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

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Basic information

Entry
Database: PDB / ID: 8u1b
TitleC-terminal LRRK2 bound to E11 DARPin
Components
  • E11 DARPin
  • Leucine-rich repeat serine/threonine-protein kinase 2
KeywordsPROTEIN BINDING / Kinase / complex / DARPins
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 / regulation of branching morphogenesis of a nerve / beta-catenin destruction complex binding / 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 / regulation of branching morphogenesis of a nerve / beta-catenin destruction complex binding / 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 mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / regulation of synaptic vesicle transport / regulation of lysosomal lumen pH / positive regulation of dopamine receptor signaling pathway / amphisome / regulation of CAMKK-AMPK signaling cascade / cytoplasmic side of mitochondrial outer membrane / co-receptor binding / mitochondrion localization / 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 / regulation of protein kinase A signaling / regulation of dendritic spine morphogenesis / striatum development / multivesicular body, internal vesicle / protein localization to mitochondrion / cellular response to dopamine / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / presynaptic cytosol / positive regulation of programmed cell death / GTP metabolic process / Wnt signalosome / regulation of canonical Wnt signaling pathway / negative regulation of protein processing / negative regulation of GTPase activity / syntaxin-1 binding / regulation of reactive oxygen species metabolic process / exploration behavior / protein kinase A binding / regulation of locomotion / regulation of synaptic vesicle exocytosis / PTK6 promotes HIF1A stabilization / clathrin binding / Golgi-associated vesicle / negative regulation of macroautophagy / neuromuscular junction development / lysosome organization / regulation of mitochondrial fission / intracellular distribution of mitochondria / autolysosome / Golgi organization / positive regulation of nitric-oxide synthase biosynthetic process / locomotory exploration behavior / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / MAP kinase kinase kinase activity / positive regulation of protein kinase activity / canonical Wnt signaling pathway / cellular response to manganese ion / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / positive regulation of autophagy / JNK cascade / phosphorylation / regulation of synaptic transmission, glutamatergic / dendrite cytoplasm / GTPase activator activity / tubulin binding / cellular response to starvation / neuron projection morphogenesis / SNARE binding / regulation of membrane potential / excitatory postsynaptic potential / negative regulation of protein phosphorylation / positive regulation of protein ubiquitination / mitochondrion organization / negative regulation of protein binding / regulation of autophagy / determination of adult lifespan / mitochondrial membrane / peptidyl-threonine phosphorylation / calcium-mediated signaling / positive regulation of MAP kinase activity / trans-Golgi network / regulation of protein stability / terminal bouton
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 ...: / 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. / Protein kinase domain / WD40/YVTN repeat-like-containing domain superfamily / 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSanz-Murillo, M. / Mathea, S. / Dederer, V. / Knapp, S. / Leschziner, A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other privateASAP-000519 United States
CitationJournal: To Be Published
Title: Development and characterization of DARPins that bind to LRRK2 WD40 domain
Authors: Mathea, S. / Dederer, V. / Sanz-Murillo, M. / Leschziner, A. / Knapp, S.
History
DepositionAug 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Leucine-rich repeat serine/threonine-protein kinase 2
C: E11 DARPin


Theoretical massNumber of molelcules
Total (without water)155,8282
Polymers155,8282
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 136060.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRK2, PARK8 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q5S007, non-specific serine/threonine protein kinase, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#2: Protein E11 DARPin


Mass: 19766.912 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Nterminal corresponds to HisTag. Some amino acids are not modeled due to the lack of density
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: C-terminal LRRK2 bound to E11 DARPin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.156 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMSodium ChlorideNaCl1
32.5 mMMagnesium ChlorideMgCl21
40.5 mMTCEP1
55 %Glycerol1
620 uMGDP1
SpecimenConc.: 0.68 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2468

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7UCSF ChimeraX1.5model fitting
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
12cryoSPARC43D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 341041
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185601 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6VP7

6vp7


Pdb chain-ID: A / Accession code: 6VP7 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044956
ELECTRON MICROSCOPYf_angle_d0.7746716
ELECTRON MICROSCOPYf_dihedral_angle_d4.919653
ELECTRON MICROSCOPYf_chiral_restr0.045802
ELECTRON MICROSCOPYf_plane_restr0.005844

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