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- EMDB-18886: Focused map on the Roc-COR domains of the Roco protein from C. te... -

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Basic information

Entry
Database: EMDB / ID: EMD-18886
TitleFocused map on the Roc-COR domains of the Roco protein from C. tepidum in the GTP state bound to the activating Nanobody NbRoco1
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
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
KeywordsGTPase / Nanobody / Parkinson's Disease / Allostery activator / HYDROLASE
Function / homology
Function and homology information


GTP binding / identical protein binding
Similarity search - Function
C-terminal of Roc (COR) domain / C-terminal of Roc, COR, domain / Ras of Complex, Roc, domain of DAPkinase / Leucine rich repeat, ribonuclease inhibitor type / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / Roc domain profile. / Roc domain / Leucine Rich repeat / Leucine-rich repeats, bacterial type ...C-terminal of Roc (COR) domain / C-terminal of Roc, COR, domain / Ras of Complex, Roc, domain of DAPkinase / Leucine rich repeat, ribonuclease inhibitor type / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / Roc domain profile. / Roc domain / 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
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
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 13, 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_18886.png

Downloads & links

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Map

FileDownload / File: emd_18886.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.4381 Å
Density
Contour LevelBy AUTHOR: 1.1
Minimum - Maximum-8.299124000000001 - 13.506817
Average (Standard dev.)0.00016136756 (±0.15301211)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 241.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18886_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18886_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18886_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
  • 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-#2
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: Rab family protein

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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: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 3 / 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
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: 3.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 38260
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6hlu

source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8r4d:
Focused map on the Roc-COR domains of the Roco protein from C. tepidum in the GTP state bound to the activating Nanobody NbRoco1

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