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- PDB-9cho: Autoinhibited full-length LRRK2(I2020T) on microtubules with MLi-2 -

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Basic information

Entry
Database: PDB / ID: 9cho
TitleAutoinhibited full-length LRRK2(I2020T) on microtubules with MLi-2
ComponentsLeucine-rich repeat serine/threonine-protein kinase 2
KeywordsPROTEIN BINDING / Kinase / GTPase
Function / homology
Function and homology information


caveola neck / 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 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 ...caveola neck / 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 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 SNARE complex assembly / 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 / regulation of dopamine receptor signaling pathway / regulation of retrograde transport, endosome to Golgi / regulation of neuron maturation / positive regulation of microglial cell activation / positive regulation of synaptic vesicle endocytosis / negative regulation of autophagosome assembly / cytoplasmic side of mitochondrial outer membrane / negative regulation of excitatory postsynaptic potential / regulation of cAMP/PKA signal transduction / JUN kinase kinase kinase activity / olfactory bulb development / neuron projection arborization / striatum development / multivesicular body, internal vesicle / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / cellular response to dopamine / positive regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway / 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 / negative regulation of GTPase activity / regulation of canonical Wnt signaling pathway / regulation of reactive oxygen species metabolic process / lysosome organization / Golgi-associated vesicle / clathrin binding / regulation of locomotion / negative regulation of macroautophagy / PTK6 promotes HIF1A stabilization / protein kinase A binding / neuromuscular junction development / regulation of mitochondrial fission / Golgi organization / regulation of synaptic vesicle exocytosis / exploration behavior / microvillus / intracellular distribution of mitochondria / autolysosome / locomotory exploration behavior / endoplasmic reticulum exit site / negative regulation of Notch signaling pathway / regulation of synaptic vesicle endocytosis / cellular response to manganese ion / MAP kinase kinase kinase activity / Rho protein signal transduction / canonical Wnt signaling pathway / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / presynaptic cytosol / regulation of synaptic transmission, glutamatergic / phagocytic vesicle / JNK cascade / positive regulation of autophagy / tubulin binding / dendrite cytoplasm / GTPase activator activity / SNARE binding / cellular response to starvation / neuron projection morphogenesis / positive regulation of protein ubiquitination / excitatory postsynaptic potential / regulation of membrane potential / determination of adult lifespan / cellular response to reactive oxygen species / mitochondrion organization / trans-Golgi network / calcium-mediated signaling / mitochondrial membrane / regulation of protein stability / small GTPase binding / autophagy / terminal bouton
Similarity search - Function
: / : / : / LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / C-terminal of Roc, COR-B domain / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain ...: / : / : / LRRK2 ARM repeat / LRRK2 ANK repeat / LRRK2 beta propeller / : / C-terminal of Roc, COR-B domain / 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
Chem-A1N / GUANOSINE-5'-DIPHOSPHATE / Leucine-rich repeat serine/threonine-protein kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.8 Å
AuthorsChen, S. / Villa, E. / Leschziner, A.E.
Funding support United States, 2items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP)ASAP-000519 United States
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: bioRxiv / Year: 2024
Title: Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2.
Authors: Siyu Chen / Tamar Basiashvili / Joshua Hutchings / Marta Sanz Murillo / Amalia Villagran Suarez / Jaime Alegrio Louro / Andres E Leschziner / Elizabeth Villa /
Abstract: Parkinson's Disease (PD) is the second most common neurodegenerative disorder. Mutations in leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a ...Parkinson's Disease (PD) is the second most common neurodegenerative disorder. Mutations in leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a leading cause of the familial form of PD. Pathogenic LRRK2 mutations increase LRRK2 kinase activity. While the bulk of LRRK2 is found in the cytosol, the protein associates with membranes where its Rab GTPase substrates are found, and under certain conditions, with microtubules. Integrative structural studies using single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have revealed the architecture of microtubule-associated LRRK2 filaments, and that formation of these filaments requires LRRK2's kinase to be in the active-like conformation. However, whether LRRK2 can interact with and form filaments on microtubules in its autoinhibited state, where the kinase domain is in the inactive conformation and the N-terminal LRR domain covers the kinase active site, was not known. Using cryo-ET, we show that full-length LRRK2 can oligomerize on microtubules in its autoinhibited state. Both WT-LRRK2 and PD-linked LRRK2 mutants formed filaments on microtubules. While these filaments are stabilized by the same interfaces seen in the active-LRRK2 filaments, we observed a new interface involving the N-terminal repeats that were disordered in the active-LRRK2 filaments. The helical parameters of the autoinhibited-LRRK2 filaments are different from those reported for the active-LRRK2 filaments. Finally, the autoinhibited-LRRK2 filaments are shorter and less regular, suggesting they are less stable.
#1: Journal: Elife / Year: 2024
Title: Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2
Authors: Chen, S. / Basiashvili, T. / Hutchings, J. / Sanz Murillo, M. / Villagran Suarez, A. / Alegrio Louro, J. / Leschziner, A.E. / Villa, E.
History
DepositionJul 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)225,7283
Polymers224,9051
Non-polymers8232
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Leucine-rich repeat serine/threonine-protein kinase 2 / Dardarin


Mass: 224904.875 Da / Num. of mol.: 1 / Fragment: UNP residues 543-2527 / Mutation: I2020T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRK2, PARK8 / Cell line (production host): Sf9 / 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
#2: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#3: Chemical ChemComp-A1N / (2~{R},6~{S})-2,6-dimethyl-4-[6-[5-(1-methylcyclopropyl)oxy-1~{H}-indazol-3-yl]pyrimidin-4-yl]morpholine


Mass: 379.456 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H25N5O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Full-length autoinhibited LRRK2 I2020T on microtubules
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.286103 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
280 mMsodium chlorideNaCl1
30.5 mMTCEP1
42.5 mMmagnesium chlorideMgCl21
510 uMTAXOL1
SpecimenConc.: 1.43 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 42000 X / Calibrated magnification: 42000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 3000 nm / Calibrated defocus min: 3500 nm / Calibrated defocus max: 5500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 0.65 sec. / Electron dose: 3.24 e/Å2 / Avg electron dose per subtomogram: 120 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 1
Image scansWidth: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategory
1Dynamovolume selection
2SerialEM4.6image acquisition
3PACEtomo1.6image acquisition
5WarpCTF correction
8UCSF ChimeraX1.7model fitting
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
14PHENIX1.20.1_4487:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60556 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 131 / Num. of volumes extracted: 65612 / Reference model: ab-initio
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311791
ELECTRON MICROSCOPYf_angle_d0.72216104
ELECTRON MICROSCOPYf_dihedral_angle_d4.8061655
ELECTRON MICROSCOPYf_chiral_restr0.0442008
ELECTRON MICROSCOPYf_plane_restr0.0052013

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