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Yorodumi- EMDB-21021: MicroED structure of an oxindole product from modified trpB at 0.... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-21021 | |||||||||
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Title | MicroED structure of an oxindole product from modified trpB at 0.9A resolution | |||||||||
Map data | MicroED 2Fo-Fc map for an oxindole product from modified trpB at 0.9A resolution | |||||||||
Sample |
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Biological species | Escherichia coli (E. coli) | |||||||||
Method | electron crystallography / cryo EM / Resolution: 0.9 Å | |||||||||
Authors | Dick M / Sarai NS / Martynowycz MW / Gonen T / Arnold FH | |||||||||
Funding support | United States, 1 items
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Citation | Journal: J Am Chem Soc / Year: 2019 Title: Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation. Authors: Markus Dick / Nicholas S Sarai / Michael W Martynowycz / Tamir Gonen / Frances H Arnold / Abstract: We previously engineered the β-subunit of tryptophan synthase (TrpB), which catalyzes the condensation of l-serine and indole to l-tryptophan, to synthesize a range of noncanonical amino acids from ...We previously engineered the β-subunit of tryptophan synthase (TrpB), which catalyzes the condensation of l-serine and indole to l-tryptophan, to synthesize a range of noncanonical amino acids from l-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds on N of the substrate. Protecting N encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereocenter at the γ-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the γ-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L culture, TrpB offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_21021.map.gz | 558 KB | EMDB map data format | |
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Header (meta data) | emd-21021-v30.xml emd-21021.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
Images | emd_21021.png | 43.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-21021 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-21021 | HTTPS FTP |
-Validation report
Summary document | emd_21021_validation.pdf.gz | 79.7 KB | Display | EMDB validaton report |
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Full document | emd_21021_full_validation.pdf.gz | 78.8 KB | Display | |
Data in XML | emd_21021_validation.xml.gz | 492 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-21021 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-21021 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_21021.map.gz / Format: CCP4 / Size: 4.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | MicroED 2Fo-Fc map for an oxindole product from modified trpB at 0.9A resolution | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X: 0.28929 Å / Y: 0.28063 Å / Z: 0.28556 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
Entire | Name: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid |
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Components |
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-Supramolecule #1: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
Supramolecule | Name: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid type: complex / ID: 1 / Parent: 0 / Details: Oxindole product from TrpB |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Experimental: 215 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron crystallography |
Aggregation state | 3D array |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7 |
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: NITROGEN |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 28.0 µm / Number grids imaged: 2 / Number real images: 120 / Number diffraction images: 120 / Average exposure time: 1.0 sec. / Average electron dose: 0.01 e/Å2 Details: Frames were recorded every 0.5 degrees for ~60 degrees with a 1 second integration time. |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1053 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: 0, 60 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 0.9 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES |
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Crystal parameters | Unit cell - A: 12.15 Å / Unit cell - B: 8.98 Å / Unit cell - C: 10.28 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 99.41 ° / Space group: 14 |
Crystallography statistics | Number intensities measured: 2524 / Number structure factors: 1028 / Fourier space coverage: 65.1 / R sym: 12.8 / R merge: 10.1 / Overall phase error: 20 / Overall phase residual: 20 / Phase error rejection criteria: 0 / High resolution: 0.9 Å / Shell - Shell ID: 1 / Shell - High resolution: 10.0 Å / Shell - Low resolution: 0.9 Å / Shell - Number structure factors: 1024 / Shell - Phase residual: 20 / Shell - Fourier space coverage: 65.1 / Shell - Multiplicity: 2.4 |
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 6 / Target criteria: Least Squares |
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