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- EMDB-21250: Cryo-EM structure of the C-terminal half of the Parkinson's Disea... -

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Entry
Database: EMDB / ID: EMD-21250
TitleCryo-EM structure of the C-terminal half of the Parkinson's Disease-linked protein Leucine Rich Repeat Kinase 2 (LRRK2)
Map data3.8A signal subtracted locally filtered cryo-EM map of C-terminal half of Leucine Rich Repeat Kinase 2 (LRRK2)
Sample
  • Complex: The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) protein.
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
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 / regulation of synaptic vesicle transport / 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 lysosomal lumen pH / regulation of CAMKK-AMPK signaling cascade / amphisome / mitochondrion localization / cytoplasmic side of mitochondrial outer membrane / co-receptor binding / regulation of retrograde transport, endosome to Golgi / negative regulation of excitatory postsynaptic potential / negative regulation of autophagosome assembly / regulation of dopamine receptor signaling pathway / 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 dendritic spine morphogenesis / regulation of protein kinase A signaling / multivesicular body, internal vesicle / striatum development / protein localization to mitochondrion / cellular response to dopamine / presynaptic cytosol / positive regulation of protein autoubiquitination / endoplasmic reticulum organization / Wnt signalosome / positive regulation of programmed cell death / 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 / regulation of locomotion / autolysosome / protein kinase A binding / exploration behavior / regulation of synaptic vesicle exocytosis / Golgi-associated vesicle / PTK6 promotes HIF1A stabilization / clathrin binding / negative regulation of macroautophagy / lysosome organization / regulation of mitochondrial fission / neuromuscular junction development / intracellular distribution of mitochondria / Golgi organization / positive regulation of nitric-oxide synthase biosynthetic process / locomotory exploration behavior / microvillus / Rho protein signal transduction / cellular response to organic cyclic compound / MAP kinase kinase kinase activity / canonical Wnt signaling pathway / positive regulation of protein kinase activity / cellular response to manganese ion / endoplasmic reticulum exit site / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / positive regulation of autophagy / JNK cascade / regulation of synaptic transmission, glutamatergic / excitatory postsynaptic potential / regulation of membrane potential / cellular response to starvation / dendrite cytoplasm / GTPase activator activity / mitochondrion organization / tubulin binding / SNARE binding / neuron projection morphogenesis / negative regulation of protein phosphorylation / negative regulation of protein binding / positive regulation of protein ubiquitination / regulation of autophagy / determination of adult lifespan / calcium-mediated signaling / mitochondrial membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / peptidyl-threonine phosphorylation / regulation of protein stability / positive regulation of MAP kinase activity / trans-Golgi network
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 / Leucine-rich repeat profile. ...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. / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLeschziner A / Deniston C / Lahiri I
Funding support United States, 3 items
OrganizationGrant numberCountry
Michael J. Fox Foundation11425 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107214 United States
Michael J. Fox Foundation11425.02 United States
CitationJournal: Nature / Year: 2020
Title: Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.
Authors: C K Deniston / J Salogiannis / S Mathea / D M Snead / I Lahiri / M Matyszewski / O Donosa / R Watanabe / J Böhning / A K Shiau / S Knapp / E Villa / S L Reck-Peterson / A E Leschziner /
Abstract: Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease and is also linked to its idiopathic form. LRRK2 has been proposed to function in membrane ...Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease and is also linked to its idiopathic form. LRRK2 has been proposed to function in membrane trafficking and colocalizes with microtubules. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.
History
DepositionJan 29, 2020-
Header (metadata) releaseAug 26, 2020-
Map releaseAug 26, 2020-
UpdateDec 23, 2020-
Current statusDec 23, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0267
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  • Surface view colored by radius
  • Surface level: 0.0267
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  • Surface view with fitted model
  • Atomic models: PDB-6vno
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6vp6
  • Surface level: 0.03
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  • Surface view with fitted model
  • Atomic models: PDB-6vp7
  • Surface level: 0.06
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  • Surface view with fitted model
  • Atomic models: PDB-6vp8
  • Surface level: 0.0267
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vno
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vp6
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vp7
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vp8
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21250.png

Downloads & links

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Map

FileDownload / File: emd_21250.map.gz / Format: CCP4 / Size: 172.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3.8A signal subtracted locally filtered cryo-EM map of C-terminal half of Leucine Rich Repeat Kinase 2 (LRRK2)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0267 / Movie #1: 0.0267
Minimum - Maximum-0.032042872 - 0.0754334
Average (Standard dev.)8.440901e-05 (±0.0022088308)
SymmetrySpace group: 0
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions356356356
Spacing356356356
CellA=B=C: 380.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z356356356
origin x/y/z0.0000.0000.000
length x/y/z380.920380.920380.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS356356356
D min/max/mean-0.0320.0750.000

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Supplemental data

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Additional map: 3.47A C3 symmetric locally filtered cryo-EM map of...

