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- EMDB-25672: Structure of the C-terminal half of Leucine Rich Repeat Kinase 1 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-25672
TitleStructure of the C-terminal half of Leucine Rich Repeat Kinase 1 (LRRK1)
Map dataCatalytic C-terminus of Leucine Rich Repeat Kinase 1 (LRRK1)
Sample
  • Complex: Catalytic C-terminus of Leucine Rich Repeat Kinase 1
    • Protein or peptide: C-terminal half of Leucine Rich Repeat Kinase 1
Keywordskinase / gtpase / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsMatyszewski M / Snead DM / Leschziner AE
Funding support United States, 1 items
OrganizationGrant numberCountry
Other privateASAP-000519 United States
CitationJournal: 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
DepositionDec 9, 2021-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25672.png

Downloads & links

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Map

FileDownload / File: emd_25672.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCatalytic C-terminus of Leucine Rich Repeat Kinase 1 (LRRK1)
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.144
Minimum - Maximum-0.30536932 - 0.77849686
Average (Standard dev.)0.000021988697 (±0.015852444)
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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Mask #1

Fileemd_25672_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non-sharpened map of LRRK1

Fileemd_25672_additional_1.map
AnnotationNon-sharpened map of LRRK1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_25672_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_25672_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Catalytic C-terminus of Leucine Rich Repeat Kinase 1

EntireName: Catalytic C-terminus of Leucine Rich Repeat Kinase 1
Components
  • Complex: Catalytic C-terminus of Leucine Rich Repeat Kinase 1
    • Protein or peptide: C-terminal half of Leucine Rich Repeat Kinase 1

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Supramolecule #1: Catalytic C-terminus of Leucine Rich Repeat Kinase 1

SupramoleculeName: Catalytic C-terminus of Leucine Rich Repeat Kinase 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 155 KDa

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Macromolecule #1: C-terminal half of Leucine Rich Repeat Kinase 1

MacromoleculeName: C-terminal half of Leucine Rich Repeat Kinase 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: RKAEKCKLMK MIIVGPPRQG KSTLLEILQT GRAPQVVHGE ATIRTTKWEL QRPAGSRAKV ESVEFNVWD IGGPASMATV NQCFFTDKAL YVVVWNLALG EEAVANLQFW LLNIEAKAPN A VVLVVGTH LDLIEAKFRV ERIATLRAYV LALCRSPSGS RATGFPDITF ...String:
RKAEKCKLMK MIIVGPPRQG KSTLLEILQT GRAPQVVHGE ATIRTTKWEL QRPAGSRAKV ESVEFNVWD IGGPASMATV NQCFFTDKAL YVVVWNLALG EEAVANLQFW LLNIEAKAPN A VVLVVGTH LDLIEAKFRV ERIATLRAYV LALCRSPSGS RATGFPDITF KHLHEISCKS LE GQEGLRQ LIFHVTCSMK DVGSTIGCQR LAGRLIPRSY LSLQEAVLAE QQRRSRDDDV QYL TDRQLE QLVEQTPDND IKDYEDLQSA ISFLIETGTL LHFPDTSHGL RNLYFLDPIW LSEC LQRIF NIKGSRSVAK NGVIRAEDLR MLLVGTGFTQ QTEEQYFQFL AKFEIALPVA NDSYL LPHL LPSKPGLDTH GMRHPTANTI QRVFKMSFVP VGFWQRFIAR MLISLAEMDL QLFENK KNT KSRNRKVTIY SFTGNQRNRC STFRVKRNQT IYWQEGLLVT FDGGYLSVES SDVNWKK KK SGGMKIVCQS EVRDFSAMAF ITDHVNSLID QWFPALTATE SDGTPLMEQY VPCPVCET A WAQHTDPSEK SEDVQYFDME DCVLTAIERD FISCPRHPDL PVPLQELVPE LFMTDFPAR LFLENSKLEH SEDEGSVLGQ GGSGTVIYRA RYQGQPVAVK RFHIKKFKNF ANVPADTMLR HLRATDAMK NFSEFRQEAS MLHALQHPCI VALIGISIHP LCFALELAPL SSLNTVLSEN A RDSSFIPL GHMLTQKIAY QIASGLAYLH KKNIIFCDLK SDNILVWSLD VKEHINIKLS DY GISRQSF HEGALGVEGT PGYQAPEIRP RIVYDEKVDM FSYGMVLYEL LSGQRPALGH HQL QIAKKL SKGIRPVLGQ PEEVQFRRLQ ALMMECWDTK PEKRPLALSV VSQMKDPTFA TFMY ELCCG KQTAFFSSQG QEYTVVFWDG KEESRNYTVV NTEKGLMEVQ RMCCPGMKVS CQLQV QRSL WTATEDQKIY IYTLKGMCPL NTPQQALDTP AVVTCFLAVP VIKKNSYLVL AGLADG LVA VFPVVRGTPK DSCSYLCSHT ANRSKFSIAD EDARQNPYPV KAMEVVNSGS EVWYSNG PG LLVIDCASLE ICRRLEPYMA PSMVTSVVCS SEGRGEEVVW CLDDKANSLV MYHSTTYQ L CARYFCGVPS PLRDMFPVRP LDTEPPAASH TANPKVPEGD SIADVSIMYS EELGTQILI HQESLTDYCS MSSYSSSPPR QAARSPSSLP SSPASSSSVP FSTDCEDSDM LHTPGAASDR SEHDLTPMD GETFSQHLQA VKILAVRDLI WVPRRGGDVI VIGLEKDSGA QRGRVIAVLK A RELTPHGV LVDAAVVAKD TVVCTFENEN TEWCLAVWRG WGAREFDIFY QSYEELGRLE AC TRKRR

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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

BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES
80.0 mMSodium ChlorideNaClSodium chloride
0.5 mMTCEP
2.5 mMMagnesium ChlorideMgCl2
20.0 uMGDP
GridModel: UltrAuFoil R1.2/1.3 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsConcentrations varied from 2 to 6 uM depending on the grid.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Details1 out of the 4 datasets collected was tilted by 20 degrees
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 4 / Average electron dose: 55.0 e/Å2
Details: 1 dataset was tilted by 20 degrees. 250 ms frames used
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 645743
Details: crYOLO was more specific with picks than blob picking, but after 2D classification, results were almost identical. Blob picker was used during on-the-fly analysis and if results looked good, ...Details: crYOLO was more specific with picks than blob picking, but after 2D classification, results were almost identical. Blob picker was used during on-the-fly analysis and if results looked good, the particles were kept. Otherwise, crYOLO was used with a previous trained model for LRRK2 RCKW (from Deniston et al).
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: Ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Software - details: Non-uniform refinement
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Non-uniform refinement / Number images used: 44748
FSC plot (resolution estimation)

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