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- EMDB-29533: Cryo-EM structure of Stanieria sp. CphA2 -

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Basic information

Entry
Database: EMDB / ID: EMD-29533
TitleCryo-EM structure of Stanieria sp. CphA2
Map dataStanieria sp. CphA2 locally filtered map
Sample
  • Complex: Stanieria sp. CphA2
    • Protein or peptide: RimK domain-containing protein ATP-grasp
Keywordscyanophycin / CphA2 / ligase / 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. (bacteria) / Stanieria sp. NIES-3757 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMarkus LM / Sharon I / Strauss M / Schmeing TM
Funding support Canada, 1 items
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, 2023-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateNov 8, 2023-
Current statusNov 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29533.png

Downloads & links

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Map

FileDownload / File: emd_29533.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStanieria sp. CphA2 locally filtered map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 512 pix.
= 345.6 Å		0.68 Å/pix.
x 512 pix.
= 345.6 Å		0.68 Å/pix.
x 512 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.675 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.08
Minimum - Maximum-1.4136705 - 1.9178178
Average (Standard dev.)0.0018339091 (±0.033729862)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29533_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Stanieria sp. CphA2 half map A

Fileemd_29533_half_map_1.map
AnnotationStanieria sp. CphA2 half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Stanieria sp. CphA2 half map B

Fileemd_29533_half_map_2.map
AnnotationStanieria sp. CphA2 half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Stanieria sp. CphA2

EntireName: Stanieria sp. CphA2
Components
  • Complex: Stanieria sp. CphA2
    • Protein or peptide: RimK domain-containing protein ATP-grasp

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Supramolecule #1: Stanieria sp. CphA2

SupramoleculeName: Stanieria sp. CphA2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Stanieria sp. (bacteria)

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

MacromoleculeName: RimK domain-containing protein ATP-grasp / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Stanieria sp. NIES-3757 (bacteria)
Molecular weightTheoretical: 72.886516 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: -0.0025 µm / Nominal defocus min: -0.001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 251001
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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