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- EMDB-25907: Structure of Leucine Rich Repeat Kinase 2's ROC domain interactin... -

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Basic information

Entry
Database: EMDB / ID: EMD-25907
TitleStructure of Leucine Rich Repeat Kinase 2's ROC domain interacting with the microtubule facing the plus end
Map dataSharpened map
Sample
  • Complex: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordsparkinson's disease / microtubule / kinase / gtpase / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsMatyszewski M / Leschziner AE
Funding support United States, 4 items
OrganizationGrant numberCountry
Other privateASAP-000519 United States
Michael J. Fox Foundation18321 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121772 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107214 United States
CitationJournal: 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
DepositionJan 12, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25907.png

Downloads & links

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Map

FileDownload / File: emd_25907.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 300 pix.
= 348. Å		1.16 Å/pix.
x 300 pix.
= 348. Å		1.16 Å/pix.
x 300 pix.
= 348. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.254
Minimum - Maximum-1.1120619 - 2.7757661
Average (Standard dev.)0.0058422643 (±0.059319537)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 348.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25907_msk_1.map
Projections & Slices
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Additional map: Non-sharpened map

Fileemd_25907_additional_1.map
AnnotationNon-sharpened map
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Half map: Half map 1

Fileemd_25907_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_25907_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present

EntireName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
Components
  • Complex: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present

SupramoleculeName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1

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

MacromoleculeName: Leucine-rich repeat serine/threonine-protein kinase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.191762 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: YNRMKLMIVG NTGSGKTTLL QQLMKTKKSD LGMQSATVGI DVKDWPIQIR DKRKRDLVLN VWDFAGREEF YSTHPHFMTQ RALYLAVYD LSKGQAEVDA MKPWLFNIKA RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA T EESDALAK ...String:
YNRMKLMIVG NTGSGKTTLL QQLMKTKKSD LGMQSATVGI DVKDWPIQIR DKRKRDLVLN VWDFAGREEF YSTHPHFMTQ RALYLAVYD LSKGQAEVDA MKPWLFNIKA RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA T EESDALAK LRKTIINESL NFKIRDQLVV GQLIPD

UniProtKB: Leucine-rich repeat serine/threonine-protein kinase 2

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Macromolecule #2: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES
80.0 mMSodium ChlorideNaCl
0.5 mMTCEP
2.5 mMMagnesium ChlorideMgCl2
20.0 uMGDP

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.
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
Details: EMS LC-300 lacey grid used (not homemade, but can't choose EMS as the manufacturer)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K
Details4.5 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).

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Average exposure time: 10.0 sec. / Average electron dose: 55.0 e/Å2 / Details: 250 ms frames
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 557577
Details: Filament Autopicker with templates created by manual picking. This is before symmetry expansion.
Startup modelType of model: NONE
Details: Featureless cylinder for the original helical reconstruction, then subunit from the helical reconstruction after reboxing and symmetry expansion.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2)
Software - details: local refinement, with non-uniform refinement turned off
Number images used: 99854
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsUsed 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.
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7thz:
Structure of Leucine Rich Repeat Kinase 2's ROC domain interacting with the microtubule facing the plus end

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