Fileemd_21250_additional_1.map
Annotation3.47A C3 symmetric locally filtered cryo-EM map of C-terminal half of Leucine Rich Repeat Kinase 2 (LRRK2).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map 1 of C3 symmetric map

Fileemd_21250_additional_2.map
AnnotationHalf map 1 of C3 symmetric map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map 2 of C3 symmetric map

Fileemd_21250_additional_3.map
AnnotationHalf map 2 of C3 symmetric map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of signal subtracted map

Fileemd_21250_half_map_1.map
AnnotationHalf map 1 of signal subtracted map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of signal subtracted map

Fileemd_21250_half_map_2.map
AnnotationHalf map 2 of signal subtracted map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) p...

EntireName: The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) protein.
Components
  • Complex: The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) protein.
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) p...

SupramoleculeName: The C-terminal half of the Leucine Rich Repeat Kinase 2 (LRRK2) protein.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: C-terminal half runs from residue 1327-2527.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 137 KDa

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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 / Details: C-terminal residues 1330-2527 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 136.943609 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KKAVPYNRMK LMIVGN(TPO)GSG KTTLLQQLMK TKKSDLGMQS ATVGIDVKDW PIQIRDKRKR DLVLNVWDFA GREEFY STH PHFMTQRALY LAVYDLSKGQ AEVDAMKPWL FNIKARASSS PVILVGTHLD VSDEKQRKAC MSKITKELLN KRGFPAI RD YHFVNATEES ...String:
KKAVPYNRMK LMIVGN(TPO)GSG KTTLLQQLMK TKKSDLGMQS ATVGIDVKDW PIQIRDKRKR DLVLNVWDFA GREEFY STH PHFMTQRALY LAVYDLSKGQ AEVDAMKPWL FNIKARASSS PVILVGTHLD VSDEKQRKAC MSKITKELLN KRGFPAI RD YHFVNATEES DALAKLRKTI INESLNFKIR DQLVVGQLIP DCYVELEKII LSERKNVPIE FPVIDRKRLL QLVRENQL Q LDENELPHAV HFLNESGVLL HFQDPALQLS DLYFVEPKWL CKIMAQILTV KVEGCPKHPK GIISRRDVEK FLSKKRKFP KNYMSQYFKL LEKFQIALPI GEEYLLVPSS LSDHRPVIEL PHCENSEIII RLYEMPYFPM GFWSRLINRL LEISPYMLSG RERALRPNR MYWRQGIYLN WSPEAYCLVG SEVLDNHPES FLKITVPSCR KGCILLGQVV DHIDSLMEEW FPGLLEIDIC G EGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI LADLPRNIML NNDELEFEQA PE FLLGDGS FGSVYRAAYE GEEVAVKIFN KHTSLRLLRQ ELVVLCHLHH PSLISLLAAG IRPRMLVMEL ASKGSLDRLL QQD KASLTR TLQHRIALHV ADGLRYLHSA MIIYRDLKPH NVLLFTLYPN AAIIAKIADY GIAQYCCRMG IKTSEGTPGF RAPE VARGN VIYNQQADVY SFGLLLYDIL TTGGRIVEGL KFPNEFDELE IQGKLPDPVK EYGCAPWPMV EKLIKQCLKE NPQER PTSA QVFDILNSAE LVCLTRRILL PKNVIVECMV ATHHNSRNAS IWLGCGHTDR GQLSFLDLNT EGYTSEEVAD SRILCL ALV HLPVEKESWI VSGTQSGTLL VINTEDGKKR HTLEKMTDSV TCLYCNSFSK QSKQKNFLLV GTADGKLAIF EDKTVKL KG AAPLKILNIG NVSTPLMCLS ESTNSTERNV MWGGCGTKIF SFSNDFTIQK LIETRTSQLF SYAAFSDSNI ITVVVDTA L YIAKQNSPVV EVWDKKTEKL CGLIDCVHFL REVMVKENKE SKHKMSYSGR VKTLCLQKNT ALWIGTGGGH ILLLDLSTR RLIRVIYNFC NSVRVMMTAQ LGSLKNVMLV LGYNRKNTEG TQKQKEIQSC LTVWDINLPH EVQNLEKHIE VRKELAEKMR RTSVE

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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 / Guanosine diphosphate

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES
80.0 mMNaClSodium chloride
0.5 mMTCEP
5.0 %Glycerol
2.5 mMMgCl2
20.0 uMGDP
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II
Details4uM concentration

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF 2002
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3826 / Average exposure time: 8.0 sec. / Average electron dose: 6.65 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 836956
CTF correctionSoftware - Name: Gctf (ver. 1) / Details: Per-particle CTF values
Startup modelType of model: OTHER
Details: Generated initial models from ab initio refinement in Cryosparc.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: RELION (ver. 3), cryoSPARC (ver. 2))
Details: Signal subtracted map was generated in Relion3. The C3 map however was created in Cryosparc 2.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: RELION (ver. 3), cryoSPARC (ver. 2))
Details: Signal subtracted map was generated in Relion3. The C3 map however was created in Cryosparc 2.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software:
Namedetails
RELION (ver. 3)C1
cryoSPARC (ver. 2)C3

Details: For the signal subtracted map, 105,787 (tripled) particles went into the final map that achieved 3.8A resolution.
Number images used: 70953
FSC plot (resolution estimation)

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