Japan Agency for Medical Research and Development (AMED)
JP21am0101077
Japan
Japan Agency for Medical Research and Development (AMED)
JP21am0101071
Japan
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.
Model: 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.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 15 seconds (blot force 0)..
Details
This sample was mono-disperse.
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Image recording
Film 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 beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Model: Talos Arctica / Image courtesy: FEI Company
+
Image processing
Particle selection
Number selected: 556730
Startup model
Type 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 reconstruction
Number 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 assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
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