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- EMDB-32383: Trichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-32383
TitleTrichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) with ATPgammaS, 4x(beta-Asp-Arg), and aspartate
Map dataMap without b-factor sharpening
Sample
  • Complex: TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate
    • Protein or peptide: Cyanophycin synthase
  • Protein or peptide: 4x(beta-Asp-Arg)
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ASPARTIC ACID
  • Ligand: 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 / macromolecule biosynthetic process / 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 / Mur ligase family, glutamate ligase domain / Mur ligase, C-terminal domain superfamily ...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 / 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 speciesTrichodesmium erythraeum IMS101 (bacteria) / Synthetic construct (others)
Methodsingle 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, 2 items
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, 2021-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateSep 14, 2022-
Current statusSep 14, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32383.png

Downloads & links

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Map

FileDownload / File: emd_32383.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap without b-factor sharpening
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.024024602 - 0.061544675
Average (Standard dev.)9.502058e-06 (±0.0014436833)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 345.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32383_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Map with b-factor sharpening

Fileemd_32383_additional_1.map
AnnotationMap with b-factor sharpening
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32383_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32383_half_map_2.map
Projections & Slices
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Sample components

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Entire : TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate

EntireName: TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate
Components
  • Complex: TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate
    • Protein or peptide: Cyanophycin synthase
  • Protein or peptide: 4x(beta-Asp-Arg)
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ASPARTIC ACID
  • Ligand: ARGININE

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Supramolecule #1: TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate

SupramoleculeName: TeCphA1 with ATPgammaS, 4x(beta-Asp-Arg) and aspartate
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Homotetramer
Source (natural)Organism: Trichodesmium erythraeum IMS101 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant cell: BL21(DE3) / Recombinant plasmid: pET-22b(+)
Molecular weightTheoretical: 400 KDa

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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: Trichodesmium erythraeum IMS101 (bacteria) / Strain: IMS101
Molecular weightTheoretical: 99.079914 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKILKLQTLR GPNYWSIHRH KLVVMRLDLE DLYEKYTSDI PGFYKGLTEV LPSLVEHLCS PGVKGGFLTR VEKGTLIGHV IEHVAIELQ ELAGMPVGFG RTRETSTTGV FQVVIEYENE QAGRYAARAA VRLCQSIVDT GTYPATELQQ DLEDLKELKN Q ASLGPSTE ...String:
MKILKLQTLR GPNYWSIHRH KLVVMRLDLE DLYEKYTSDI PGFYKGLTEV LPSLVEHLCS PGVKGGFLTR VEKGTLIGHV IEHVAIELQ ELAGMPVGFG RTRETSTTGV FQVVIEYENE QAGRYAARAA VRLCQSIVDT GTYPATELQQ DLEDLKELKN Q ASLGPSTE AIVKEAEARG IPWTQLGARF MIQFGYGVNQ KKIQATLSNQ TGILGVELAC DKEGTKRILK DAGVPVPRGT VA RYFDELQ DAIEYVGGYP IVIKPLDGNH GRGITIDVKN WQEAEEAYDL ARKASKTKTV IVERYYTGKD HRVLVVNGKV VAV AERVPA HVVGNGKSTI AELIEETNRD PQRGDGHDNI LTRITVDKSA LDILGKQGYS IDSIPLKGKK CFLRATANLS TGGI AVDRT DEIHPENVWL LSRVAKIIGL DIAGIDVVTE DISQPLREVE GVIVEVNAAP GFRMHVAPSR GLARNVAGAV MDMLF PGSK NGRIPILSVT GTNGKTTTTR LLAHIIKQTG KVVGYTTTDG TYIGEYLAET GDNTGPQSAH LILSDPTVEV AVLETA RGG ILRSGLGFSS CEVGIVLNVT ADHLGIGDID TIEQLAKLKS VVAESVMPKG YAVLNAEDPL VAAMADRVKG QVAYFSM DP NNELLLRHTE AGGLAAIYEN GYISILKGDW TLRIEKAVNV PITMAGKAPF MIANALAACL AVFTQGVKIE HIRKGLST F VASVDQTPGR MNMFNMGSYH ALVDYAHNPA SYEALGGFVR NWPGKRIGVV GGPGDRRDED FVSLGELAAD IFDEIIIKE DDDTRGRPRG NAAELICQGV KQFLNGIKNS ESKATYESIL DETAAINTAL DRAPIDGLVV ILPESVNRAI SLIEGRHVIQ DIELLQDSQ REPKDQEVLK SSILEHHHHH H

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Macromolecule #2: 4x(beta-Asp-Arg)

MacromoleculeName: 4x(beta-Asp-Arg) / type: protein_or_peptide / ID: 2
Details: Backbone peptide bonds are formed between Asp residues. An Arg residue forms a peptide bond with the beta-carboxy group of Asp.
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Synthetic construct (others)
Molecular weightTheoretical: 478.366 Da
SequenceString:
DDDD

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 11 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 8 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #5: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 5 / Number of copies: 1 / Formula: ASP
Molecular weightTheoretical: 133.103 Da
Chemical component information

ChemComp-ASP:
ASPARTIC ACID / Aspartic acid

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Macromolecule #6: ARGININE

MacromoleculeName: ARGININE / type: ligand / ID: 6 / Number of copies: 8 / Formula: ARG
Molecular weightTheoretical: 175.209 Da
Chemical component information

ChemComp-ARG:
ARGININE / Arginine

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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

Concentration0.40 mg/mL
BufferpH: 8.2
Component:
ConcentrationName
50.0 mMtris(hydroxymethyl)aminomethane
67.0 mMsodium chloride
20.0 mMpotassium chloride
20.0 mMmagnesium chloride
1.0 mMdithiothreitol

Details: The buffer contains 10 mM ATPgammaS, 100 mM aspartate and 20 mM 4-mer cyanophycin peptide as ligands.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 10 seconds (blot force 10)..
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.84 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3843 / Average exposure time: 4.63 sec. / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1371057
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4.1)Initial estimation
RELION (ver. 3.1)CTF refinement
Startup modelType of model: OTHER
Details: An ab initio model was generated using RELION3's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 50 A for use as an initial model for 3D classification.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 71875 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 198918
FSC plot (resolution estimation)

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