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

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Basic information

Entry
Database: PDB / ID: 6xr4
TitleIntegrative in situ structure of Parkinsons disease-linked human LRRK2
ComponentsLeucine-rich repeat serine/threonine-protein kinase 2
KeywordsSIGNALING PROTEIN / TRANSFERASE / kinase / GTPase / Parkinson's Disease / pseudo-kinase
Function / homology
Function and homology information


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 / cytoplasmic side of mitochondrial outer membrane / mitochondrion localization / 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 / multivesicular body, internal vesicle / regulation of protein kinase A signaling / striatum development / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / cellular response to dopamine / positive regulation of protein autoubiquitination / endoplasmic reticulum organization / presynaptic cytosol / positive regulation of programmed cell death / Wnt signalosome / GTP metabolic process / regulation of canonical Wnt signaling pathway / negative regulation of protein processing / syntaxin-1 binding / regulation of reactive oxygen species metabolic process / negative regulation of GTPase activity / exploration behavior / autolysosome / regulation of locomotion / protein kinase A binding / regulation of synaptic vesicle exocytosis / Golgi-associated vesicle / PTK6 promotes HIF1A stabilization / negative regulation of macroautophagy / clathrin binding / neuromuscular junction development / lysosome organization / regulation of mitochondrial fission / intracellular distribution of mitochondria / Golgi organization / positive regulation of nitric-oxide synthase biosynthetic process / locomotory exploration behavior / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / MAP kinase kinase kinase activity / canonical Wnt signaling pathway / positive regulation of protein kinase activity / cellular response to manganese ion / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / positive regulation of autophagy / JNK cascade / regulation of synaptic transmission, glutamatergic / dendrite cytoplasm / cellular response to starvation / tubulin binding / GTPase activator activity / phosphorylation / neuron projection morphogenesis / regulation of membrane potential / SNARE binding / excitatory postsynaptic potential / negative regulation of protein phosphorylation / mitochondrion organization / negative regulation of protein binding / positive regulation of protein ubiquitination / regulation of autophagy / determination of adult lifespan / mitochondrial membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / peptidyl-threonine phosphorylation / calcium-mediated signaling / positive regulation of MAP kinase activity / trans-Golgi network / regulation of protein stability
Similarity search - Function
: / C-terminal of Roc (COR) domain / C-terminal of Roc, COR, 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 ...: / C-terminal of Roc (COR) domain / C-terminal of Roc, COR, 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 / WD40-repeat-containing domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / WD40/YVTN repeat-like-containing domain superfamily / Serine/Threonine protein kinases, catalytic domain / 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)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 14 Å
AuthorsVilla, E. / Lasker, K. / Audagnotto, M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM123494-01 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
DepositionJul 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 30, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Leucine-rich repeat serine/threonine-protein kinase 2
B: Leucine-rich repeat serine/threonine-protein kinase 2


Theoretical massNumber of molelcules
Total (without water)572,9012
Polymers572,9012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models53

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Components

#1: Protein Leucine-rich repeat serine/threonine-protein kinase 2 / Dardarin / Coordinate model: Cα atoms only


