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- PDB-9ci3: Structure of the LRRK2/14-3-3 complex -

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Basic information

Entry
Database: PDB / ID: 9ci3
TitleStructure of the LRRK2/14-3-3 complex
Components
  • 14-3-3 protein gamma
  • Leucine-rich repeat serine/threonine-protein kinase 2
KeywordsTRANSFERASE/SIGNALING PROTEIN / LRRK2 / LRRK2 complex / LRRK2 14-3-3 complex / LRRK2 autoinhibited / TRANSFERASE / TRANSFERASE-SIGNALING PROTEIN complex
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 / amphisome / regulation of synaptic vesicle transport / positive regulation of cell-cell adhesion / regulation of CAMKK-AMPK signaling cascade / regulation of lysosomal lumen pH / co-receptor binding / negative regulation of GTPase activity / phosphorylation-dependent protein binding / regulation of dopamine receptor signaling pathway / mitochondrion localization / regulation of neuron maturation / positive regulation of microglial cell activation / regulation of retrograde transport, endosome to Golgi / positive regulation of synaptic vesicle endocytosis / cytoplasmic side of mitochondrial outer membrane / negative regulation of excitatory postsynaptic potential / negative regulation of autophagosome assembly / JUN kinase kinase kinase activity / olfactory bulb development / neuron projection arborization / multivesicular body, internal vesicle / striatum development / positive regulation of T cell mediated immune response to tumor cell / 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 / regulation of canonical Wnt signaling pathway / regulation of neuron differentiation / syntaxin-1 binding / regulation of reactive oxygen species metabolic process / lysosome organization / Golgi-associated vesicle / clathrin binding / protein kinase A binding / regulation of locomotion / PTK6 promotes HIF1A stabilization / negative regulation of macroautophagy / neuromuscular junction development / protein kinase C inhibitor activity / regulation of cAMP/PKA signal transduction / regulation of mitochondrial fission / Golgi organization / regulation of synaptic vesicle exocytosis / exploration behavior / microvillus / intracellular distribution of mitochondria / endoplasmic reticulum exit site / autolysosome / locomotory exploration behavior / Regulation of localization of FOXO transcription factors / negative regulation of Notch signaling pathway / Activation of BAD and translocation to mitochondria / regulation of signal transduction / MAP kinase kinase kinase activity / regulation of synaptic vesicle endocytosis / canonical Wnt signaling pathway / protein targeting / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / regulation of synaptic transmission, glutamatergic / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / negative regulation of protein kinase activity / cellular response to glucose starvation / presynaptic cytosol / Rho protein signal transduction / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / phagocytic vesicle / neuron projection morphogenesis / JNK cascade / cellular response to manganese ion / negative regulation of TORC1 signaling / insulin-like growth factor receptor binding / positive regulation of autophagy / Transcriptional and post-translational regulation of MITF-M expression and activity
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 / 14-3-3 proteins signature 2. / 14-3-3 protein, conserved site / 14-3-3 proteins signature 1. / 14-3-3 protein / 14-3-3 homologues / 14-3-3 domain / 14-3-3 domain superfamily / 14-3-3 protein / 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
GUANOSINE-5'-DIPHOSPHATE / 14-3-3 protein gamma / Leucine-rich repeat serine/threonine-protein kinase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsMartinez Fiesco, J.A. / Zhang, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nat Commun / Year: 2025
Title: 14-3-3 binding maintains the Parkinson's associated kinase LRRK2 in an inactive state.
Authors: Juliana A Martinez Fiesco / Alexandra Beilina / Astrid Alvarez de la Cruz / Ning Li / Riley D Metcalfe / Mark R Cookson / Ping Zhang /
Abstract: Leucine-rich repeat kinase 2 (LRRK2) is an essential regulator in cellular signaling and a major contributor to Parkinson's disease (PD) pathogenesis. 14-3-3 proteins are critical modulators of LRRK2 ...Leucine-rich repeat kinase 2 (LRRK2) is an essential regulator in cellular signaling and a major contributor to Parkinson's disease (PD) pathogenesis. 14-3-3 proteins are critical modulators of LRRK2 activity, yet the structural basis of their interaction has remained unclear. Here, we present the cryo-electron microscopy structure of the LRRK2:14-3-3 autoinhibitory complex, revealing how a 14-3-3 dimer stabilizes an autoinhibited LRRK2 monomer through dual-site anchoring. The dimer engages both phosphorylated S910/S935 sites and the COR-A/B subdomains within the Roc-COR GTPase region. This spatial configuration constrains LRR domain mobility, reinforces the inactive conformation, and likely impedes LRRK2 dimerization and oligomer formation. Structure-guided mutagenesis studies show that PD-associated mutations at the COR:14-3-3 interface and within the GTPase domain weaken 14-3-3 binding and impair its inhibitory effect on LRRK2 kinase activity. Furthermore, we demonstrate that type I LRRK2 kinase inhibitor, which stabilizes the kinase domain in its active conformation, reduces 14-3-3 binding and promotes dephosphorylation at pS910 and pS935. Together, these findings provide a structural basis for understanding how LRRK2 is maintained in an inactive state, elucidate the mechanistic role of 14-3-3 in LRRK2 regulation, inform the interpretation of PD biomarkers, and suggest therapeutic strategies aimed at enhancing LRRK2-14-3-3 interactions to treat PD and related disorders.
History
DepositionJul 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: 14-3-3 protein gamma
B: 14-3-3 protein gamma
A: Leucine-rich repeat serine/threonine-protein kinase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,9694
Polymers351,5263
Non-polymers4431
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 14-3-3 protein gamma / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 30436.814 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: YWHAG / Production host: Escherichia coli (E. coli) / References: UniProt: P61981
#2: Protein Leucine-rich repeat serine/threonine-protein kinase 2 / Dardarin


Mass: 290652.188 Da / Num. of mol.: 1 / Mutation: R50H variant
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRK2, PARK8 / Production host: Homo sapiens (human)
References: UniProt: Q5S007, non-specific serine/threonine protein kinase, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#3: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex of LRRK2 monomer with 14-3-3 dimerCOMPLEX#1-#20MULTIPLE SOURCES
2LRRK2COMPLEX#21RECOMBINANT
314-3-3 dimerCOMPLEX#11RECOMBINANT
Molecular weightValue: 342.7 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Escherichia coli (E. coli)562
Buffer solutionpH: 8.3
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 60.8 K

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM software
IDNameCategory
1Topazparticle selection
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 432285 / Symmetry type: POINT
RefinementHighest resolution: 3.96 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316242
ELECTRON MICROSCOPYf_angle_d0.73821970
ELECTRON MICROSCOPYf_dihedral_angle_d5.3722170
ELECTRON MICROSCOPYf_chiral_restr0.0452517
ELECTRON MICROSCOPYf_plane_restr0.0052788

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