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- PDB-7wae: Trichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) with ... -

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Basic information

Entry
Database: PDB / ID: 7wae
TitleTrichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) with ATPgammaS, 4x(beta-Asp-Arg), and aspartate
Components
  • 4x(beta-Asp-Arg)
  • Cyanophycin synthase
KeywordsLIGASE / Cyanophycin / Non-ribosomal peptide synthesis / ATP / Aspartate / Arginine
Function / homology
Function and homology information


cyanophycin synthase (L-aspartate-adding) / cyanophycin synthase (L-arginine-adding) / cyanophycin synthetase activity (L-aspartate-adding) / cyanophycin synthetase activity (L-arginine-adding) / tetrahydrofolylpolyglutamate synthase activity / ATP binding / metal ion binding
Similarity search - Function
Cyanophycin synthase-like, N-terminal / Cyanophycin synthase-like N-terminal domain / Folylpolyglutamate synthase signature 1. / Cyanophycin synthetase / Folylpolyglutamate synthetase, conserved site / ATP-grasp fold, RimK-type / RimK-like ATP-grasp domain / Mur ligase, C-terminal domain / Mur-like, catalytic domain / Mur ligase, C-terminal ...Cyanophycin synthase-like, N-terminal / Cyanophycin synthase-like N-terminal domain / Folylpolyglutamate synthase signature 1. / Cyanophycin synthetase / Folylpolyglutamate synthetase, conserved site / ATP-grasp fold, RimK-type / RimK-like ATP-grasp domain / Mur ligase, C-terminal domain / Mur-like, catalytic domain / Mur ligase, C-terminal / Mur ligase, C-terminal domain superfamily / Mur ligase, glutamate ligase domain / Mur ligase, central / Mur-like, catalytic domain superfamily / Mur ligase middle domain / UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase / ATP-grasp fold / ATP-grasp fold profile. / Protein-Tyrosine Phosphatase; Chain A / Alpha-Beta Complex / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ARGININE / ASPARTIC ACID / Cyanophycin synthetase
Similarity search - Component
Biological speciesTrichodesmium erythraeum IMS101 (bacteria)
Synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsMiyakawa, T. / Yang, J. / Kawasaki, M. / Adachi, N. / Fujii, A. / Miyauchi, Y. / Muramatsu, T. / Moriya, T. / Senda, T. / Tanokura, M.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21am0101077 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase.
Authors: Takuya Miyakawa / Jian Yang / Masato Kawasaki / Naruhiko Adachi / Ayumu Fujii / Yumiko Miyauchi / Tomonari Muramatsu / Toshio Moriya / Toshiya Senda / Masaru Tanokura /
Abstract: Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin ...Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.
History
DepositionDec 14, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cyanophycin synthase
B: Cyanophycin synthase
C: Cyanophycin synthase
D: Cyanophycin synthase
J: 4x(beta-Asp-Arg)
K: 4x(beta-Asp-Arg)
L: 4x(beta-Asp-Arg)
M: 4x(beta-Asp-Arg)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)404,22136
Polymers398,2338
Non-polymers5,98828
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein / Protein/peptide , 2 types, 8 molecules ABCDJKLM

#1: Protein
Cyanophycin synthase / Cyanophycin synthetase


Mass: 99079.914 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trichodesmium erythraeum IMS101 (bacteria)
Strain: IMS101 / Gene: Tery_1965 / Production host: Escherichia coli (E. coli)
References: UniProt: Q113V7, cyanophycin synthase (L-aspartate-adding), cyanophycin synthase (L-arginine-adding)
#2: Protein/peptide
4x(beta-Asp-Arg)


Mass: 478.366 Da / Num. of mol.: 4 / Source method: obtained synthetically
Details: Backbone peptide bonds are formed between Asp residues. An Arg residue forms a peptide bond with the beta-carboxy group of Asp.
Source: (synth.) Synthetic construct (others)

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Non-polymers , 4 types, 28 molecules

#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-ARG / ARGININE


Type: L-peptide linking / Mass: 175.209 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C6H15N4O2

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Details

Has ligand of interestY
Has protein modificationY

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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: TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate
Type: COMPLEX / Details: Homotetramer / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.40 MDa / Experimental value: NO
Source (natural)Organism: Trichodesmium erythraeum IMS101 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Cell: BL21(DE3) / Plasmid: pET-22b(+)
Buffer solutionpH: 8.2
Details: The buffer contains 10 mM ATPgammaS, 100 mM aspartate and 20 mM 4-mer cyanophycin peptide as ligands.
Buffer component
IDConc.NameBuffer-ID
150 mMtris(hydroxymethyl)aminomethane1
267 mMsodium chloride1
320 mMpotassium chloride1
420 mMmagnesium chloride1
51 mMdithiothreitol1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: The grid was washed by acetone prior to use. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 10 seconds (blot force 10).

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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 magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 840 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.63 sec. / Electron dose: 49 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3843

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4CTFFIND4.1CTF correctionInitial estimation
5RELION3.1CTF correctionCTF refinement
8UCSF Chimera1.13.1model fitting
9Coot0.8.9.2model fitting
11Coot0.8.9.2model refinement
12PHENIX1.19.2model refinement
13RELION3.1initial Euler assignment
14RELION3.1final Euler assignment
15RELION3.1classification
16RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1371057
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198918 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00425316
ELECTRON MICROSCOPYf_angle_d0.69134370
ELECTRON MICROSCOPYf_dihedral_angle_d6.6523698
ELECTRON MICROSCOPYf_chiral_restr0.0514002
ELECTRON MICROSCOPYf_plane_restr0.0044420

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