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- PDB-8fxh: Cryo-EM structure of Stanieria sp. CphA2 -

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Basic information

Entry
Database: PDB / ID: 8fxh
TitleCryo-EM structure of Stanieria sp. CphA2
ComponentsRimK domain-containing protein ATP-grasp
KeywordsLIGASE / cyanophycin / CphA2 / ATP-grasp
Function / homologyCarbamoyl-phosphate synthetase large subunit-like, ATP-binding domain / Carbamoyl-phosphate synthase L chain, ATP binding domain / ATP-grasp fold, subdomain 1 / ATP-grasp fold / ATP-grasp fold profile. / catalytic activity / ATP binding / metal ion binding / RimK domain-containing protein ATP-grasp
Function and homology information
Biological speciesStanieria sp. NIES-3757 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMarkus, L.M. / Sharon, I. / Strauss, M. / Schmeing, T.M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)178084 Canada
CitationJournal: Protein Sci / Year: 2023
Title: Structure and function of a hexameric cyanophycin synthetase 2.
Authors: Linda M D Markus / Itai Sharon / Kim Munro / Marcel Grogg / Donald Hilvert / Mike Strauss / T Martin Schmeing /
Abstract: Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store ...Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide β-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.
History
DepositionJan 24, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation_author.identifier_ORCID
Revision 1.3Nov 8, 2023Group: Data collection / Source and taxonomy / Category: chem_comp_atom / chem_comp_bond / entity_src_gen
Item: _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RimK domain-containing protein ATP-grasp
B: RimK domain-containing protein ATP-grasp
C: RimK domain-containing protein ATP-grasp
D: RimK domain-containing protein ATP-grasp
E: RimK domain-containing protein ATP-grasp
F: RimK domain-containing protein ATP-grasp


Theoretical massNumber of molelcules
Total (without water)437,3196
Polymers437,3196
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RimK domain-containing protein ATP-grasp


Mass: 72886.516 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Stanieria sp. NIES-3757 (bacteria) / Gene: STA3757_02480 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140K0M0

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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: Stanieria sp. CphA2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Stanieria sp. (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2.5 nm / Nominal defocus min: -1 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251001 / Symmetry type: POINT

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