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- EMDB-25649: Helical Reconstruction of the C-terminal Half of Leucine Rich Rep... -

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Entry
Database: EMDB / ID: EMD-25649
TitleHelical Reconstruction of the C-terminal Half of Leucine Rich Repeat Kinase 2 (LRRK2) (I2020T) bound to 11-protofilament microtubule in presence of MLi-2 kinase inhibitor
Map dataLRRK2 RCKW I2020T bound to 11-pf microtubule with MLi-2. No helical symmetry on final reconstruction due to symmetry mismatch between LRRK2 and the microtubule.
Sample
  • Complex: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
    • Protein or peptide: Catalytic C-terminus of Leucine Rich Repeat Kinase 2
    • Protein or peptide: Tubulin alpha
    • Protein or peptide: Tublin beta
Keywordsparkinson's disease / microtubule / kinase / gtpase / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodhelical reconstruction / cryo EM / Resolution: 18.0 Å
AuthorsMatyszewski M / 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 6, 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_25649.png

Downloads & links

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Map

FileDownload / File: emd_25649.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLRRK2 RCKW I2020T bound to 11-pf microtubule with MLi-2. No helical symmetry on final reconstruction due to symmetry mismatch between LRRK2 and the microtubule.
Voxel sizeX=Y=Z: 2.32 Å
Density
Contour LevelBy AUTHOR: 0.0073
Minimum - Maximum-0.024565548 - 0.03819174
Average (Standard dev.)0.00017157575 (±0.0073397607)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 760.95996 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map 2

Fileemd_25649_half_map_1.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_25649_half_map_2.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present

EntireName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
Components
  • Complex: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
    • Protein or peptide: Catalytic C-terminus of Leucine Rich Repeat Kinase 2
    • Protein or peptide: Tubulin alpha
    • Protein or peptide: Tublin beta

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Supramolecule #1: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present

SupramoleculeName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: Catalytic C-terminus of Leucine Rich Repeat Kinase 2

MacromoleculeName: Catalytic C-terminus of Leucine Rich Repeat Kinase 2 / type: protein_or_peptide / ID: 1
Details: I2020T mutation present Residues 1327-2527 of LRRK2
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KKAVPYNRMK LMIVGNTGSG KTTLLQQLMK TKKSDLGMQS ATVGIDVKDW PIQIRDKRKR DLVLNVWDF AGREEFYSTH PHFMTQRALY LAVYDLSKGQ AEVDAMKPWL FNIKARASSS P VILVGTHL DVSDEKQRKA CMSKITKELL NKRGFPAIRD YHFVNATEES ...String:
KKAVPYNRMK LMIVGNTGSG KTTLLQQLMK TKKSDLGMQS ATVGIDVKDW PIQIRDKRKR DLVLNVWDF AGREEFYSTH PHFMTQRALY LAVYDLSKGQ AEVDAMKPWL FNIKARASSS P VILVGTHL DVSDEKQRKA CMSKITKELL NKRGFPAIRD YHFVNATEES DALAKLRKTI IN ESLNFKI RDQLVVGQLI PDCYVELEKI ILSERKNVPI EFPVIDRKRL LQLVRENQLQ LDE NELPHA VHFLNESGVL LHFQDPALQL SDLYFVEPKW LCKIMAQILT VKVEGCPKHP KGII SRRDV EKFLSKKRKF PKNYMSQYFK LLEKFQIALP IGEEYLLVPS SLSDHRPVIE LPHCE NSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGRERALRPN RMYWRQGIYL NWSPEA YCL VGSEVLDNHP ESFLKITVPS CRKGCILLGQ VVDHIDSLME EWFPGLLEID ICGEGET LL KKWALYSFND GEEHQKILLD DLMKKAEEGD LLVNPDQPRL TIPISQIAPD LILADLPR N IMLNNDELEF EQAPEFLLGD GSFGSVYRAA YEGEEVAVKI FNKHTSLRLL RQELVVLCH LHHPSLISLL AAGIRPRMLV MELASKGSLD RLLQQDKASL TRTLQHRIAL HVADGLRYLH SAMIIYRDL KPHNVLLFTL YPNAAIIAKI ADYGTAQYCC RMGIKTSEGT PGFRAPEVAR G NVIYNQQA DVYSFGLLLY DILTTGGRIV EGLKFPNEFD ELEIQGKLPD PVKEYGCAPW PM VEKLIKQ CLKENPQERP TSAQVFDILN SAELVCLTRR ILLPKNVIVE CMVATHHNSR NAS IWLGCG HTDRGQLSFL DLNTEGYTSE EVADSRILCL ALVHLPVEKE SWIVSGTQSG TLLV INTED GKKRHTLEKM TDSVTCLYCN SFSKQSKQKN FLLVGTADGK LAIFEDKTVK LKGAA PLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT SQLFSY AAF SDSNIITVVV DTALYIAKQN SPVVEVWDKK TEKLCGLIDC VHFLREVMVK ENKESKH KM SYSGRVKTLC LQKNTALWIG TGGGHILLLD LSTRRLIRVI YNFCNSVRVM MTAQLGSL K NVMLVLGYNR KNTEGTQKQK EIQSCLTVWD INLPHEVQNL EKHIEVRKEL AEKMRRTSV E

