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- EMDB-20825: In situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-20825 | |||||||||
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Title | In situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound_LRRK2(I2020T)_Tight Mask_A | |||||||||
![]() | In situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound_LRRK2(I2020T)_Tight Mask_A | |||||||||
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![]() | kinase / GTPase / Parkinson's Disease / pseudo-kinase / SIGNALING PROTEIN / CYTOSOLIC PROTEIN | |||||||||
Function / homology | ![]() peroxidase inhibitor activity / caveola neck / negative regulation of thioredoxin peroxidase activity by peptidyl-threonine phosphorylation / negative regulation of protein processing involved in protein targeting to mitochondrion / Wnt signalosome assembly / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / regulation of kidney size / regulation of neuron maturation / tangential migration from the subventricular zone to the olfactory bulb ...peroxidase inhibitor activity / caveola neck / negative regulation of thioredoxin peroxidase activity by peptidyl-threonine phosphorylation / negative regulation of protein processing involved in protein targeting to mitochondrion / Wnt signalosome assembly / beta-catenin destruction complex binding / regulation of branching morphogenesis of a nerve / regulation of kidney size / regulation of neuron maturation / tangential migration from the subventricular zone to the olfactory bulb / protein localization to endoplasmic reticulum exit site / GTP-dependent protein kinase activity / regulation of neuroblast proliferation / regulation of ER to Golgi vesicle-mediated transport / negative regulation of late endosome to lysosome transport / regulation of synaptic vesicle transport / regulation of mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / positive regulation of dopamine receptor signaling pathway / regulation of lysosomal lumen pH / regulation of CAMKK-AMPK signaling cascade / amphisome / mitochondrion localization / cytoplasmic side of mitochondrial outer membrane / co-receptor binding / regulation of retrograde transport, endosome to Golgi / negative regulation of excitatory postsynaptic potential / regulation of dopamine receptor signaling pathway / negative regulation of autophagosome assembly / positive regulation of microglial cell activation / neuron projection arborization / positive regulation of synaptic vesicle endocytosis / JUN kinase kinase kinase activity / olfactory bulb development / regulation of protein kinase A signaling / multivesicular body, internal vesicle / striatum development / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / cellular response to dopamine / presynaptic cytosol / positive regulation of protein autoubiquitination / endoplasmic reticulum organization / positive regulation of programmed cell death / Wnt signalosome / regulation of canonical Wnt signaling pathway / GTP metabolic process / negative regulation of protein processing / syntaxin-1 binding / regulation of reactive oxygen species metabolic process / negative regulation of GTPase activity / exploration behavior / autolysosome / protein kinase A binding / regulation of locomotion / Golgi-associated vesicle / neuromuscular junction development / regulation of synaptic vesicle exocytosis / PTK6 promotes HIF1A stabilization / clathrin binding / negative regulation of macroautophagy / regulation of mitochondrial fission / lysosome organization / intracellular distribution of mitochondria / positive regulation of nitric-oxide synthase biosynthetic process / locomotory exploration behavior / Golgi organization / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / MAP kinase kinase kinase activity / positive regulation of protein kinase activity / canonical Wnt signaling pathway / cellular response to manganese ion / positive regulation of autophagy / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / JNK cascade / regulation of synaptic transmission, glutamatergic / excitatory postsynaptic potential / dendrite cytoplasm / cellular response to starvation / tubulin binding / mitochondrion organization / GTPase activator activity / neuron projection morphogenesis / SNARE binding / negative regulation of protein phosphorylation / negative regulation of protein binding / positive regulation of protein ubiquitination / regulation of autophagy / regulation of membrane potential / determination of adult lifespan / mitochondrial membrane / calcium-mediated signaling / peptidyl-threonine phosphorylation / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / positive regulation of MAP kinase activity / regulation of protein stability / protein localization / trans-Golgi network Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 14.0 Å | |||||||||
![]() | Boehning J / Buschauer R / Watanabe R / Villa E | |||||||||
Funding support | ![]()
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![]() | ![]() Title: The In Situ Structure of Parkinson's Disease-Linked LRRK2. Authors: Reika Watanabe / Robert Buschauer / Jan Böhning / Martina Audagnotto / Keren Lasker / Tsan-Wen Lu / Daniela Boassa / Susan Taylor / Elizabeth Villa / ![]() Abstract: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light ...Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 7.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.7 KB 13.7 KB | Display Display | ![]() |
Images | ![]() | 55.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 451.1 KB | Display | ![]() |
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Full document | ![]() | 450.7 KB | Display | |
Data in XML | ![]() | 5.4 KB | Display | |
Data in CIF | ![]() | 6.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6xr4MC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | In situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound_LRRK2(I2020T)_Tight Mask_A | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : FIB-milled cellular samples expressing Parkinson's disease mutant...
