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Yorodumi- EMDB-29534: Cryo-EM structure of Stanieria sp. CphA2 in complex with ADPCP an... -
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-Basic information
Entry | Database: EMDB / ID: EMD-29534 | |||||||||
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Title | Cryo-EM structure of Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg) | |||||||||
Map data | Locally filtered map of Stanieria sp. CphA2 with ADPCP and 4x(Asp-Arg) | |||||||||
Sample |
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Keywords | cyanophycin / CphA2 / ligase / ATP-grasp | |||||||||
Function / homology | Carbamoyl-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 species | Stanieria sp. (bacteria) / Stanieria sp. NIES-3757 / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.7 Å | |||||||||
Authors | Markus LM / Sharon I / Strauss M / Schmeing TM | |||||||||
Funding support | Canada, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29534.map.gz | 22.2 MB | EMDB map data format | |
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Header (meta data) | emd-29534-v30.xml emd-29534.xml | 16 KB 16 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_29534_fsc.xml | 16.9 KB | Display | FSC data file |
Images | emd_29534.png | 64.8 KB | ||
Masks | emd_29534_msk_1.map | 512 MB | Mask map | |
Filedesc metadata | emd-29534.cif.gz | 6.1 KB | ||
Others | emd_29534_half_map_1.map.gz emd_29534_half_map_2.map.gz | 474.5 MB 474.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29534 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29534 | HTTPS FTP |
-Related structure data
Related structure data | 8fxiMC 8fxhC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29534.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Locally filtered map of Stanieria sp. CphA2 with ADPCP and 4x(Asp-Arg) | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.675 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_29534_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Half map A of Stanieria sp. CphA2 with ADPCP and 4x(Asp-Arg)
File | emd_29534_half_map_1.map | ||||||||||||
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Annotation | Half map A of Stanieria sp. CphA2 with ADPCP and 4x(Asp-Arg) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map B of Stanieria sp. CphA2 with ADPCP and 4x(Asp-Arg)
File | emd_29534_half_map_2.map | ||||||||||||
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Annotation | Half map B of Stanieria sp. CphA2 with ADPCP and 4x(Asp-Arg) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg)
Entire | Name: Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg) |
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Components |
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-Supramolecule #1: Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg)
Supramolecule | Name: Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Stanieria sp. (bacteria) |
-Macromolecule #1: RimK domain-containing protein ATP-grasp
Macromolecule | Name: RimK domain-containing protein ATP-grasp / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Stanieria sp. NIES-3757 |
Molecular weight | Theoretical: 72.886516 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MLTKQAVEPV RINARTTDVF DIFNVKQYVG ANPYLNQAAL VFDFAFTESY QPLPIENYLA VVGDRYPRLK EIEYQSYAEL FASTVAEVN KLEMDLHLKG WNVKPIEEIN RIAIESLHHR TTKEVVYCVW DWFEFITQGE EFDLSKQIAI LQQLFRNSVY G GPTVYALL ...String: MLTKQAVEPV RINARTTDVF DIFNVKQYVG ANPYLNQAAL VFDFAFTESY QPLPIENYLA VVGDRYPRLK EIEYQSYAEL FASTVAEVN KLEMDLHLKG WNVKPIEEIN RIAIESLHHR TTKEVVYCVW DWFEFITQGE EFDLSKQIAI LQQLFRNSVY G GPTVYALL RTANEKHIPA FYLWDEGLMQ YGYGKQQVRG IATTFDVDSH IDSDFTTQKD DCKKFLQELG FPVPQGDVVF SL AEAKEVA AEIGYPVAVK PVAGHKGIGV TADVQDEIEL EAAYDRAVAG IPLEEKICII VENSIAGHDY RLLCVNGRFV AAT ERKPAY VVGDGYSTIA ELIEKENFSP NRSDTPTSPM GKIRTDEAMH LYLEEQGLDL DSVIDRDRTI YLRKVANLSS GGFS IDATN RVHPDNIILA QDIAQHFRLT CLGIDIITND IGRSWKETSF GIIEINAAPG VYMHLKPAIG EPVDVTARIL ETFFE TEKN ARIPIITFNR VSIRQLQKLS DRILMSHPDW TIGAVCREGI LINRSEKILN RHYNTNVLNL LRNPKLDLLI AEYDED ALE AEGMFYHGSN LVVLEDPSEI EMILTRDVFS DSTVIIKQGR EITIKRKGLL EQYELEAEEL IEQVYLKEIG TISENLY FQ UniProtKB: RimK domain-containing protein ATP-grasp |
-Macromolecule #2: 4x(beta-Asp-Arg)
Macromolecule | Name: 4x(beta-Asp-Arg) / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 1.103106 KDa |
Sequence | String: (7ID)(7ID)(7ID)(7ID) |
-Macromolecule #3: 1-[(4-aminopyrimidin-5-yl)amino]-2,5-anhydro-1-deoxy-6-O-[(S)-hyd...
Macromolecule | Name: 1-[(4-aminopyrimidin-5-yl)amino]-2,5-anhydro-1-deoxy-6-O-[(S)-hydroxy{[(R)-hydroxy(phosphonomethyl)phosphoryl]oxy}phosphoryl]-D-allitol type: ligand / ID: 3 / Number of copies: 1 / Formula: YHZ |
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Molecular weight | Theoretical: 494.225 Da |
Chemical component information | ChemComp-YHZ: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #5: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
Macromolecule | Name: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: ACP |
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Molecular weight | Theoretical: 505.208 Da |
Chemical component information | ChemComp-ACP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.0025 µm / Nominal defocus min: -0.001 µm |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 80.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |