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Yorodumi- PDB-8pn7: Engineered glycolyl-CoA carboxylase (G20R variant) with bound CoA -
+Open data
-Basic information
Entry | Database: PDB / ID: 8pn7 | ||||||||||||
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Title | Engineered glycolyl-CoA carboxylase (G20R variant) with bound CoA | ||||||||||||
Components |
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Keywords | LIGASE / glycolyl-CoA carboxylase | ||||||||||||
Function / homology | Function and homology information propionyl-CoA carboxylase / propionyl-CoA carboxylase activity / lipid catabolic process / ATP binding / metal ion binding Similarity search - Function | ||||||||||||
Biological species | Methylorubrum extorquens AM1 (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.03 Å | ||||||||||||
Authors | Zarzycki, J. / Marchal, D.G. / Schulz, L. / Prinz, S. / Erb, T.J. | ||||||||||||
Funding support | European Union, Germany, 3items
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Citation | Journal: ACS Synth Biol / Year: 2023 Title: Machine Learning-Supported Enzyme Engineering toward Improved CO-Fixation of Glycolyl-CoA Carboxylase. Authors: Daniel G Marchal / Luca Schulz / Ingmar Schuster / Jelena Ivanovska / Nicole Paczia / Simone Prinz / Jan Zarzycki / Tobias J Erb / Abstract: Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve ...Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency compared to its template PCC and suffers from futile ATP hydrolysis during CO fixation. To further improve upon GCC M5, we developed a machine learning-supported workflow that reduces screening efforts for identifying improved enzymes. Using this workflow, we present two novel GCC variants with 2-fold increased carboxylation rate and 60% reduced energy demand, respectively, which are able to address kinetic and thermodynamic limitations of the TaCo pathway. Our work highlights the potential of combining machine learning and directed evolution strategies to reduce screening efforts in enzyme engineering. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8pn7.cif.gz | 867.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8pn7.ent.gz | 704.2 KB | Display | PDB format |
PDBx/mmJSON format | 8pn7.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pn/8pn7 ftp://data.pdbj.org/pub/pdb/validation_reports/pn/8pn7 | HTTPS FTP |
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-Related structure data
Related structure data | 17777MC 8pn8C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 56064.066 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Methylorubrum extorquens AM1 (bacteria) Gene: pccB, MexAM1_META1p0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C5AP75, propionyl-CoA carboxylase #2: Protein | Mass: 71986.961 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Methylorubrum extorquens AM1 (bacteria) Gene: pccA, MexAM1_META1p3203 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C5AWU5, propionyl-CoA carboxylase #3: Chemical | ChemComp-COA / #4: Chemical | ChemComp-BTI / #5: Water | ChemComp-HOH / | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: glycolyl-CoA carboxylase with bound CoA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Units: MEGADALTONS / Experimental value: NO |
Source (natural) | Organism: Methylorubrum extorquens AM1 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm |
Image recording | Electron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: D3 (2x3 fold dihedral) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 647870 / Symmetry type: POINT |