrybus,\tK.\tM.\tUnusual\tdynamics\tof\tthe\tdiver-\ngent\tmalaria\tparasite\tPfAct1\tactin\tfilament.\t Proc.\tNatl.\tAcad.\tSci.\tU.\tS.\t\nA.\t116,\t20418–20427\t(2019).\t\n7.\t Kumpula,\tE.-P.\tet\tal.\tApicomplexan\tactin\tpolymerization\tdepends\ton\t\nnucleation.\tSci.\tRep.\t7,\t12137\t(2017).\t\n8.\t Yee,\tM.,\tWalther,\tT.,\tFrischknecht,\tF.\t&\tDouglas,\tR.\tG.\tDivergent\tPlas-\nmodium\t actin\t residues\t are\t essential\t for\t filament\t localization,\t mos-\nquito\tsalivary\tgland\tinvasion\tand\tmalaria\ttransmission.\t PLOS\tPathog.\t\n18,\te1010779\t(2022).\t\n9.\t Del\t Rosario,\t M.\tet\t al.\t Apicomplexan\t F-actin\t is\t required\t for\t efficient\t\nnuclear\t entry\t during\t host\t cell\t invasion.\t EMBO\t Rep. \t 20,\t e48896\t\n(2019).\t\n10.\t Tosetti,\tN.,\tDos\tSantos\tPacheco,\tN.,\tSoldati -Favre,\tD.\t&\tJacot,\tD.\tThree\t\nF-actin\t assembly\t centers\t regulate\t organelle\t inheritance,\t cell -cell\t\ncommunication\t and\t motility\t in\t Toxoplasma\t gondii.\teLife\t 8,\t e42669\t\n(2019).\t\n11.\t Martinez,\tM.\tet\tal.\tOrigin\tand\tarrangement\tof\tactin\tfilaments\tfor\tglid-\ning\tmotility\tin\tapicomplexan\tparasites\trevealed\tby\tcryo -electron\tto-\nmography.\tNat.\tCommun.\t14,\t4800\t(2023).\t\n12.\t Bane,\t K.\t S.\tet\t al.\t The\t Actin\t Filament-Binding\t Protein\t Coronin\t Regu-\nlates\tMotility\tin\tPlasmodium\tSporozoites.\tPLOS\tPathog.\t12,\te1005710\t\n(2016).\t\n13.\t Douglas,\tR.\tG.\tet\tal.\tInter-subunit\tinteractions\tdrive\tdivergent\tdynam-\nics\t in\t mammalian\t and\t Plasmodium\t actin\t filaments.\t PLOS\t Biol.\t 16,\t\ne2005345\t(2018).\t\n14.\t De\t Niz,\t M.\tet\t al.\t Progress\t in\t imaging\t methods:\t insights\t gained\t into\t\nPlasmodium\tbiology.\tNat.\tRev.\tMicrobiol.\t15,\t37–54\t(2017).\t\n15.\t Vahokoski,\tJ.\t et\tal.\tHigh-resolution\tstructures\tof\tmalaria\tparasite\tac-\ntomyosin\tand\tactin\tfilaments.\t PLOS\tPathog.\t18,\te1010408\t(2022).\t\n16.\t Harding,\tC.\tR.\t et\tal.\tAlveolar\tproteins\tstabilize\tcortical\tmicrotubules\t\nin\tToxoplasma\tgondii.\tNat.\tCommun.\t10,\t401\t(2019).\t\n17.\t Schaffer,\tM.\tet\tal.\tCryo-focused\tIon\tBeam\tSample\tPreparation\tfor\tIm-\naging\t Vitreous\t Cells\t by\t Cryo -electron\t Tomography.\t Bio-Protoc.\t 5,\t\ne1575\t(2015).\t\n18.\t Mastronarde,\tD.\tN.\tDual -Axis\tTomography:\tAn\tApproach\twith\tAlign-\nment\tMethods\tThat\tPreserve\tResolution.\t J.\tStruct.\tBiol.\t120,\t343–352\t\n(1997).\t\n19.\t Kremer,\tJ.\tR.,\tMastronarde,\tD.\tN.\t&\tMcIntosh,\tJ.\tR.\tComputer\tvisualiza-\ntion\tof\tthree-dimensional\timage\tdata\tusing\tIMOD.\t J.\tStruct.\tBiol.\t116,\t\n71–76\t(1996).\t\n20.\t Heymann,\tJ.\tB.\tBsoft:\tImage\tand\tMolecular\tProcessing\tin\tElectron\tMi-\ncroscopy.\tJ.\tStruct.