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- EMDB-45591: Autoinhibited full-length LRRK2(I2020T) on microtubules with MLi-2 -

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Basic information

Entry
Database: EMDB / ID: EMD-45591
TitleAutoinhibited full-length LRRK2(I2020T) on microtubules with MLi-2
Map dataIT_Mli2
Sample
  • Complex: Full-length autoinhibited LRRK2 I2020T on microtubules
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: (2~{R},6~{S})-2,6-dimethyl-4-[6-[5-(1-methylcyclopropyl)oxy-1~{H}-indazol-3-yl]pyrimidin-4-yl]morpholine
KeywordsKinase / GTPase / PROTEIN BINDING
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)
Methodsubtomogram averaging / cryo EM / Resolution: 7.8 Å
AuthorsChen S / Villa E / Leschziner AE
Funding support United States, 2 items
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 AE / Villa E
History
DepositionJul 1, 2024-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateJan 1, 2025-
Current statusJan 1, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45591.png

Downloads & links

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Map

FileDownload / File: emd_45591.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIT_Mli2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 192 pix.
= 414.912 Å		2.16 Å/pix.
x 192 pix.
= 414.912 Å		2.16 Å/pix.
x 192 pix.
= 414.912 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.161 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-2.6347623 - 5.085097
Average (Standard dev.)0.18537861 (±0.23316269)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 414.912 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_45591_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_45591_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Full-length autoinhibited LRRK2 I2020T on microtubules

EntireName: Full-length autoinhibited LRRK2 I2020T on microtubules
Components
  • Complex: Full-length autoinhibited LRRK2 I2020T on microtubules
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: (2~{R},6~{S})-2,6-dimethyl-4-[6-[5-(1-methylcyclopropyl)oxy-1~{H}-indazol-3-yl]pyrimidin-4-yl]morpholine

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Supramolecule #1: Full-length autoinhibited LRRK2 I2020T on microtubules

