{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/DesignPRIDE.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8729LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8784LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8730LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8725LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8733LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8724LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8734LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8722LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8723LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8726LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8732LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8728LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8727LD.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/X8731LD.raw"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063391/20240523_143909_Ines_Drinnenberg_Quanti_07052024.sne"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["damarys.loew@curie.fr"],"submitter":["Vanessa Masson"],"technology_type":["Affinity purification coupled with mass spectrometry proteomics","Mass Spectrometry"],"software":[""],"submitter_keywords":["Kinetochore","Insects","Structure"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD063391"],"tissue":["Cell Culture"],"sample_protocol":["Cultures of the Sf9 strains expressing full-length S. frugiperda kinetochore proteins fused to a C-terminal 3XFLAG tags or control wild-type Sf9 cells were grown to exponential phase in Sf-900 II SFM medium (Gibco Cat#10902-088). For each strain, 100 ml of 5-6 x 109 cells were harvested by centrifuging for 10 min at 300 xg, and the pellets were washed twice in cold PBS. The pellets were resuspended in 3 mL HDG150 Buffer (20 mM HEPES pH 7.0, 150 mM KCl, 10% glycerol, 0.5 mM DTT, 1 tablet cOmplete Protease Inhibitor), and then cells were disrupted with 50 strokes in a dounce homogenizer at 4 °C. The dounced fraction was centrifuged at 1700 xg for 10 min at 4 °C, and the nuclei (lower fraction) were gently resuspended with 2 mL HDG150 Buffer and re-centrifuged in the same conditions. The nuclei were resuspended in a final volume of 2 mL using HDG150 Buffer. Nuclear extracts were prepared by passing the nuclear fraction 10 times through a 20G 1 ½” needle (0.9 x 38 mm) and then 5 times through a 25G 3/8” needle (0.5 x 16 mm). The nuclear extracts were centrifuged at 20 000 xg for 10 min at 4 °C in microcentrifuge tubes. The pellets were resuspended in HDG150 Buffer and pooled together at a final volume of 2 mL. To prepare the chromatin, the nuclear extracts were digested with ~10 units MNase (Sigma Cat#N3755-500UN) for 1 hour at 4 °C on a roller, 3 mM CaCl2 was added to the digestions. The MNase digestions were stopped by adding 50 µL of 0.2 M EGTA. To solubilize the digested chromatin, 4 mL of HDG400 Buffer (20 mM HEPES pH 7.0, 400 mM KCl, 10% glycerol, 1 mM DTT, 0.05% NP-40, 1 tablet cOmplete Protease Inhibitor) was added to the samples and incubated for 2 h at 4 °C on a roller. The samples were centrifuged at 8000 xg for 10 min at 4 °C. The supernatants were saved to bind to the anti-FLAG M2 beads (Sigma Cat#M8823; RRID:AB_2637089). The M2 magnetic beads were prepared according to the manufacturer’s recommendations. The digested chromatin samples were incubated with 30 µL anti-FLAG M2 beads. The beads were washed four times with 1 mL HDGN320 Buffer (20 mM HEPES pH 7.0, 320 mM KCl, 10% glycerol, 1 mM DTT, 0.05% NP-40, 1 tablet cOmplete Protease Inhibitor). For proteomic analyses, IP samples were washed trice with 100 µL of 100 mM NH4HCO3 (ABC) and proteins were trypsin/lysC (0.2 µg, Promega) digested directly on beads in a total volume of 100 µL of 100 mM ABC for one hour at 37 °C while vortexed at 1000 rpm. Samples were then cleaned on homemade C18 StageTips (AttractSPE Disk Bio C18-100.47.20 Affinisep), before being eluted using 40/60 CH3CN/H2O + 0.1% formic acid and vacuum concentrated to dryness. Peptides were reconstituted in 10µL of injection buffer in 0.3% trifluoroacetic acid before liquid chromatography-tandem mass spectrometry analysis."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["For identification, the data were searched against the Spodoptera frugiperda corn annotation OGS6.1 database (https://bipaa.genouest.org/sp/spodoptera_frugiperda_pub/) using Pulsar search engine through Spectronaut v18.7 (Biognosys) by directDIA analysis using default search settings. Enzyme specificity was set to trypsin and a maximum of two missed cleavage sites was allowed.  