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- EMDB-32381: Trichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) -

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Basic information

Entry
Database: EMDB / ID: EMD-32381
TitleTrichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1)
Map dataMap without b-factor sharpening
Sample
  • Complex: TeCphA1
    • Protein or peptide: Cyanophycin synthase
KeywordsCyanophycin / Non-ribosomal peptide synthesis / ATP / Aspartate / Arginine / LIGASE
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 / Mur ligase, C-terminal domain superfamily / Mur ligase, glutamate ligase domain ...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, C-terminal domain superfamily / Mur ligase, glutamate ligase domain / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsKawasaki M / Miyakawa T
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-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32381.png

Downloads & links

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Map

FileDownload / File: emd_32381.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap without b-factor sharpening
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 200 pix.
= 176. Å		0.88 Å/pix.
x 200 pix.
= 176. Å		0.88 Å/pix.
x 200 pix.
= 176. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.033281293 - 0.09006895
Average (Standard dev.)-0.00014666127 (±0.006353744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 176.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32381_msk_1.map
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Additional map: Map with b-factor sharpening

Fileemd_32381_additional_1.map
AnnotationMap with b-factor sharpening
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Half map: #2

Fileemd_32381_half_map_1.map
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Half map: #1

Fileemd_32381_half_map_2.map
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Sample components

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Entire : TeCphA1

EntireName: TeCphA1
Components
  • Complex: TeCphA1
    • Protein or peptide: Cyanophycin synthase

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Supramolecule #1: TeCphA1

SupramoleculeName: TeCphA1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Homotetramer
Source (natural)Organism: Trichodesmium erythraeum IMS101 (bacteria)
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

UniProtKB: Cyanophycin synthetase

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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

Concentration1.19 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMtris(hydroxymethyl)aminomethane
400.0 mMsodium chloride
1.0 mMdithiothreitol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / 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 15 seconds (blot force 0)..
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2109 / Average exposure time: 55.75 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 556730
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 60 A for use as an initial model for 3D classification.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 161823
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 2 / Avg.num./class: 213286 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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