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- EMDB-41806: C-terminal LRRK2 bound to E11 DARPin -

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Basic information

Entry
Database: EMDB / ID: EMD-41806
TitleC-terminal LRRK2 bound to E11 DARPin
Map data
Sample
  • Complex: C-terminal LRRK2 bound to E11 DARPin
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
    • Protein or peptide: E11 DARPin
KeywordsKinase / complex / DARPins / 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSanz-Murillo M / Mathea S / Dederer V / Knapp S / Leschziner A
Funding support United States, 1 items
OrganizationGrant numberCountry
Other privateASAP-000519 United States
CitationJournal: J Biol Chem / Year: 2024
Title: A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.
Authors: Verena Dederer / Marta Sanz Murillo / Eva P Karasmanis / Kathryn S Hatch / Deep Chatterjee / Franziska Preuss / Kamal R Abdul Azeez / Landon Vu Nguyen / Christian Galicia / Birgit Dreier / ...Authors: Verena Dederer / Marta Sanz Murillo / Eva P Karasmanis / Kathryn S Hatch / Deep Chatterjee / Franziska Preuss / Kamal R Abdul Azeez / Landon Vu Nguyen / Christian Galicia / Birgit Dreier / Andreas Plückthun / Wim Versees / Sebastian Mathea / Andres E Leschziner / Samara L Reck-Peterson / Stefan Knapp /
Abstract: Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The ...Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
History
DepositionAug 31, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41806.png

Downloads & links

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Map

FileDownload / File: emd_41806.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 288 pix.
= 334.08 Å		1.16 Å/pix.
x 288 pix.
= 334.08 Å		1.16 Å/pix.
x 288 pix.
= 334.08 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: 0.212
Minimum - Maximum-1.2509565 - 2.939302
Average (Standard dev.)-0.00001639309 (±0.021459064)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 334.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : C-terminal LRRK2 bound to E11 DARPin

EntireName: C-terminal LRRK2 bound to E11 DARPin
Components
  • Complex: C-terminal LRRK2 bound to E11 DARPin
    • Protein or peptide: Leucine-rich repeat serine/threonine-protein kinase 2
    • Protein or peptide: E11 DARPin

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Supramolecule #1: C-terminal LRRK2 bound to E11 DARPin

SupramoleculeName: C-terminal LRRK2 bound to E11 DARPin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 156 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: 136.060656 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: RMKLMIVGNT GSGKTTLLQQ LMKTKKSDLG MQSATVGIDV KDWPIQIRDK RKRDLVLNVW DFAGREEFYS THPHFMTQRA LYLAVYDLS KGQAEVDAMK PWLFNIKARA SSSPVILVGT HLDVSDEKQR KACMSKITKE LLNKRGFPAI RDYHFVNATE E SDALAKLR ...String:
RMKLMIVGNT GSGKTTLLQQ LMKTKKSDLG MQSATVGIDV KDWPIQIRDK RKRDLVLNVW DFAGREEFYS THPHFMTQRA LYLAVYDLS KGQAEVDAMK PWLFNIKARA SSSPVILVGT HLDVSDEKQR KACMSKITKE LLNKRGFPAI RDYHFVNATE E SDALAKLR KTIINESLNF KIRDQLVVGQ LIPDCYVELE KIILSERKNV PIEFPVIDRK RLLQLVRENQ LQLDENELPH AV HFLNESG VLLHFQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KLL EKFQIA LPIGEEYLLV PSSLSDHRPV IELPHCENSE IIIRLYEMPY FPMGFWSRLI NRLLEISPYM LSGRERALRP NRMY WRQGI YLNWSPEAYC LVGSEVLDNH PESFLKITVP SCRKGCILLG QVVDHIDSLM EEWFPGLLEI DICGEGETLL KKWAL YSFN DGEEHQKILL DDLMKKAEEG DLLVNPDQPR LTIPISQIAP DLILADLPRN IMLNNDELEF EQAPEFLLGD GSFGSV YRA AYEGEEVAVK IFNKHTSLRL LRQELVVLCH LHHPSLISLL AAGIRPRMLV MELASKGSLD RLLQQDKASL TRTLQHR IA LHVADGLRYL HSAMIIYRDL KPHNVLLFTL YPNAAIIAKI ADYGIAQYCC RMGIKTSEGT PGFRAPEVAR GNVIYNQQ A DVYSFGLLLY DILTTGGRIV EGLKFPNEFD ELEIQGKLPD PVKEYGCAPW PMVEKLIKQC LKENPQERPT SAQVFDILN SAELVCLTRR ILLPKNVIVE CMVATHHNSR NASIWLGCGH TDRGQLSFLD LNTEGYTSEE VADSRILCLA LVHLPVEKES WIVSGTQSG TLLVINTEDG KKRHTLEKMT DSVTCLYCNS FSKQSKQKNF LLVGTADGKL AIFEDKTVKL KGAAPLKILN I GNVSTPLM CLSESTNSTE RNVMWGGCGT KIFSFSNDFT IQKLIETRTS QLFSYAAFSD SNIITVVVDT ALYIAKQNSP VV EVWDKKT EKLCGLIDCV HFLREVMVKE NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCN SVRVMM TAQLGSLKNV MLVLGYNRKN TEGTQKQKEI QSCLTVWDIN LPHEVQNLEK HIEVRKELAE KMRRTSVE

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

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Macromolecule #2: E11 DARPin

MacromoleculeName: E11 DARPin / type: protein_or_peptide / ID: 2
Details: Nterminal corresponds to HisTag. Some amino acids are not modeled due to the lack of density
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.766912 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MRGSHHHHHH HHGSDLGKKL LEAARAGQDD EVRILMANGA DVNATDEAGV TPLHLAADSG HLEIVEVLLK TGADVNAWDH YGFTPLHLA AHVGHLEIVE VLLKAGADVN AQDHAGWTPL HLAALYGHLE IVEVLLKHGA DVNAQDMWGE TPFDLAIDNG N EDIAEVLQ KAAKLNDYKD DDDK

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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

Concentration0.68 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMSodium ChlorideNaCl
2.5 mMMagnesium ChlorideMgCl2
0.5 mMTCEP
5.0 %Glycerol
20.0 uMGDP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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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: 1 / Number real images: 2468 / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 341041
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6vp7

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 185601
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6vp7


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8u1b:
C-terminal LRRK2 bound to E11 DARPin

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