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- EMDB-23328: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP... -

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Basic information

Entry
Database: EMDB / ID: EMD-23328
TitleCyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-AsnCyanophycin synthase (L-aspartate-adding)
Map dataCyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-Asn mapCyanophycin synthase (L-aspartate-adding)
Sample
  • Complex: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-AsnCyanophycin synthase (L-aspartate-adding)
    • Protein or peptide: Cyanophycin synthase
    • Protein or peptide: 8x(Asp-Arg)-Asn
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
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) / macromolecule biosynthetic process / ATP binding / metal ion binding
Similarity search - Function
Cyanophycin synthase-like, N-terminal / Cyanophycin synthase-like N-terminal domain / Cyanophycin synthetase / ATP-grasp fold, RimK-type / RimK-like ATP-grasp domain / Mur ligase, C-terminal / Mur ligase family, glutamate ligase domain / Mur ligase, C-terminal domain superfamily / Mur ligase, central / Mur-like, catalytic domain superfamily ...Cyanophycin synthase-like, N-terminal / Cyanophycin synthase-like N-terminal domain / Cyanophycin synthetase / ATP-grasp fold, RimK-type / RimK-like ATP-grasp domain / Mur ligase, C-terminal / Mur ligase family, glutamate ligase domain / Mur ligase, C-terminal domain superfamily / Mur ligase, central / Mur-like, catalytic domain superfamily / Mur ligase middle domain / ATP-grasp fold / ATP-grasp fold profile.
Similarity search - Domain/homology
Cyanophycin synthetase
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6714 (bacteria) / Synechocystis sp. (strain PCC 6714) (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSharon I / Grogg M / Hilvert D / Schmeing TM
CitationJournal: Nat Chem Biol / Year: 2021
Title: Structures and function of the amino acid polymerase cyanophycin synthetase.
Authors: Itai Sharon / Asfarul S Haque / Marcel Grogg / Indrajit Lahiri / Dieter Seebach / Andres E Leschziner / Donald Hilvert / T Martin Schmeing /
Abstract: Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the β-carboxylate sidechains by isopeptide ...Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the β-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.
History
DepositionJan 20, 2021-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.65
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lgq
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23328.png

Downloads & links

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Map

FileDownload / File: emd_23328.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-Asn map
Voxel sizeX=Y=Z: 0.855 Å
Density
Contour LevelBy AUTHOR: 0.65 / Movie #1: 0.65
Minimum - Maximum-2.7651517 - 4.533293
Average (Standard dev.)0.006049364 (±0.12242548)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8550.8550.855
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z342.000342.000342.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-2.7654.5330.006

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Supplemental data

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Mask #1

Fileemd_23328_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with...

Fileemd_23328_half_map_1.map
AnnotationCyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-Asn half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with...

Fileemd_23328_half_map_2.map
AnnotationCyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-Asn half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP...

EntireName: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-AsnCyanophycin synthase (L-aspartate-adding)
Components
  • Complex: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-AsnCyanophycin synthase (L-aspartate-adding)
    • Protein or peptide: Cyanophycin synthase
    • Protein or peptide: 8x(Asp-Arg)-Asn
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP...

SupramoleculeName: Cyanophycin synthetase 1 from Synechocystis sp. UTEX2470 with ATP and 8x(Asp-Arg)-Asn
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria)

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Macromolecule #1: Cyanophycin synthase

MacromoleculeName: Cyanophycin synthase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: cyanophycin synthase (L-aspartate-adding)
Source (natural)Organism: Synechocystis sp. (strain PCC 6714) (bacteria) / Strain: PCC 6714
Molecular weightTheoretical: 95.758836 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKILKTLTLR GPNYWSIRRK KLIVMRLDLE DLAERPSNSI PGFYEGLIKV LPSLVEHFCS PGYQGGFLER VKEGTYMGHI VEHVALELQ ELVGMTAGFG RTRETSTPGV YNVVYEYVDE QAGRYAGRAA VRLCRSLVDT GDYPRLELEK DLEDLRDLGA N SALGPSTE ...String:
MKILKTLTLR GPNYWSIRRK KLIVMRLDLE DLAERPSNSI PGFYEGLIKV LPSLVEHFCS PGYQGGFLER VKEGTYMGHI VEHVALELQ ELVGMTAGFG RTRETSTPGV YNVVYEYVDE QAGRYAGRAA VRLCRSLVDT GDYPRLELEK DLEDLRDLGA N SALGPSTE TIVTEAEARK IPWMLLSARA MVQLGYGVYQ QRIQATLSSH SGILGVELAC DKEGTKTILQ DAGIPVPRGT TI QYFDDLE EAINDVGGYP VVIKPLDGNH GRGITINVRH WQEAIAAYDL AAEESKSRAI IVERYYEGSD HRVLVVNGKL VAV AERIPA HVTGDGSSTI SELIEKTNQD PNRGDGHDNI LTKIVVNKTA IDVMERQGYN LDSVLPKDEV VYLRATANLS TGGI AIDRT DDIHPENIWL MERVAKVIGL DIAGIDVVTS DISKPLRETN GVIVEVNAAP GFRMHVAPSQ GLPRNVAAPV LDMLF PPGT PSRIPILAVT GTNGKTTTTR LLAHIYRQTG KTVGYTSTDA IYINEYCVEK GDNTGPQSAG VILRDPTVEV AVLETA RGG ILRAGLAFDS CDVGVVLNVA ADHLGLGDID TIEQMAKVKS VIAEVVDPSG YAVLNADDPL VAAMADKVKA KVAYFSM NP DNPIIQAHVR RNGIAAVYES GYLSILEGSW TLRVEQAKLI PMTMGGMAPF MIANALAACL AAFVNGLDVE VIRQGVRT F TTSAEQTPGR MNLFNLGQHH ALVDYAHNPA GYRAVGDFVK NWQGQRFGVV GGPGDRRDSD LIELGQIAAQ VFDRIIVKE DDDKRGRSEG ETADLIVKGI LQENPGASYE VILDETIALN KALDQVEEKG LVVVFPESVT RAIDLIKVRN PIGENLYFQ

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Macromolecule #2: 8x(Asp-Arg)-Asn

MacromoleculeName: 8x(Asp-Arg)-Asn / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.3023 KDa
SequenceString:
(7ID)(7ID)(7ID)(7ID)(7ID)(7ID)(7ID)(7ID)N

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration3 mg/mL
BufferpH: 8
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 135110
FSC plot (resolution estimation)

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