SupramoleculeName: Full-length autoinhibited LRRK2 I2020T on microtubules
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 286.103 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 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 224.904875 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: HKLVLAALNR FIGNPGIQKC GLKVISSIVH FPDALEMLSL EGAMDSVLHT LQMYPDDQEI QCLGLSLIGY LITKKNVFIG TGHLLAKIL VSSLYRFKDV AEIQTKGFQT ILAILKLSAS FSKLLVHHSF DLVIFHQMSS NIMEQKDQQF LNLCCKCFAK V AMDDYLKN ...String:
HKLVLAALNR FIGNPGIQKC GLKVISSIVH FPDALEMLSL EGAMDSVLHT LQMYPDDQEI QCLGLSLIGY LITKKNVFIG TGHLLAKIL VSSLYRFKDV AEIQTKGFQT ILAILKLSAS FSKLLVHHSF DLVIFHQMSS NIMEQKDQQF LNLCCKCFAK V AMDDYLKN VMLERACDQN NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG KG DSQIISL LLRRLALDVA NNSICLGGFC IGKVEPSWLG PLFPDKTSNL RKQTNIASTL ARMVIRYQMK SAVEEGTASG SDG NFSEDV LSKFDEWTFI PDSSMDSVFA QSDDLDSEGS EGSFLVKKKS NSISVGEFYR DAVLQRCSPN LQRHSNSLGP IFDH EDLLK RKRKILSSDD SLRSSKLQSH MRHSDSISSL ASEREYITSL DLSANELRDI DALSQKCCIS VHLEHLEKLE LHQNA LTSF PQQLCETLKS LTHLDLHSNK FTSFPSYLLK MSCIANLDVS RNDIGPSVVL DPTVKCPTLK QFNLSYNQLS FVPENL TDV VEKLEQLILE GNKISGICSP LRLKELKILN LSKNHISSLS ENFLEACPKV ESFSARMNFL AAMPFLPPSM TILKLSQ NK FSCIPEAILN LPHLRSLDMS SNDIQYLPGP AHWKSLNLRE LLFSHNQISI LDLSEKAYLW SRVEKLHLSH NKLKEIPP E IGCLENLTSL DVSYNLELRS FPNEMGKLSK IWDLPLDELH LNFDFKHIGC KAKDIIRFLQ QRLKKAVPYN RMKLMIVGN TGSGKTTLLQ QLMKTKKSDL GMQSATVGID VKDWPIQIRD KRKRDLVLNV WDFAGREEFY STHPHFMTQR ALYLAVYDLS KGQAEVDAM KPWLFNIKAR ASSSPVILVG THLDVSDEKQ RKACMSKITK ELLNKRGFPA IRDYHFVNAT EESDALAKLR K TIINESLN FKIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN QLQLDENELP HAVHFLNESG VL LHFQDPA LQLSDLYFVE PKWLCKIMAQ ILTVKVEGCP KHPKGIISRR DVEKFLSKKR KFPKNYMSQY FKLLEKFQIA LPI GEEYLL VPSSLSDHRP VIELPHCENS EIIIRLYEMP YFPMGFWSRL INRLLEISPY MLSGRERALR PNRMYWRQGI YLNW SPEAY CLVGSEVLDN HPESFLKITV PSCRKGCILL GQVVDHIDSL MEEWFPGLLE IDICGEGETL LKKWALYSFN DGEEH QKIL LDDLMKKAEE GDLLVNPDQP RLTIPISQIA PDLILADLPR NIMLNNDELE FEQAPEFLLG DGSFGSVYRA AYEGEE VAV KIFNKHTSLR LLRQELVVLC HLHHPSLISL LAAGIRPRML VMELASKGSL DRLLQQDKAS LTRTLQHRIA LHVADGL RY LHSAMIIYRD LKPHNVLLFT LYPNAAIIAK IADYGTAQYC CRMGIKTSEG TPGFRAPEVA RGNVIYNQQA DVYSFGLL L YDILTTGGRI VEGLKFPNEF DELEIQGKLP DPVKEYGCAP WPMVEKLIKQ CLKENPQERP TSAQVFDILN SAELVCLTR RILLPKNVIV ECMVATHHNS RNASIWLGCG HTDRGQLSFL DLNTEGYTSE EVADSRILCL ALVHLPVEKE SWIVSGTQSG TLLVINTED GKKRHTLEKM TDSVTCLYCN SFSKQSKQKN FLLVGTADGK LAIFEDKTVK LKGAAPLKIL NIGNVSTPLM C LSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EK LCGLIDC VHFLREVMVK ENKESKHKMS YSGRVKTLCL QKNTALWIGT GGGHILLLDL STRRLIRVIY NFCNSVRVMM TAQ LGSLKN VMLVLGYNRK NTEGTQKQKE IQSCLTVWDI NLPHEVQNLE KHIEVRKELA EKMRRTSVE

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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Macromolecule #3: (2~{R},6~{S})-2,6-dimethyl-4-[6-[5-(1-methylcyclopropyl)oxy-1~{H}...

MacromoleculeName: (2~{R},6~{S})-2,6-dimethyl-4-[6-[5-(1-methylcyclopropyl)oxy-1~{H}-indazol-3-yl]pyrimidin-4-yl]morpholine
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1N
Molecular weightTheoretical: 379.456 Da
Chemical component information

ChemComp-A1N:
(2~{R},6~{S})-2,6-dimethyl-4-[6-[5-(1-methylcyclopropyl)oxy-1~{H}-indazol-3-yl]pyrimidin-4-yl]morpholine

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

Concentration1.43 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES
80.0 mMsodium chlorideNaCl
0.5 mMTCEP
2.5 mMmagnesium chlorideMgCl2
10.0 uMTAXOL
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 90.0 K
SoftwareName: SerialEM (ver. 4.6)
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number real images: 1 / Average exposure time: 0.65 sec. / Average electron dose: 3.24 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.5 µm / Calibrated defocus min: 3.5 µm / Calibrated magnification: 42000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number subtomograms used: 60556
ExtractionNumber tomograms: 131 / Number images used: 65612 / Reference model: ab-initio / Software - Name: Dynamo
Final 3D classificationNumber classes: 10 / Avg.num./class: 30000 / Software - Name: RELION (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: UCSF ChimeraX (ver. 1.7)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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