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- EMDB-21021: MicroED structure of an oxindole product from modified trpB at 0.... -

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Basic information

Entry
Database: EMDB / ID: EMD-21021
TitleMicroED structure of an oxindole product from modified trpB at 0.9A resolution
Map dataMicroED 2Fo-Fc map for an oxindole product from modified trpB at 0.9A resolution
Sample
  • Complex: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
Biological speciesEscherichia coli (E. coli)
Methodelectron crystallography / cryo EM / Resolution: 0.9 Å
AuthorsDick M / Sarai NS / Martynowycz MW / Gonen T / Arnold FH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125887 United States
CitationJournal: 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
DepositionNov 21, 2019-
Header (metadata) releaseDec 11, 2019-
Map releaseDec 11, 2019-
UpdateJan 1, 2020-
Current statusJan 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.454545
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.454545
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21021.map.gz / Format: CCP4 / Size: 4.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicroED 2Fo-Fc map for an oxindole product from modified trpB at 0.9A resolution
Voxel sizeX: 0.28929 Å / Y: 0.28063 Å / Z: 0.28556 Å
Density
Contour LevelBy AUTHOR: 0.454545 / Movie #1: 0.454545
Minimum - Maximum-0.7543616 - 1.9895042
Average (Standard dev.)-0.00000640520 (±0.30302677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-42-38-29
Dimensions107109109
Spacing107109109
CellA: 30.953571 Å / B: 30.588123 Å / C: 31.125553 Å
α: 90.0 ° / β: 99.411 ° / γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.289289719626170.280623853211010.28555963302752
M x/y/z107109109
origin x/y/z0.0000.0000.000
length x/y/z30.95430.58831.126
α/β/γ90.00099.41190.000
start NX/NY/NZ-42-38-29
NX/NY/NZ107109109
MAP C/R/S213
start NC/NR/NS-38-42-29
NC/NR/NS109107109
D min/max/mean-0.7541.990-0.000

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Supplemental data

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Sample components

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Entire : (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid

EntireName: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
Components
  • Complex: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid

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Supramolecule #1: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid

SupramoleculeName: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
type: complex / ID: 1 / Parent: 0 / Details: Oxindole product from TrpB
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 215 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

Concentration1 mg/mL
BufferpH: 7
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1053 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: 0, 60
Image recordingFilm 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.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit 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 statisticsNumber 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
Final reconstructionResolution.type: BY AUTHOR / Resolution: 0.9 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 6 / Target criteria: Least Squares

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