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- EMDB-20825: In situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound... -

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Basic information

Entry
Database: EMDB / ID: EMD-20825
TitleIn situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound_LRRK2(I2020T)_Tight Mask_A
Map dataIn situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound_LRRK2(I2020T)_Tight Mask_A
Sample
  • Cell: FIB-milled cellular samples expressing Parkinson's disease mutant LRRK2 (I2020T) in HEK293 cells
    • Protein or peptide: Leucine Rich Repeat Kinase 2
Keywordskinase / GTPase / Parkinson's Disease / pseudo-kinase / SIGNALING PROTEIN / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


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 / regulation of cell projection organization / tangential migration from the subventricular zone to the olfactory bulb ...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 / regulation of cell projection organization / 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 / peroxidase inhibitor activity / negative regulation of late endosome to lysosome transport / regulation of mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / positive regulation of dopamine receptor signaling pathway / regulation of synaptic vesicle transport / regulation of CAMKK-AMPK signaling cascade / regulation of lysosomal lumen pH / amphisome / co-receptor binding / mitochondrion localization / negative regulation of excitatory postsynaptic potential / regulation of retrograde transport, endosome to Golgi / regulation of dopamine receptor signaling pathway / positive regulation of microglial cell activation / positive regulation of synaptic vesicle endocytosis / negative regulation of autophagosome assembly / cytoplasmic side of mitochondrial outer membrane / neuron projection arborization / regulation of cAMP/PKA signal transduction / olfactory bulb development / striatum development / multivesicular body, internal vesicle / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / JUN kinase kinase kinase activity / cellular response to dopamine / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / Wnt signalosome / positive regulation of programmed cell death / negative regulation of protein processing / GTP metabolic process / syntaxin-1 binding / regulation of canonical Wnt signaling pathway / negative regulation of GTPase activity / exploration behavior / regulation of reactive oxygen species metabolic process / lysosome organization / clathrin binding / Golgi-associated vesicle / regulation of locomotion / protein kinase A binding / phosphorylation / negative regulation of macroautophagy / PTK6 promotes HIF1A stabilization / neuromuscular junction development / regulation of synaptic vesicle exocytosis / regulation of mitochondrial fission / Golgi organization / intracellular distribution of mitochondria / locomotory exploration behavior / regulation of synaptic vesicle endocytosis / endoplasmic reticulum exit site / autolysosome / microvillus / MAP kinase kinase kinase activity / negative regulation of Notch signaling pathway / positive regulation of protein kinase activity / Rho protein signal transduction / cellular response to manganese ion / canonical Wnt signaling pathway / presynaptic cytosol / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / regulation of synaptic transmission, glutamatergic / phagocytic vesicle / JNK cascade / positive regulation of autophagy / dendrite cytoplasm / negative regulation of protein binding / peptidyl-threonine phosphorylation / positive regulation of MAP kinase activity / tubulin binding / GTPase activator activity / SNARE binding / neuron projection morphogenesis / cellular response to starvation / positive regulation of protein ubiquitination / regulation of membrane potential / excitatory postsynaptic potential / determination of adult lifespan / cellular response to reactive oxygen species / mitochondrion organization / trans-Golgi network / calcium-mediated signaling / mitochondrial membrane
Similarity search - Function
LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain / Ras of Complex, Roc, domain of DAPkinase / Roc domain profile. / Roc domain / Leucine-rich repeats, bacterial type ...LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain / Ras of Complex, Roc, domain of DAPkinase / Roc domain profile. / Roc domain / Leucine-rich repeats, bacterial type / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Rab subfamily of small GTPases / Leucine-rich repeat domain superfamily / Ankyrin repeat-containing domain superfamily / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40-repeat-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Leucine-rich repeat serine/threonine-protein kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 14.0 Å
AuthorsBoehning J / Buschauer R / Watanabe R / Villa E
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM123494 United States
Michael J. Fox Foundation11425 United States
CitationJournal: Cell / Year: 2020
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
DepositionOct 14, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseAug 19, 2020-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6xr4
  • Surface level: 0.05
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6xr4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20825.png

Downloads & links

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Map

FileDownload / File: emd_20825.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ structure of LRRK2(I2020T)-Microtubule: Microtubule_bound_LRRK2(I2020T)_Tight Mask_A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 128 pix.
= 281.6 Å		2.2 Å/pix.
x 128 pix.
= 281.6 Å		2.2 Å/pix.
x 128 pix.
= 281.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.12805797 - 0.20926288
Average (Standard dev.)0.0029853692 (±0.030524667)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z281.600281.600281.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1280.2090.003

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Supplemental data

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Sample components

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Entire : FIB-milled cellular samples expressing Parkinson's disease mutant...

EntireName: FIB-milled cellular samples expressing Parkinson's disease mutant LRRK2 (I2020T) in HEK293 cells
Components
  • Cell: FIB-milled cellular samples expressing Parkinson's disease mutant LRRK2 (I2020T) in HEK293 cells
    • Protein or peptide: Leucine Rich Repeat Kinase 2

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Supramolecule #1: FIB-milled cellular samples expressing Parkinson's disease mutant...

SupramoleculeName: 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)
Source (natural)Organism: Homo sapiens (human) / Cell: HEK293

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Macromolecule #1: Leucine Rich Repeat Kinase 2

MacromoleculeName: Leucine Rich Repeat Kinase 2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Details: unspecified
VitrificationCryogen name: ETHANE-PROPANE
DetailsFIB-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

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied 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
ExtractionNumber tomograms: 12 / Number images used: 11508
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-6xr4:
Integrative in situ structure of Parkinsons disease-linked human LRRK2

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