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Yorodumi- EMDB-25906: Structure of Leucine Rich Repeat Kinase 2's ROC domain interactin... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25906 | |||||||||||||||
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Title | Structure of Leucine Rich Repeat Kinase 2's ROC domain interacting with the microtubule facing the minus end | |||||||||||||||
Map data | Sharpened map | |||||||||||||||
Sample |
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Keywords | parkinson's disease / microtubule / kinase / gtpase / CYTOSOLIC PROTEIN | |||||||||||||||
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 / negative regulation of late endosome to lysosome transport / regulation of synaptic vesicle transport / regulation of mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / positive regulation of dopamine receptor signaling pathway / regulation of lysosomal lumen pH / PTK6 promotes HIF1A stabilization / regulation of CAMKK-AMPK signaling cascade / amphisome / cytoplasmic side of mitochondrial outer membrane / mitochondrion localization / co-receptor binding / regulation of retrograde transport, endosome to Golgi / negative regulation of excitatory postsynaptic potential / regulation of dopamine receptor signaling pathway / negative regulation of autophagosome assembly / positive regulation of microglial cell activation / neuron projection arborization / positive regulation of synaptic vesicle endocytosis / JUN kinase kinase kinase activity / olfactory bulb development / multivesicular body, internal vesicle / regulation of protein kinase A signaling / striatum development / regulation of dendritic spine morphogenesis / protein localization to mitochondrion / cellular response to dopamine / positive regulation of protein autoubiquitination / endoplasmic reticulum organization / presynaptic cytosol / positive regulation of programmed cell death / Wnt signalosome / 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 / exploration behavior / MAP kinase kinase kinase activity / autolysosome / regulation of locomotion / protein kinase A binding / regulation of synaptic vesicle exocytosis / Golgi-associated vesicle / negative regulation of macroautophagy / clathrin binding / neuromuscular junction development / lysosome organization / regulation of mitochondrial fission / intracellular distribution of mitochondria / Golgi organization / positive regulation of nitric-oxide synthase biosynthetic process / locomotory exploration behavior / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / canonical Wnt signaling pathway / positive regulation of protein kinase activity / cellular response to manganese ion / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / positive regulation of autophagy / neuron projection morphogenesis / JNK cascade / regulation of synaptic transmission, glutamatergic / dendrite cytoplasm / cellular response to starvation / phosphorylation / tubulin binding / GTPase activator activity / regulation of membrane potential / SNARE binding / positive regulation of MAP kinase activity / excitatory postsynaptic potential / negative regulation of protein phosphorylation / mitochondrion organization / positive regulation of protein ubiquitination / negative regulation of protein binding / regulation of autophagy / determination of adult lifespan / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / peptidyl-threonine phosphorylation / calcium-mediated signaling / cellular response to reactive oxygen species / trans-Golgi network / regulation of protein stability Similarity search - Function | |||||||||||||||
Biological species | Homo sapiens (human) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.2 Å | |||||||||||||||
Authors | Matyszewski M / Leschziner AE | |||||||||||||||
Funding support | United States, 4 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_25906.map.gz | 97.1 MB | EMDB map data format | |
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Header (meta data) | emd-25906-v30.xml emd-25906.xml | 22.1 KB 22.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_25906_fsc.xml | 10.4 KB | Display | FSC data file |
Images | emd_25906.png | 88.1 KB | ||
Masks | emd_25906_msk_1.map | 103 MB | Mask map | |
Filedesc metadata | emd-25906.cif.gz | 6.6 KB | ||
Others | emd_25906_additional_1.map.gz emd_25906_half_map_1.map.gz emd_25906_half_map_2.map.gz | 50.9 MB 95.7 MB 95.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25906 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25906 | HTTPS FTP |
-Validation report
Summary document | emd_25906_validation.pdf.gz | 950.3 KB | Display | EMDB validaton report |
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Full document | emd_25906_full_validation.pdf.gz | 949.9 KB | Display | |
Data in XML | emd_25906_validation.xml.gz | 17.9 KB | Display | |
Data in CIF | emd_25906_validation.cif.gz | 23.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25906 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25906 | HTTPS FTP |
-Related structure data
Related structure data | 7thyMC 7thzC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25906.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Sharpened map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.16 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_25906_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Non-sharpened map
File | emd_25906_additional_1.map | ||||||||||||
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Annotation | Non-sharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 1
File | emd_25906_half_map_1.map | ||||||||||||
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Annotation | Half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2
File | emd_25906_half_map_2.map | ||||||||||||
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Annotation | Half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
Entire | Name: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present |
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Components |
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-Supramolecule #1: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
Supramolecule | Name: 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
Macromolecule | Name: 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 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 22.191762 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: 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 |
-Macromolecule #2: GUANOSINE-5'-DIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: GDP |
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Molecular weight | Theoretical: 443.201 Da |
Chemical component information | ChemComp-GDP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | filament |
-Sample preparation
Buffer | pH: 7.4 Component:
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. | ||||||||||||||||||
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Grid | Model: Homemade / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. Details: Using EMS LC-300 Lacey Carbon grids (Not homemade, EMS not a choice for grid company) | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K | ||||||||||||||||||
Details | 4.5 uM of LRRK2RCKW 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). |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film 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 beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Details | Used 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. |
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Refinement | Protocol: FLEXIBLE FIT |
Output model | PDB-7thy: |