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- EMDB-25908: 2 microtubule protofilaments locally refined from Leucine Rich Re... -

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Basic information

Entry
Database: EMDB / ID: EMD-25908
Title2 microtubule protofilaments locally refined from Leucine Rich Repeat Kinase 2 filament structure bound to 11-protofilament microtubules
Map dataSharpened map, recommend masking with provided refinement mask to remove partially resolved LRRK2 from the background.
Sample
  • Complex: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present. This map focuses on the microtubule only
    • Protein or peptide: Tubulin alpha
    • Protein or peptide: Tubulin beta
Keywordsparkinson's disease / microtubule / CYTOSOLIC PROTEIN
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.4 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
DepositionJan 12, 2022-
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_25908.png

Downloads & links

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Map

FileDownload / File: emd_25908.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map, recommend masking with provided refinement mask to remove partially resolved LRRK2 from the background.
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.354
Minimum - Maximum-0.4932689 - 1.4042832
Average (Standard dev.)0.006528491 (±0.06615328)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 348.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25908_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

Fileemd_25908_additional_1.map
AnnotationNon-sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_25908_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_25908_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 : LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 presen...

EntireName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present. This map focuses on the microtubule only
Components
  • Complex: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present. This map focuses on the microtubule only
    • Protein or peptide: Tubulin alpha
    • Protein or peptide: Tubulin beta

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

SupramoleculeName: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present. This map focuses on the microtubule only
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: Tubulin alpha

MacromoleculeName: Tubulin alpha / type: protein_or_peptide / ID: 1 / 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 #2: Tubulin beta

MacromoleculeName: Tubulin beta / type: protein_or_peptide / ID: 2 / 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
Processingsingle particle 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: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: Using EMS LC-300 lacey grids (Not homemade)
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: 2 / 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

Particle selectionNumber selected: 557577
Details: Filament Autopicker with templates created by manual picking. This is before symmetry expansion.
Startup modelType of model: NONE
Details: Featureless cylinder for the original helical reconstruction, then subunit from the helical reconstruction after reboxing and symmetry expansion.
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2)
Software - details: local refinement, with non-uniform refinement turned off
Details: Local refinement focused on the microtubule from the full LRRK2-MT complex
Number images used: 133246
FSC plot (resolution estimation)

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