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- EMDB-18884: Ensemble map of the Roco protein from C. tepidum in the GTP state... -

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Basic information

Entry
Database: EMDB / ID: EMD-18884
TitleEnsemble map of the Roco protein from C. tepidum in the GTP state bound to the activating Nanobodies NbRoco1 and NbRoco2
Map data
Sample
  • Complex: CtRoco in the active GTP state bound to the two activating Nanobodies NbRoco1 and NbRoco2
    • Protein or peptide: Rab family protein
    • Protein or peptide: NbRoco1
    • Protein or peptide: NbRoco2
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
KeywordsGTPase / Nanobody / Parkinson's Disease / Allostery activator / HYDROLASE
Function / homology
Function and homology information


non-specific serine/threonine protein kinase / GTP binding / identical protein binding
Similarity search - Function
: / C-terminal of Roc, COR-B domain / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain / Ras of Complex, Roc, domain of DAPkinase / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / Roc domain profile. / Roc domain / Leucine rich repeat, ribonuclease inhibitor type ...: / C-terminal of Roc, COR-B domain / C-terminal of Roc (COR) domain / C-terminal of Roc, COR-A domain / Ras of Complex, Roc, domain of DAPkinase / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / Roc domain profile. / Roc domain / Leucine rich repeat, ribonuclease inhibitor type / Leucine Rich repeat / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Rab subfamily of small GTPases / Leucine-rich repeat domain superfamily / Small GTP-binding protein domain / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
non-specific serine/threonine protein kinase
Similarity search - Component
Biological speciesChlorobaculum tepidum (bacteria) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.88 Å
AuthorsGalicia C / 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
CitationJournal: 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.
History
DepositionNov 13, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18884.png

Downloads & links

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Map

FileDownload / File: emd_18884.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.51 Å/pix.
x 256 pix.
= 386.56 Å		1.51 Å/pix.
x 256 pix.
= 386.56 Å		1.51 Å/pix.
x 256 pix.
= 386.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.51 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.05
Minimum - Maximum-0.45193458 - 10.187048000000001
Average (Standard dev.)0.0021323336 (±0.06338506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 386.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : CtRoco in the active GTP state bound to the two activating Nanobo...

EntireName: CtRoco in the active GTP state bound to the two activating Nanobodies NbRoco1 and NbRoco2
Components
  • Complex: CtRoco in the active GTP state bound to the two activating Nanobodies NbRoco1 and NbRoco2
    • Protein or peptide: Rab family protein
    • Protein or peptide: NbRoco1
    • Protein or peptide: NbRoco2
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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Supramolecule #1: CtRoco in the active GTP state bound to the two activating Nanobo...

SupramoleculeName: CtRoco in the active GTP state bound to the two activating Nanobodies NbRoco1 and NbRoco2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Chlorobaculum tepidum (bacteria)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Rab family protein

MacromoleculeName: Rab family protein / type: protein_or_peptide / ID: 1 / Details: CtRoco bound to GTPgammaS / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlorobaculum tepidum (bacteria)
Molecular weightTheoretical: 124.313453 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMGSMSDLD VIRQIEQELG MQLEPVDKLK WYSKGYKLDK DQRVTAIGLY DCGSDTLDRI IQPLESLKSL SELSLSSNQI TDISPLASL NSLSMLWLDR NQITDIAPLA SLNSLSMLWL FGNKISDIAP LESLKSLTEL QLSSNQITDI APLASLKSLT E LSLSGNNI ...String:
GAMGSMSDLD VIRQIEQELG MQLEPVDKLK WYSKGYKLDK DQRVTAIGLY DCGSDTLDRI IQPLESLKSL SELSLSSNQI TDISPLASL NSLSMLWLDR NQITDIAPLA SLNSLSMLWL FGNKISDIAP LESLKSLTEL QLSSNQITDI APLASLKSLT E LSLSGNNI SDIAPLESLK SLTELSLSSN QITDIAPLAS LKSLTELSLS SNQISDIAPL ESLKSLTELQ LSRNQISDIA PL ESLKSLT ELQLSSNQIT DIAPLASLKS LTELQLSRNQ ISDIAPLESL NSLSKLWLNG NQITDIAPLA SLNSLTELEL SSN QITDIA PLASLKSLST LWLSSNQISD IAPLASLESL SELSLSSNQI SDISPLASLN SLTGFDVRRN PIKRLPETIT GFDM EILWN DFSSSGFITF FDNPLESPPP EIVKQGKEAV RQYFQSIEEA RSKGEALVHL QEIKVHLIGD GMAGKTSLLK QLIGE TFDP KESQTHGLNV VTKQAPNIKG LENDDELKEC LFHFWDFGGQ EIMHASHQFF MTRSSVYMLL LDSRTDSNKH YWLRHI EKY GGKSPVIVVM NKIDENPSYN IEQKKINERF PAIENRFHRI SCKNGDGVES IAKSLKSAVL HPDSIYGTPL APSWIKV KE KLVEATTAQR YLNRTEVEKI CNDSGITDPG ERKTLLGYLN NLGIVLYFEA LDLSEIYVLD PHWVTIGVYR IINSSKTK N GHLNTSALGY ILNEEQIRCD EYDPAKNNKF TYTLLEQRYL LDIMKQFELC YDEGKGLFII PSNLPTQIDN EPEITEGEP LRFIMKYDYL PSTIIPRLMI AMQHQILDRM QWRYGMVLKS QDHEGALAKV VAETKDSTIT IAIQGEPRCK REYLSIIWYE IKKINANFT NLDVKEFIPL PGHPDELVEY KELLGLEKMG RDEYVSGKLE KVFSVSKMLD SVISKEERNK ERLMGDINIK L ENIGNPTI PIHQQVEVNV SQETVQHVEN LQGFFENLKA DILREAELEI DDPKERKRLA NELELAENAI TKMDAAVKSG KN KLKPDVK DRLGEFIDNL ANENSRLRKG IALVMNGAEK VQKLARYYNN VAPFFDLPSV PPVLLGKEKT

UniProtKB: non-specific serine/threonine protein kinase

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Macromolecule #2: NbRoco1

MacromoleculeName: NbRoco1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 15.206718 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCANSGLTFS TYTMGWFRQA PGKEREFVAA IRWSGTSTYY QDHADSVKGR FTISRDNAKN TVYLQMNSL KPEDTAVYYC AASRLRAGVK APSEYDYWGQ GTQVTVSSHH HHHHEPEA

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Macromolecule #3: NbRoco2

MacromoleculeName: NbRoco2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.180744 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGKLLS INVMGWYRQA PGKQRELVAS ITTGGRINYA DSVKGRFTIT RDNAKNTVYL QMDSLKPED TAVYYCNADG IIAGLYQNDH WGQGTQVTVS SHHHHHHEPE A

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Macromolecule #4: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GSP
Molecular weightTheoretical: 539.246 Da
Chemical component information

ChemComp-GSP:
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 90 %

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Electron microscopy

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

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 193185
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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