Mass: 286450.719 Da / Num. of mol.: 2 / Mutation: I2020T / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293
References: UniProt: Q5S007, non-specific serine/threonine protein kinase, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: HEK293 cell / Type: CELL
Details: Dimer within the oligomer of microtubule-bound human LRRK2-I2020T in HEK cells
Entity ID: all / Source: NATURAL
Source (natural)Organism: Homo sapiens (human) / Cell: HEK293 / Cellular location: cytosol
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: FIB-milled cellular samples expressing Parkinson's disease-related mutant LRRK2 (I2020T) proteins in human embryonic kidney cells (HEK293 cells)
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum SE / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
7IMPmodel fitting
9IMPmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4307
Details: Each model in the conformation ensemble represents a cluster. Models are arranged sequentially from lowest to highest cluster RMSD. Model 1 is the best representative model. Model-Cluster ...Details: Each model in the conformation ensemble represents a cluster. Models are arranged sequentially from lowest to highest cluster RMSD. Model 1 is the best representative model. Model-Cluster correpondence is as follows: Model 1 = Cluster 20 Model 2 = Cluster 16 Model 3 = Cluster 24 Model 4 = Cluster 32 Model 5 = Cluster 14 Model 6 = Cluster 1 Model 7 = Cluster 44 Model 8 = Cluster 11 Model 9 = Cluster 3 Model 10 = Cluster 22 Model 11 = Cluster 4 Model 12 = Cluster 12 Model 13 = Cluster 19 Model 14 = Cluster 7 Model 15 = Cluster 31 Model 16 = Cluster 26 Model 17 = Cluster 52 Model 18 = Cluster 2 Model 19 = Cluster 45 Model 20 = Cluster 8 Model 21 = Cluster 5 Model 22 = Cluster 23 Model 23 = Cluster 42 Model 24 = Cluster 25 Model 25 = Cluster 43 Model 26 = Cluster 27 Model 27 = Cluster 39 Model 28 = Cluster 6 Model 29 = Cluster 13 Model 30 = Cluster 28 Model 31 = Cluster 21 Model 32 = Cluster 10 Model 33 = Cluster 53 Model 34 = Cluster 30 Model 35 = Cluster 35 Model 36 = Cluster 38 Model 37 = Cluster 33 Model 38 = Cluster 40 Model 39 = Cluster 37 Model 40 = Cluster 29 Model 41 = Cluster 51 Model 42 = Cluster 41 Model 43 = Cluster 15 Model 44 = Cluster 9 Model 45 = Cluster 17 Model 46 = Cluster 50 Model 47 = Cluster 48 Model 48 = Cluster 49 Model 49 = Cluster 18 Model 50 = Cluster 46 Model 51 = Cluster 36 Model 52 = Cluster 47 Model 53 = Cluster 34
Symmetry type: POINT
EM volume selectionNum. of tomograms: 12 / Num. of volumes extracted: 11508
Atomic model buildingProtocol: OTHER
RefinementDetails: Each model in the conformation ensemble represents a cluster. Models are arranged sequentially from lowest to highest cluster RMSD. Model 1 is the best representative model. Model-Cluster ...Details: Each model in the conformation ensemble represents a cluster. Models are arranged sequentially from lowest to highest cluster RMSD. Model 1 is the best representative model. Model-Cluster correpondence is as follows: Model 1 = Cluster 20 Model 2 = Cluster 16 Model 3 = Cluster 24 Model 4 = Cluster 32 Model 5 = Cluster 14 Model 6 = Cluster 1 Model 7 = Cluster 44 Model 8 = Cluster 11 Model 9 = Cluster 3 Model 10 = Cluster 22 Model 11 = Cluster 4 Model 12 = Cluster 12 Model 13 = Cluster 19 Model 14 = Cluster 7 Model 15 = Cluster 31 Model 16 = Cluster 26 Model 17 = Cluster 52 Model 18 = Cluster 2 Model 19 = Cluster 45 Model 20 = Cluster 8 Model 21 = Cluster 5 Model 22 = Cluster 23 Model 23 = Cluster 42 Model 24 = Cluster 25 Model 25 = Cluster 43 Model 26 = Cluster 27 Model 27 = Cluster 39 Model 28 = Cluster 6 Model 29 = Cluster 13 Model 30 = Cluster 28 Model 31 = Cluster 21 Model 32 = Cluster 10 Model 33 = Cluster 53 Model 34 = Cluster 30 Model 35 = Cluster 35 Model 36 = Cluster 38 Model 37 = Cluster 33 Model 38 = Cluster 40 Model 39 = Cluster 37 Model 40 = Cluster 29 Model 41 = Cluster 51 Model 42 = Cluster 41 Model 43 = Cluster 15 Model 44 = Cluster 9 Model 45 = Cluster 17 Model 46 = Cluster 50 Model 47 = Cluster 48 Model 48 = Cluster 49 Model 49 = Cluster 18 Model 50 = Cluster 46 Model 51 = Cluster 36 Model 52 = Cluster 47 Model 53 = Cluster 34

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