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- PDB-8p50: Photorhabdus luminescens Makes caterpillars floppy (Mcf) toxin wi... -

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Basic information

Entry
Database: PDB / ID: 8p50
TitlePhotorhabdus luminescens Makes caterpillars floppy (Mcf) toxin with the C-terminal deletion in complex with Arf3
Components
  • ADP-ribosylation factor 3ADP ribosylation factor
  • Toxin protein
KeywordsTOXIN / Bacterial toxin / Arf
Function / homology
Function and homology information


Synthesis of PIPs at the Golgi membrane / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / vesicle-mediated transport / intracellular protein transport / Golgi membrane / GTPase activity / GTP binding / perinuclear region of cytoplasm ...Synthesis of PIPs at the Golgi membrane / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / vesicle-mediated transport / intracellular protein transport / Golgi membrane / GTPase activity / GTP binding / perinuclear region of cytoplasm / extracellular exosome / plasma membrane
Similarity search - Function
ADP-ribosylation factor 1-5 / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / ARF-like small GTPases; ARF, ADP-ribosylation factor / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / ADP-ribosylation factor 3 / Toxin protein
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.04 Å
AuthorsBelyy, A. / Heilen, P. / Hofnagel, O. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2023
Title: Structure and activation mechanism of the Makes caterpillars floppy 1 toxin.
Authors: Alexander Belyy / Philipp Heilen / Philine Hagel / Oliver Hofnagel / Stefan Raunser /
Abstract: The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1 intoxication is poorly ...The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1 intoxication is poorly understood. Here, we present the cryo-EM structure of Mcf1 from Photorhabdus luminescens, revealing a seahorse-like shape with a head and tail. While the three head domains contain two effectors, as well as an activator-binding domain (ABD) and an autoprotease, the tail consists of two putative translocation and three putative receptor-binding domains. Rearrangement of the tail moves the C-terminus away from the ABD and allows binding of the host cell ADP-ribosylation factor 3, inducing conformational changes that position the cleavage site closer to the protease. This distinct activation mechanism that is based on a hook-loop interaction results in three autocleavage reactions and the release of two toxic effectors. Unexpectedly, the BH3-like domain containing ABD is not an active effector. Our findings allow us to understand key steps of Mcf1 intoxication at the molecular level.
History
DepositionMay 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toxin protein
B: ADP-ribosylation factor 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)346,6954
Polymers346,1472
Non-polymers5472
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Toxin protein /


Mass: 325211.562 Da / Num. of mol.: 1 / Mutation: C1397A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria) / Gene: mcf / Production host: Escherichia coli (E. coli) / References: UniProt: Q8KT65
#2: Protein ADP-ribosylation factor 3 / ADP ribosylation factor


Mass: 20935.895 Da / Num. of mol.: 1 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARF3 / Production host: Escherichia coli (E. coli) / References: UniProt: P61204
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Photorhabdus luminescens Makes caterpillars floppy (Mcf) toxin with the C-terminal deletion in complex with Arf3COMPLEX#1-#20RECOMBINANT
2Toxin proteinCOMPLEX#11RECOMBINANT
3ADP-ribosylation factor 3ADP ribosylation factorCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Photorhabdus luminescens (bacteria)29488
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 62.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18671

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Processing

EM software
IDNameCategory
4CTFFINDCTF correction
7ISOLDEmodel fitting
8iMODFITmodel fitting
10PHENIXmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101942 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
16II6

6ii6

C6II6C1PDBexperimental model
2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413967
ELECTRON MICROSCOPYf_angle_d0.87418916
ELECTRON MICROSCOPYf_dihedral_angle_d14.2515206
ELECTRON MICROSCOPYf_chiral_restr0.0482100
ELECTRON MICROSCOPYf_plane_restr0.012488

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