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- EMDB-26768: MicroED structure of Aeropyrum pernix protoglobin mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-26768
TitleMicroED structure of Aeropyrum pernix protoglobin mutant
Map data
Sample
  • Complex: Homotetramer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
    • Protein or peptide: Protogloblin ApPgb
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: IMIDAZOLE
  • Ligand: water
KeywordsMicroED / protoglobin / heme / METAL BINDING PROTEIN
Function / homologyProtoglobin / Globin-sensor domain / Protoglobin / Globin/Protoglobin / Globin-like superfamily / oxygen binding / heme binding / Protogloblin ApPgb
Function and homology information
Biological speciesAeropyrum pernix (archaea)
Methodelectron crystallography / cryo EM
AuthorsDanelius E / Gonen T / Unge JT
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Am Chem Soc / Year: 2022
Title: Biocatalytic Carbene Transfer Using Diazirines.
Authors: Nicholas J Porter / Emma Danelius / Tamir Gonen / Frances H Arnold /
Abstract: Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. ...Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of protoglobin (Pgb) that use diazirines as carbene precursors. While the enhanced stability of diazirines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered Pgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an Pgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly stable carbene precursors in biocatalytic cyclopropanation, N-H insertion, and Si-H insertion reactions.
History
DepositionApr 26, 2022-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26768.png

Downloads & links

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Map

FileDownload / File: emd_26768.map.gz / Format: CCP4 / Size: 25.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX: 0.51384 Å / Y: 0.48587 Å / Z: 0.50458 Å
Density
Contour LevelBy AUTHOR: 1.417605
Minimum - Maximum-3.4176717 - 10.64161
Average (Standard dev.)-0.0044281874 (±0.945067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-118-136-90
Dimensions175200190
Spacing190175200
CellA: 97.63024 Å / B: 85.02783 Å / C: 100.91512 Å
α: 104.084 ° / β: 98.5927 ° / γ: 90.1041 °

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Supplemental data

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Sample components

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Entire : Homotetramer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L,...

EntireName: Homotetramer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
Components
  • Complex: Homotetramer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
    • Protein or peptide: Protogloblin ApPgb
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: IMIDAZOLE
  • Ligand: water

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Supramolecule #1: Homotetramer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L,...

SupramoleculeName: Homotetramer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Aeropyrum pernix (archaea)

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Macromolecule #1: Protogloblin ApPgb

MacromoleculeName: Protogloblin ApPgb / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Aeropyrum pernix (archaea)
Molecular weightTheoretical: 22.718887 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTPSDIPGYD YGRVEKSPIT DLEFDLLKKT VMLGEKDVMY LKKAGDVLKD QVDEILDLLV GWRASNEHLI YYFSNPDTGE PIKEYLERV RARFGAWILD TTSRDYNREW LDYQYEVGLR HHRSKKGVTD GVRTVPHIPL RYLIAQIYPL TATIKPFLAK K GGSPEDIE ...String:
MTPSDIPGYD YGRVEKSPIT DLEFDLLKKT VMLGEKDVMY LKKAGDVLKD QVDEILDLLV GWRASNEHLI YYFSNPDTGE PIKEYLERV RARFGAWILD TTSRDYNREW LDYQYEVGLR HHRSKKGVTD GVRTVPHIPL RYLIAQIYPL TATIKPFLAK K GGSPEDIE GMYNAWFKSV VLQVAIWSHP YTKENDW

UniProtKB: Protogloblin ApPgb

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Macromolecule #2: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 2 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

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Macromolecule #3: IMIDAZOLE

MacromoleculeName: IMIDAZOLE / type: ligand / ID: 3 / Number of copies: 4 / Formula: IMD
Molecular weightTheoretical: 69.085 Da
Chemical component information

ChemComp-IMD:
IMIDAZOLE / Imidazole

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 240 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration20 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 2460 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 0.00025 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystallography statisticsNumber intensities measured: 33825 / Number structure factors: 33825 / Fourier space coverage: 72 / R merge: 0.43 / Overall phase residual: 0 / Phase error rejection criteria: not applicable / High resolution: 2.1 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.1 Å / Shell - Low resolution: 24.26 Å / Shell - Number structure factors: 33825 / Shell - Phase residual: 29.25 / Shell - Fourier space coverage: 72 / Shell - Multiplicity: 4.1
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX (ver. 1.20.1-4487-000)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 18.2 / Target criteria: maximum likelihood
Output model

PDB-7ute:
MicroED structure of Aeropyrum pernix protoglobin mutant

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