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- EMDB-18879: Roco protein from C. tepidum in the GTP state bound to an activat... -

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Basic information

Entry
Database: EMDB / ID: EMD-18879
TitleRoco protein from C. tepidum in the GTP state bound to an activating Nanobody
Map data
Sample
  • Complex: CtRoco in the active GTP state bound to the activating Nanobody NbRoco1
    • Protein or peptide: CtRoco
    • Protein or peptide: NbRoco1
KeywordsGTPase / Nanobody / Parkinson's Disease / Allostery activator / HYDROLASE
Biological speciesChlorobaculum tepidum (bacteria) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsGalicia C / Fislage M / Versees W
Funding support Belgium, 3 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G005219N Belgium
Research Foundation - Flanders (FWO)G003322N Belgium
Vrije Universiteit BrusselSRP50 Belgium
Citation
Journal: Elife / Year: 2024
Title: Structural insights into the GTP-driven monomerization and activation of a bacterial LRRK2 homolog using allosteric nanobodies.
Authors: Christian Galicia / Giambattista Guaitoli / Marcus Fislage / Christian Johannes Gloeckner / Wim Versées /
Abstract: Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson's disease. LRRK2 is a large and complex protein combining a GTPase and ...Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson's disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: Nb and Nb. This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how Nb and Nb collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.
#1: Journal: Elife / Year: 2023
Title: Structural insights in the GTP-driven monomerization and activation of a bacterial LRRK2 homologue using allosteric nanobodies
Authors: Galicia C / Guaitoli G / Fislage M / Gloeckner CJ / Versees W
History
DepositionNov 10, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18879.png

Downloads & links

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Map

FileDownload / File: emd_18879.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 256 pix.
= 317.824 Å		1.24 Å/pix.
x 256 pix.
= 317.824 Å		1.24 Å/pix.
x 256 pix.
= 317.824 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2415 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.127
Minimum - Maximum-0.15683718 - 0.43985894
Average (Standard dev.)-0.00016380222 (±0.015680347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 317.824 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18879_msk_1.map
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Half map: #1

Fileemd_18879_half_map_1.map
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Half map: #2

Fileemd_18879_half_map_2.map
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Sample components

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Entire : CtRoco in the active GTP state bound to the activating Nanobody N...

EntireName: CtRoco in the active GTP state bound to the activating Nanobody NbRoco1
Components
  • Complex: CtRoco in the active GTP state bound to the activating Nanobody NbRoco1
    • Protein or peptide: CtRoco
    • Protein or peptide: NbRoco1

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Supramolecule #1: CtRoco in the active GTP state bound to the activating Nanobody N...

SupramoleculeName: CtRoco in the active GTP state bound to the activating Nanobody NbRoco1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chlorobaculum tepidum (bacteria)
Molecular weightTheoretical: 140 KDa

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Macromolecule #1: CtRoco

MacromoleculeName: CtRoco / type: protein_or_peptide / ID: 1 / Details: CtRoco bound to GTPgammaS / Enantiomer: LEVO
Source (natural)Organism: Chlorobaculum tepidum (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMGSMSDLD VIRQIEQELG MQLEPVDKLK WYSKGYKLDK DQRVTAIGLY DCGSDTLDRI IQP LESLKS LSELSLSSNQ ITDISPLASL NSLSMLWLDR NQITDIAPLA SLNSLSMLWL FGNKISDIAP L ESLKSLTE LQLSSNQITD IAPLASLKSL TELSLSGNNI ...String:
GAMGSMSDLD VIRQIEQELG MQLEPVDKLK WYSKGYKLDK DQRVTAIGLY DCGSDTLDRI IQP LESLKS LSELSLSSNQ ITDISPLASL NSLSMLWLDR NQITDIAPLA SLNSLSMLWL FGNKISDIAP L ESLKSLTE LQLSSNQITD IAPLASLKSL TELSLSGNNI SDIAPLESLK SLTELSLSSN QITDIAPLAS L KSLTELSL SSNQISDIAP LESLKSLTEL QLSRNQISDI APLESLKSLT ELQLSSNQIT DIAPLASLKS L TELQLSRN QISDIAPLES LNSLSKLWLN GNQITDIAPL ASLNSLTELE LSSNQITDIA PLASLKSLS TLWLSSNQIS DIAPLASLES LSELSLSSNQ ISDISPLASL NSLTGFDVRR NPIKRLPETI TGFDMEIL W NDFSSSGFIT FFDNPLESPP PEIVKQGKEA VRQYFQSIEE ARSKGEALVH LQEIKVHLIG DGMA GKTSL LKQLIGETFD PKESQTHGLN VVTKQAPNIK GLENDDELKE CLFHFWDFGG QEIMHASH Q FFMTRSSVYM LLLDSRTDSN KHYWLRHIEK YGGKSPVIVV MNKIDENPSY NIEQKKINER FPA IENRFH RISCKNGDGV ESIAKSLKSA VLHPDSIYGT PLAPSWIKVK EKLVEATTAQ RYLNRTEVE KICNDSGITD PGERKTLLGY LNNLGIVLYF EALDLSEIYV LDPHWVTIGV YRIINSSKTK NGHLN TSAL GYILNEEQIR CDEYDPAKNN KFTYTLLEQR YLLDIMKQFE LCYDEGKGLF IIPSNLPTQI D NEPEITEG EPLRFIMKYD YLPSTIIPRL MIAMQHQILD RMQWRYGMVL KSQDHEGALA KVVAE TKDS TITIAIQGEP RCKREYLSII WYEIKKINAN FTNLDVKEFI PLPGHPDELV EYKELLGLEK MG RDEYVSG KLEKVFSVSK MLDSVISKEE RNKERLMGDI NIKLENIGNP TIPIHQQVEV NVSQETV QH VENLQGFFEN LKADILREAE LEIDDPKERK RLANELELAE NAITKMDAAV KSGKNKLKPD V KDRLGEFI DNLANENSRL RKGIALVMNG AEKVQKLARY YNNVAPFFDL PSVPPVLLGK EKT

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Macromolecule #2: NbRoco1

MacromoleculeName: NbRoco1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCANSGLTFS TYTMGWFRQA PGKEREFVAA IRWSGTSTYY QDHADSVKGR FTISRDNAKN TVYLQMNSLK PEDTAVYYCA ASRLRAGVKA PSEYDYWGQG TQVTVSSHHH HHHEPEA

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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

Concentration0.08 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 90 %

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.55 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 63.0 e/Å2

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99460
FSC plot (resolution estimation)

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