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Yorodumi- EMDB-32382: Trichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) with ... -
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-Basic information
Entry | Database: EMDB / ID: EMD-32382 | |||||||||
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Title | Trichodesmium erythraeum cyanophycin synthetase 1 (TeCphA1) with ATPgammaS | |||||||||
Map data | Map without b-factor sharpening | |||||||||
Sample |
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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 | |||||||||
Biological species | Trichodesmium erythraeum IMS101 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.96 Å | |||||||||
Authors | Kawasaki M / Miyakawa T / Yang J / Adachi N / Fujii A / Miyauchi Y / Muramatsu T / Moriya T / Senda T / Tanokura M | |||||||||
Funding support | Japan, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_32382.map.gz | 28.2 MB | EMDB map data format | |
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Header (meta data) | emd-32382-v30.xml emd-32382.xml | 23.6 KB 23.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_32382_fsc.xml | 14.2 KB | Display | FSC data file |
Images | emd_32382.png | 183.3 KB | ||
Masks | emd_32382_msk_1.map | 244.1 MB | Mask map | |
Others | emd_32382_additional_1.map.gz emd_32382_half_map_1.map.gz emd_32382_half_map_2.map.gz | 28.4 MB 27.2 MB 27.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-32382 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-32382 | HTTPS FTP |
-Related structure data
Related structure data | 7wadMC 7wacC 7waeC 7wafC 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_32382.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Map without b-factor sharpening | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.88 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_32382_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Map with b-factor sharpening
File | emd_32382_additional_1.map | ||||||||||||
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Annotation | Map with b-factor sharpening | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_32382_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_32382_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : TeCphA1 with ATPgammaS
Entire | Name: TeCphA1 with ATPgammaS |
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Components |
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-Supramolecule #1: TeCphA1 with ATPgammaS
Supramolecule | Name: TeCphA1 with ATPgammaS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Homotetramer |
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Source (natural) | Organism: Trichodesmium erythraeum IMS101 (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant cell: BL21(DE3) / Recombinant plasmid: pET-22b(+) |
Molecular weight | Theoretical: 400 KDa |
-Macromolecule #1: Cyanophycin synthase
Macromolecule | Name: Cyanophycin synthase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: cyanophycin synthase (L-aspartate-adding) |
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Source (natural) | Organism: Trichodesmium erythraeum IMS101 (bacteria) / Strain: IMS101 |
Molecular weight | Theoretical: 99.079914 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 8 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ChemComp-AGS: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.20 mg/mL | ||||||||||||
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Buffer | pH: 8.2 Component:
Details: The buffer contains 4 mM ATPgammaS, 5 mM aspartate, 0.5 mM arginine and 10 mM 4-mer cyanophycin peptide as ligands. | ||||||||||||
Grid | Model: 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. | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 15 seconds (blot force 10).. | ||||||||||||
Details | This sample was mono-disperse. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1730 / Average exposure time: 56.18 sec. / Average electron dose: 50.0 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |