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- EMDB-21311: Cryo-EM map of C-terminal half of Leucine Rich Repeat Kinase 2 as... -

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Basic information

Entry
Database: EMDB / ID: EMD-21311
TitleCryo-EM map of C-terminal half of Leucine Rich Repeat Kinase 2 as a COR-COR domain dimer in the presence of the MLi-2 kinase inhibitor at 9.0 angstroms
Map data9.04A map of a Leucine Rich Repeat Kinase 2 truncate as a COR mediated dimer
Sample
  • Organelle or cellular component: Truncated Leucine Rich Repeat Kinase 2 as a COR-COR domain mediated dimer in the presence of the MLi-2 kinase inhibitor.
    • Protein or peptide: Leucine Rich Repeat Kinase 2
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 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 Center for Research ResourcesR01GM107214 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
DepositionFeb 3, 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: 0.448
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.448
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21311.map.gz / Format: CCP4 / Size: 19.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation9.04A map of a Leucine Rich Repeat Kinase 2 truncate as a COR mediated dimer
Voxel sizeX=Y=Z: 2.32 Å
Density
Contour LevelBy AUTHOR: 0.448 / Movie #1: 0.448
Minimum - Maximum-0.5732527 - 1.4175295
Average (Standard dev.)0.0005919619 (±0.06729745)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions172172172
Spacing172172172
CellA=B=C: 399.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.322.322.32
M x/y/z172172172
origin x/y/z0.0000.0000.000
length x/y/z399.040399.040399.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS172172172
D min/max/mean-0.5731.4180.001

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Supplemental data

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Half map: Half map 1 of COR mediated dimer map

Fileemd_21311_half_map_1.map
AnnotationHalf map 1 of COR mediated dimer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2 of COR mediated dimer map

Fileemd_21311_half_map_2.map
AnnotationHalf map 2 of COR mediated dimer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Truncated Leucine Rich Repeat Kinase 2 as a COR-COR domain mediat...

EntireName: Truncated Leucine Rich Repeat Kinase 2 as a COR-COR domain mediated dimer in the presence of the MLi-2 kinase inhibitor.
Components
  • Organelle or cellular component: Truncated Leucine Rich Repeat Kinase 2 as a COR-COR domain mediated dimer in the presence of the MLi-2 kinase inhibitor.
    • Protein or peptide: Leucine Rich Repeat Kinase 2

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Supramolecule #1: Truncated Leucine Rich Repeat Kinase 2 as a COR-COR domain mediat...

SupramoleculeName: Truncated Leucine Rich Repeat Kinase 2 as a COR-COR domain mediated dimer in the presence of the MLi-2 kinase inhibitor.
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: A COR-COR (9.0A) mediated dimer of truncated Leucine Rich Repeat Kinase 2 starting at residue 1327 all the way to the C-terminal in the presence of the MLi-2 kinase inhibitor at 5uM.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 274 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Leucine Rich Repeat Kinase 2

MacromoleculeName: Leucine Rich Repeat Kinase 2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KKAVPYNRMK LMIVGNTGSG KTTLLQQLMK TKKSDLGMQS ATVGIDVKDW PIQIRDKRKR DLVLNVWDFA GREE FYSTH PHFMTQRALY LAVYDLSKGQ AEVDAMKPWL FNIKARASSS PVILVGTHLD VSDEKQRKAC MSKIT KELL NKRGFPAIRD YHFVNATEES ...String:
KKAVPYNRMK LMIVGNTGSG KTTLLQQLMK TKKSDLGMQS ATVGIDVKDW PIQIRDKRKR DLVLNVWDFA GREE FYSTH PHFMTQRALY LAVYDLSKGQ AEVDAMKPWL FNIKARASSS PVILVGTHLD VSDEKQRKAC MSKIT KELL NKRGFPAIRD YHFVNATEES DALAKLRKTI INESLNFKIR DQLVVGQLIP DCYVELEKII LSERKN VPI EFPVIDRKRL LQLVRENQLQ LDENELPHAV HFLNESGVLL HFQDPALQLS DLYFVEPKWL CKIMAQI LT VKVEGCPKHP KGIISRRDVE KFLSKKRKFP KNYMSQYFKL LEKFQIALPI GEEYLLVPSS LSDHRPVI E LPHCENSEII IRLYEMPYFP MGFWSRLINR LLEISPYMLS GRERALRPNR MYWRQGIYLN WSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI CGEGETLLKK WALYSFNDGE EHQKILLDD LMKKAEEGDL LVNPDQPRLT IPISQIAPDL ILADLPRNIM LNNDELEFEQ APEFLLGDGS F GSVYRAAY EGEEVAVKIF NKHTSLRLLR QELVVLCHLH HPSLISLLAA GIRPRMLVME LASKGSLDRL LQ QDKASLT RTLQHRIALH VADGLRYLHS AMIIYRDLKP HNVLLFTLYP NAAIIAKIAD YGIAQYCCRM GIK TSEGTP GFRAPEVARG NVIYNQQADV YSFGLLLYDI LTTGGRIVEG LKFPNEFDEL EIQGKLPDPV KEYG CAPWP MVEKLIKQCL KENPQERPTS AQVFDILNSA ELVCLTRRIL LPKNVIVECM VATHHNSRNA SIWLG CGHT DRGQLSFLDL NTEGYTSEEV ADSRILCLAL VHLPVEKESW IVSGTQSGTL LVINTEDGKK RHTLEK MTD SVTCLYCNSF SKQSKQKNFL LVGTADGKLA IFEDKTVKLK GAAPLKILNI GNVSTPLMCL SESTNST ER NVMWGGCGTK IFSFSNDFTI QKLIETRTSQ LFSYAAFSDS NIITVVVDTA LYIAKQNSPV VEVWDKKT E KLCGLIDCVH FLREVMVKEN KESKHKMSYS GRVKTLCLQK NTALWIGTGG GHILLLDLST RRLIRVIYN FCNSVRVMMT AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKELAEK MRRTSVE

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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
5.0 uMMLi-2
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK II
DetailsThis sample was imaged over a number of datasets. Specimen concentrations ranged from 0.375(3uM)-0.5(4uM) mg/mL, however the average was 0.5 and thus was entered as the reported specimen concentration.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
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 real images: 4030 / Average exposure time: 9.5 sec. / Average electron dose: 5.5 e/Å2
Details: Image lengths of either 9 seconds or 10 seconds were collected.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 706807
Details: Cryolo Ver.1 {Wagner,2019} was used to pick particles.
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: Initial model was built from our reconstruction of truncated Leucine Rich Repeat Kinase 2 fit into an ab initio map of the dimer out of Cryosparc2. See the paper for more details.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 36727
FSC plot (resolution estimation)

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