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- PDB-9bhw: Septin Tetrameric Complex SEPT7/SEPT9 of Ciona intestinalis by Cryo-EM -

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Basic information

Entry
Database: PDB / ID: 9bhw
TitleSeptin Tetrameric Complex SEPT7/SEPT9 of Ciona intestinalis by Cryo-EM
Components
  • CiSeptin-7
  • Neuronal-specific septin-3
KeywordsCELL CYCLE / Complex / SPA / Cytoskeleton
Function / homology
Function and homology information


septin complex / cytoskeleton-dependent cytokinesis / septin ring / cell division site / cleavage furrow / cilium assembly / kinetochore / cilium / microtubule cytoskeleton / molecular adaptor activity ...septin complex / cytoskeleton-dependent cytokinesis / septin ring / cell division site / cleavage furrow / cilium assembly / kinetochore / cilium / microtubule cytoskeleton / molecular adaptor activity / GTPase activity / GTP binding / cytosol
Similarity search - Function
Septin 7 / Septin-type guanine nucleotide-binding (G) domain / Septin / Septin-type guanine nucleotide-binding (G) domain profile. / Septin / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / Neuronal-specific septin-3 / Septin
Similarity search - Component
Biological speciesCiona intestinalis (vase tunicate)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsMendonca, D.C. / Pereira, H.M. / Garratt, R.C.
Funding support Brazil, 3items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)2018/20209-5 Brazil
Sao Paulo Research Foundation (FAPESP)2020/02897-1 Brazil
Sao Paulo Research Foundation (FAPESP)2023/06866-1 Brazil
CitationJournal: J Mol Biol / Year: 2024
Title: Structural Insights into Ciona intestinalis Septins: Complexes Suggest a Mechanism for Nucleotide-dependent Interfacial Cross-talk.
Authors: Deborah C Mendonça / Sinara T B Morais / Heloísa Ciol / Andressa P A Pinto / Diego A Leonardo / Humberto D'Muniz Pereira / Napoleão F Valadares / Rodrigo V Portugal / Bruno P Klaholz / ...Authors: Deborah C Mendonça / Sinara T B Morais / Heloísa Ciol / Andressa P A Pinto / Diego A Leonardo / Humberto D'Muniz Pereira / Napoleão F Valadares / Rodrigo V Portugal / Bruno P Klaholz / Richard C Garratt / Ana P U Araujo /
Abstract: Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a ...Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7 Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.
History
DepositionApr 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _em_admin.last_update
Revision 1.2Jul 24, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.title ..._citation.journal_volume / _citation.title / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CiSeptin-7
B: Neuronal-specific septin-3
C: Neuronal-specific septin-3
D: CiSeptin-7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,4308
Polymers180,6574
Non-polymers1,7734
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CiSeptin-7


Mass: 48769.039 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ciona intestinalis (vase tunicate) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: H2Y169
#2: Protein Neuronal-specific septin-3 / CiSeptin-9


Mass: 41559.363 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ciona intestinalis (vase tunicate) / Gene: LOC100180404 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: F7AQE7
#3: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
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

ComponentName: Ciona intestinalis Septin Tetrameric Complex SEPT7/SEPT9
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Ciona intestinalis (vase tunicate)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 41.8 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 529597 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058936
ELECTRON MICROSCOPYf_angle_d0.6212087
ELECTRON MICROSCOPYf_dihedral_angle_d8.2251204
ELECTRON MICROSCOPYf_chiral_restr0.0461376
ELECTRON MICROSCOPYf_plane_restr0.0051531

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