N-terminal acetylation and oxidation of methionine were set as variable modifications. The resulting files were further processed using myProMS v3.10. https://github.com/bioinfo-pf-curie/myproms [PMID: 17610305] For protein quantification, MS2 ion XICs (sum and fragment peak areas) from proteotypic peptides shared between compared conditions (TopN matching) were used with missed cleavages allowed. Median and scale normalization at peptide level was applied on the total signal to correct the XICs for each biological replicate (N =2 or 3). To evaluate the statistical significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed, and a two sided T-test was apply on the fold change estimated by the model. The p-values were then adjusted using the Benjamini–Hochberg FDR procedure."],"omics_type":["Proteomics"],"labhead":["Damarys Loew"],"instrument_platform":[""],"labhead_affiliation":["Head of the Curie Institute Mass Spectrometry Platform"],"submission_type":["PARTIAL"],"species":["Spodoptera Frugiperda"],"submitter_mail":["vanessa.masson@curie.fr"],"publication":["Not available"],"submitter_affiliation":["Institut Curie - Centre de Recherche"],"submitter_country":["France"],"additional_accession":[]},"is_claimable":false,"name":"Architecture and Function of Holocentric CENP-A-Independent Inner Kinetochores","description":"Kinetochores are essential macromolecular complexes anchoring chromosomes to the mitotic spindle, ensuring faithful cell division. Despite their critical role, the structural organization of kinetochores over large centromeric regions and across diverse species remains poorly understood. We present the inner kinetochore (CCAN) structure of the silkmoth Bombyx mori, an insect without the canonical centromeric CENP-A gene and with chromosome-wide centromeric activity (holocentric). Our analyses reveal a ring-shaped complex with structural parallels to the known human and yeast kinetochores. Notably, B. mori CCAN incorporates four previously uncharacterized proteins, Centromeric Subunits 1-4, which have unexpected evolutionary relationships to the outer kinetochore Dam1/DASH complex. We demonstrate that CCAN binds DNA as a distinctive head-to-head dimer, folding the DNA into a loop and generating an alternative point-centromere-like architecture poised for chromosome segregation. Our work establishes this self-contained CCAN dimer as a key structural unit that forms the basis of a holocentric organization and suggests that large-scale centromere architectures can emerge from the modular arrangement of such discrete kinetochore units.","dates":{"publication":"2026-04-20","submission":"2025-04-28"},"accession":"PXD063391","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:5722","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","NEWT:1211601","NEWT:876138","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:1096976","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:84645","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:197","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:286","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:1148","NEWT:580240","NEWT:294128","NEWT:11676","NEWT:55571","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:243277","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:460519","NEWT:1187947","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:432359","NEWT:46835","NEWT:1182263","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:253","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:32644","NEWT:499175","NEWT:109779","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:357","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NEWT:28038","NEWT:1663","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:327159","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:303","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1121114","NEWT:663","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:941442","NEWT:220668","NEWT:13076","NEWT:1249668","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:550","NEWT:675060","NEWT:4081","NEWT:334542","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:1715256","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:507601","NEWT:643680","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:102169","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NEWT:1908","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:353152","NEWT:2829","NEWT:366581","NEWT:216599","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:334747","NEWT:61235","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:170187","NEWT:260707","NCBITaxon:6191","NEWT:1836","NEWT:185431","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:2697049","NEWT:105231","NEWT:1216981","NCBITaxon:6073","NEWT:884204","NEWT:6279","NEWT:1123869","NEWT:9544","NEWT:7370","NEWT:83906","NEWT:607699","NEWT:6282","NEWT:208964","NEWT:1134506","NEWT:575584","NEWT:38783","NEWT:8727","NEWT:4006","NEWT:8726","NEWT:6426","NEWT:6669","NEWT:10090","NEWT:4120","NEWT:51515","NEWT:5693","NEWT:8724","NEWT:51511","NEWT:92867","NEWT:8723","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