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Macromolecule #2: Tubulin alpha

MacromoleculeName: Tubulin alpha / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL D RIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL D RIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE FSIYPAPQVS TA VVEPYNS ILTTHTTLEH SDCAFMVDNE AIYDICRRNL DIERPTYTNL NRLIGQIVSS ITA SLRFDG ALNVDLTEFQ TNLVPYPRIH FPLATYAPVI SAEKAYHEQL SVAEITNACF EPAN QMVKC DPRHGKYMAC CLLYRGDVVP KDVNAAIATI KTKRTIQFVD WCPTGFKVGI NYQPP TVVP GGDLAKVQRA VCMLSNTTAI AEAWARLDHK FDLMYAKRAF VHWYVGEGME EGEFSE ARE DMAALEKDYE EVGVDSVEGE GEEEGEEY

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Macromolecule #3: Tublin beta

MacromoleculeName: Tublin beta / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
SequenceString: MREIVHIQIG QCGNQIGAKF WEVIGEEHGI DWAGSYCGDS ALQLERISVY YNEAHGKKYV PRAVLVDLE PGTMDSIRSS RVGALFQPDS FVHGNSGAGN NWAKGYYTEG AELVDRVLDA V RTEAEGCD CLQGFQLVHS LGGGTGSGMG TLLLGKIREE YPDRILNSFS ...String:
MREIVHIQIG QCGNQIGAKF WEVIGEEHGI DWAGSYCGDS ALQLERISVY YNEAHGKKYV PRAVLVDLE PGTMDSIRSS RVGALFQPDS FVHGNSGAGN NWAKGYYTEG AELVDRVLDA V RTEAEGCD CLQGFQLVHS LGGGTGSGMG TLLLGKIREE YPDRILNSFS VMPSPKVSDT VV EPYNAVL ALHQLVLNSD ACFCIDNEAL YDICFRTLRL STPTYGDLNH LVSLTMSGIT TSL RFPGQL NADLRKLAVN MVPFPRLHFF MPGFAPLTAQ GSQQYRALTV AELTQQMFDA RNTM AACDP RRGRYLTVAC IFRGRMSTKE VDEQLLNVQT RNSSCFVEWI PNNVKVAVCD IPPRG LSMA ATFIGNNTAI QELFSRISEH FSAMFKRKAF VHWYTGEGMD INEFTEAESN IQDLVS EYQ QFQDARADVE EEEIGGEAEV EPADKEH

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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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

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.
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: EMS (LC-300)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K
Details4.5 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).

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 55.0 e/Å2 / Details: 250 ms frames
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 354271 / Software - Name: RELION
Details: Autopicker with templates created by manual picking.
Startup modelType of model: NONE / Details: Featureless cylinder
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 33.7 Å
Applied symmetry - Helical parameters - Δ&Phi: 32.5 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Second to final reconstruction used helical symmetry as listed. Final reconstruction did not apply symmetry to recover additional microtubule detail.
Number images used: 14350
FSC plot (resolution estimation)

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