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Yorodumi- PDB-7thz: Structure of Leucine Rich Repeat Kinase 2's ROC domain interactin... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7thz | |||||||||||||||
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Title | Structure of Leucine Rich Repeat Kinase 2's ROC domain interacting with the microtubule facing the plus end | |||||||||||||||
Components | Leucine-rich repeat serine/threonine-protein kinase 2 | |||||||||||||||
Keywords | CYTOSOLIC PROTEIN / parkinson's disease / microtubule / kinase / gtpase | |||||||||||||||
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 mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / regulation of synaptic vesicle transport / regulation of lysosomal lumen pH / positive regulation of dopamine receptor signaling pathway / amphisome / regulation of CAMKK-AMPK signaling cascade / cytoplasmic side of mitochondrial outer membrane / co-receptor binding / mitochondrion localization / 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 / regulation of dendritic spine morphogenesis / striatum development / multivesicular body, internal vesicle / protein localization to mitochondrion / cellular response to dopamine / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / presynaptic cytosol / positive regulation of programmed cell death / Wnt signalosome / GTP metabolic process / negative regulation of protein processing / regulation of canonical Wnt signaling pathway / syntaxin-1 binding / negative regulation of GTPase activity / regulation of reactive oxygen species metabolic process / exploration behavior / regulation of locomotion / protein kinase A binding / regulation of synaptic vesicle exocytosis / PTK6 promotes HIF1A stabilization / Golgi-associated vesicle / negative regulation of macroautophagy / clathrin binding / neuromuscular junction development / lysosome organization / regulation of mitochondrial fission / intracellular distribution of mitochondria / Golgi organization / autolysosome / : / locomotory exploration behavior / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / MAP kinase kinase kinase activity / positive regulation of protein kinase activity / cellular response to manganese ion / canonical Wnt signaling pathway / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / phosphorylation / positive regulation of autophagy / JNK cascade / regulation of synaptic transmission, glutamatergic / dendrite cytoplasm / GTPase activator activity / tubulin binding / cellular response to starvation / neuron projection morphogenesis / regulation of membrane potential / SNARE binding / excitatory postsynaptic potential / negative regulation of protein phosphorylation / negative regulation of protein binding / positive regulation of protein ubiquitination / regulation of autophagy / mitochondrion organization / determination of adult lifespan / peptidyl-threonine phosphorylation / mitochondrial membrane / calcium-mediated signaling / positive regulation of MAP kinase activity / trans-Golgi network / regulation of protein stability / terminal bouton Similarity search - Function | |||||||||||||||
Biological species | Homo sapiens (human) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 Å | |||||||||||||||
Authors | Matyszewski, M. / Leschziner, A.E. | |||||||||||||||
Funding support | United States, 4items
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Citation | Journal: Nat Struct Mol Biol / Year: 2022 Title: Structural basis for Parkinson's disease-linked LRRK2's binding to microtubules. Authors: David M Snead / Mariusz Matyszewski / Andrea M Dickey / Yu Xuan Lin / Andres E Leschziner / Samara L Reck-Peterson / Abstract: Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an ...Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-EM structure of the catalytic half of LRRK2, containing its kinase, in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2 but is not linked to PD. Although LRRK1's structure is similar to that of LRRK2, we find that LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2's GTPase that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2's kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors. | |||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7thz.cif.gz | 148.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7thz.ent.gz | 107.2 KB | Display | PDB format |
PDBx/mmJSON format | 7thz.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7thz_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 7thz_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7thz_validation.xml.gz | 37.5 KB | Display | |
Data in CIF | 7thz_validation.cif.gz | 51.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/th/7thz ftp://data.pdbj.org/pub/pdb/validation_reports/th/7thz | HTTPS FTP |
-Related structure data
Related structure data | 25907MC 7thyC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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Number of models | 5 |
-Components
#1: Protein | Mass: 22191.762 Da / Num. of mol.: 1 / Fragment: ROC domain Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: LRRK2, PARK8 / Production host: Spodoptera frugiperda (fall armyworm) 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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#2: Chemical | ChemComp-GDP / |
Has ligand of interest | N |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.4 Details: This is the final dilution buffer. The incubation buffer consisted of 1x BRB80, 10% glycerol, 1mM DTT, 1mM GTP, 1mM MgCl2, 10 uM taxol, and 5 uM MLi-2. Sample was diluted 3-fold right before ...Details: This is the final dilution buffer. The incubation buffer consisted of 1x BRB80, 10% glycerol, 1mM DTT, 1mM GTP, 1mM MgCl2, 10 uM taxol, and 5 uM MLi-2. Sample was diluted 3-fold right before freezing with the final buffer. | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 4.5 uM of LRRK2RCKW (I2020T) was allowed to incubate with 2.25 uM of tubulin dimer, causing both to co-polymerize. 5 uM of MLi-2 was present as well. The sample was diluted 3-fold right ...Details: 4.5 uM of LRRK2RCKW (I2020T) was allowed to incubate with 2.25 uM of tubulin dimer, causing both to co-polymerize. 5 uM of MLi-2 was present as well. The sample was diluted 3-fold right before freezing (1.5 uM LRRK2RCKW concentration final). | ||||||||||||||||||||||||||||||
Specimen support | Details: EMS LC-300 lacey grid used (not homemade, but can't choose EMS as the manufacturer) Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Homemade | ||||||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN |
Image recording | Average exposure time: 10 sec. / Electron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Details: 250 ms frames |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 557577 Details: Filament Autopicker with templates created by manual picking. This is before symmetry expansion. | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99854 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT Details: Used AlphaFold model as initial model (Q5S007) using only the ROC domain. TUB1 was added to the initial refinement to prevent ROC model from entering density reserved for the microtubule. ...Details: Used AlphaFold model as initial model (Q5S007) using only the ROC domain. TUB1 was added to the initial refinement to prevent ROC model from entering density reserved for the microtubule. TUB1 was discarded after the initial refinement. |