Entire | Name: FIB-milled cellular samples expressing Parkinson's disease mutant LRRK2 (I2020T) in HEK293 cells |
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Components |
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-Supramolecule #1: FIB-milled cellular samples expressing Parkinson's disease mutant...
Supramolecule | Name: FIB-milled cellular samples expressing Parkinson's disease mutant LRRK2 (I2020T) in HEK293 cells type: cell / ID: 1 / Parent: 0 / Macromolecule list: all Details: FIB-milled cellular samples expressing Parkinson's disease-related mutant LRRK2 (I2020T) proteins in human embryonic kidney cells (HEK293 cells) |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Leucine Rich Repeat Kinase 2
Macromolecule | Name: Leucine Rich Repeat Kinase 2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Sequence | String: MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSER ASKLFQGKNI HVPLLIVLD SYMRVASVQQ VGWSLLCKLI EVCPGTMQSL MGPQDVGNDW EVLGVHQLIL K MLTVHNAS VNLSVIGLKT LDLLLTSGKI TLLILDEESD IFMLIFDAMH ...String: MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSER ASKLFQGKNI HVPLLIVLD SYMRVASVQQ VGWSLLCKLI EVCPGTMQSL MGPQDVGNDW EVLGVHQLIL K MLTVHNAS VNLSVIGLKT LDLLLTSGKI TLLILDEESD IFMLIFDAMH SFPANDEVQK LG CKALHVL FERVSEEQLT EFVENKDYMI LLSALTNFKD EEEIVLHVLH CLHSLAIPCN NVE VLMSGN VRCYNIVVEA MKAFPMSERI QEVSCCLLHR LTLGNFFNIL VLNEVHEFVV KAVQ QYPEN AALQISALSC LALLTETIFL NQDLEEKNEN QENDDEGEED KLFWLEACYK ALTWH RKNK HVQEAACWAL NNLLMYQNSL HEKIGDEDGH FPAHREVMLS MLMHSSSKEV FQASAN ALS TLLEQNVNFR KILLSKGIHL NVLELMQKHI HSPEVAESGC KMLNHLFEGS NTSLDIM AA VVPKILTVMK RHETSLPVQL EALRAILHFI VPGMPEESRE DTEFHHKLNM VKKQCFKN D IHKLVLAALN RFIGNPGIQK CGLKVISSIV HFPDALEMLS LEGAMDSVLH TLQMYPDDQ EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA SFSKLLVHH SFDLVIFHQM SSNIMEQKDQ QFLNLCCKCF AKVAMDDYLK NVMLERACDQ N NSIMVECL LLLGADANQA KEGSSLICQV CEKESSPKLV ELLLNSGSRE QDVRKALTIS IG KGDSQII SLLLRRLALD VANNSICLGG FCIGKVEPSW LGPLFPDKTS NLRKQTNIAS TLA RMVIRY QMKSAVEEGT ASGSDGNFSE DVLSKFDEWT FIPDSSMDSV FAQSDDLDSE GSEG SFLVK KKSNSISVGE FYRDAVLQRC SPNLQRHSNS LGPIFDHEDL LKRKRKILSS DDSLR SSKL QSHMRHSDSI SSLASEREYI TSLDLSANEL RDIDALSQKC CISVHLEHLE KLELHQ NAL TSFPQQLCET LKSLTHLDLH SNKFTSFPSY LLKMSCIANL DVSRNDIGPS VVLDPTV KC PTLKQFNLSY NQLSFVPENL TDVVEKLEQL ILEGNKISGI CSPLRLKELK ILNLSKNH I SSLSENFLEA CPKVESFSAR MNFLAAMPFL PPSMTILKLS QNKFSCIPEA ILNLPHLRS LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI PPEIGCLEN LTSLDVSYNL ELRSFPNEMG KLSKIWDLPL DELHLNFDFK HIGCKAKDII R FLQQRLKK AVPYNRMKLM IVGNTGSGKT TLLQQLMKTK KSDLGMQSAT VGIDVKDWPI QI RDKRKRD LVLNVWDFAG REEFYSTHPH FMTQRALYLA VYDLSKGQAE VDAMKPWLFN IKA RASSSP VILVGTHLDV SDEKQRKACM SKITKELLNK RGFPAIRDYH FVNATEESDA LAKL RKTII NESLNFKIRD QLVVGQLIPD CYVELEKIIL SERKNVPIEF PVIDRKRLLQ LVREN QLQL DENELPHAVH FLNESGVLLH FQDPALQLSD LYFVEPKWLC KIMAQILTVK VEGCPK HPK GIISRRDVEK FLSKKRKFPK NYMSQYFKLL EKFQIALPIG EEYLLVPSSL SDHRPVI EL PHCENSEIII RLYEMPYFPM GFWSRLINRL LEISPYMLSG RERALRPNRM YWRQGIYL N WSPEAYCLVG SEVLDNHPES FLKITVPSCR KGCILLGQVV DHIDSLMEEW FPGLLEIDI CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI LADLPRNIM LNNDELEFEQ APEFLLGDGS FGSVYRAAYE GEEVAVKIFN KHTSLRLLRQ E LVVLCHLH HPSLISLLAA GIRPRMLVME LASKGSLDRL LQQDKASLTR TLQHRIALHV AD GLRYLHS AMIIYRDLKP HNVLLFTLYP NAAIIAKIAD YGTAQYCCRM GIKTSEGTPG FRA PEVARG NVIYNQQADV YSFGLLLYDI LTTGGRIVEG LKFPNEFDEL EIQGKLPDPV KEYG CAPWP MVEKLIKQCL KENPQERPTS AQVFDILNSA ELVCLTRRIL LPKNVIVECM VATHH NSRN ASIWLGCGHT DRGQLSFLDL NTEGYTSEEV ADSRILCLAL VHLPVEKESW IVSGTQ SGT LLVINTEDGK KRHTLEKMTD SVTCLYCNSF SKQSKQKNFL LVGTADGKLA IFEDKTV KL KGAAPLKILN IGNVSTPLMC LSESTNSTER NVMWGGCGTK IFSFSNDFTI QKLIETRT S QLFSYAAFSD SNIITVVVDT ALYIAKQNSP VVEVWDKKTE KLCGLIDCVH FLREVMVKE NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT AQLGSLKNV MLVLGYNRKN TEGTQKQKEI QSCLTVWDIN LPHEVQNLEK HIEVRKELAE K MRRTSVE |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | subtomogram averaging |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 7 |
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Grid | Model: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Details: unspecified |
Vitrification | Cryogen name: ETHANE-PROPANE |
Details | FIB-milled cellular samples expressing Parkinson's disease-related mutant LRRK2 (I2020T) proteins in human embryonic kidney cells (HEK293 cells) |
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Electron microscopy
Microscope | FEI POLARA 300 |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 4307 |
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Extraction | Number tomograms: 12 / Number images used: 11508 |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
-Atomic model buiding 1
Refinement | Protocol: OTHER |
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Output model | ![]() PDB-6xr4: |