\tBiol.\t133,\t156–169\t(2001).\t\n21.\t Heumann,\tJ.\tM.,\tHoenger,\tA.\t&\tMastronarde,\tD.\tN.\tClustering\tand\tvari-\nance\tmaps\tfor\tcryo-electron\ttomography\tusing\twedge-masked\tdiffer-\nences.\tJ.\tStruct.\tBiol.\t175,\t288–299\t(2011).\t\n22.\t Ferreira,\tJ.\tL.\t et\tal. \tVariable\tmicrotubule\tarchitecture\tin\tthe\tmalaria\t\nparasite.\tNat.\tCommun.\t14,\t1216\t(2023).\t\n23.\t Cragnolini,\tT.\tet\tal.\tTEMPy2:\ta\tPython\tlibrary\twith\timproved\t3D\telec-\ntron\tmicroscopy\tdensity -fitting\tand\tvalidation\tworkflows.\t Acta\tCrys-\ntallogr.\tSect.\tStruct.\tBiol. \t77,\t41–47\t(2021).\t\n24.\t Virtanen,\tP.\tet\tal.\tSciPy\t1.0:\tfundamental\talgorithms\tfor\tscientific\tcom-\nputing\tin\tPython.\tNat.\tMethods\t17,\t261–272\t(2020).\t\n25.\t Buitinck,\tL.\t et\t al.\tAPI\tdesign\tfor\tmachine\tlearning\tsoftware:\texperi-\nences\t from\t the\t scikit -learn\t project.\t Preprint\t at\t\nhttps://doi.org/10.48550/arXiv.1309.0238\t(2013). \t\n26.\t Hunter,\tJ.\tD.\tMatplotlib:\tA\t2D\tGraphics\tEnvironment.\tComput.\tSci.\tEng.\t\n9,\t90–95\t(2007).\t\n27.\t Harris,\tC.\tR.\tet\tal.\tArray\tprogramming\twith\tNumPy.\t Nature\t585,\t357–\n362\t(2020).\t\n\t\n\t\nAcknowledgements \nWe\t thank\t Lindsay\t Baker\t for\t helpful\t discussions\t and\t Carolyn\t\nMoores for her continued support and critical reading of the \nmanuscript. Thank you to the CSSB EM facility team for their \nsupport. We gratefully acknowledge funding by HFSP long-term \npostdoctoral fellowship LT000024/2020-L (JLF), Infrastructures \nfor the control of vector -borne diseases (Infravec2) funded by \nthe EU’s Horizon 2 020 programme (grant agreement No \n731060) (JLF), Wellcome Career Development award \n227774/Z/23/Z (JLF), LOEWE Centre DRUID (“Novel Drugs \nTargets against Poverty-related and Neglected Tropical Infec-\ntious Diseases”) within the Hessian Excellence Program (RGD). \nFor the purpose of open access, the author has applied a Crea-\ntive Commons Attribution (CC BY) licence to any Author Ac-\ncepted Manuscript version arising. Biorxiv template from:\t\nwww.github.com/chrelli/bioRxiv-word-template \nAuthor contributions \nVP and JLF designed the study and experiments. JLF generated \nsamples and acquired data. JLF and VP processed and ana-\nlysed data. VP, JLF, RGD, DV, KG performed critical analysis of \nfindings. JLF, VP, RGD wrote the manuscript. VP generated fig-\nures.  \n \nData availability \nAll data are available on request. Subvolume average was de-\nposited on the EMDB. \n \nCode availability \nScripts are available on request. \n \nCompeting interests \nThe authors declare no competing interests. \n \n\t\n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.22.590301doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}