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- EMDB-28616: MicroED structure of an Aeropyrum pernix protoglobin metallo-carb... -

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Basic information

Entry
Database: EMDB / ID: EMD-28616
TitleMicroED structure of an Aeropyrum pernix protoglobin metallo-carbene complex
Map data
Sample
  • Complex: Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
    • Protein or peptide: Protogloblin ApPgb
  • Ligand: benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-diyl-kappa~4~N~21~,N~22~,N~23~,N~24~)di(propanoato)(2-)]iron
  • Ligand: water
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: 2023
Title: MicroED Structure of a Protoglobin Reactive Carbene Intermediate.
Authors: Emma Danelius / Nicholas J Porter / Johan Unge / Frances H Arnold / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of ...Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an protoglobin (Pgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of Pgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered Pgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.
History
DepositionOct 19, 2022-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateApr 19, 2023-
Current statusApr 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28616.png

Downloads & links

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Map

FileDownload / File: emd_28616.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX: 0.60539 Å / Y: 0.57201 Å / Z: 0.60526 Å
Density
Contour LevelBy AUTHOR: 1.47716
Minimum - Maximum-3.8714905 - 6.338665
Average (Standard dev.)0.001670977 (±0.9847681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-54-24-79
Dimensions129109120
Spacing120129109
CellA: 72.646805 Å / B: 73.78928 Å / C: 65.97334 Å
α: 90.0 ° / β: 105.72 ° / γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y6...

EntireName: Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
Components
  • Complex: Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
    • Protein or peptide: Protogloblin ApPgb
  • Ligand: benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-diyl-kappa~4~N~21~,N~22~,N~23~,N~24~)di(propanoato)(2-)]iron
  • Ligand: water

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Supramolecule #1: Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y6...

SupramoleculeName: Homodimer of Aeropyrum pernix protoglobin (ApePgb) C45G, W59L, Y60V, V63R, C102S, F145Q, I149L mutant
type: complex / ID: 1 / Chimera: Yes / 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: 2 / 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

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Macromolecule #2: benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-d...

MacromoleculeName: benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-diyl-kappa~4~N~21~,N~22~,N~23~,N~24~)di(propanoato)(2-)]iron
type: ligand / ID: 2 / Number of copies: 2 / Formula: WUF
Molecular weightTheoretical: 707.618 Da
Chemical component information

ChemComp-WUF:
benzyl[3,3'-(7,12-diethenyl-3,8,13,17-tetramethylporphyrin-2,18-diyl-kappa~4~N~21~,N~22~,N~23~,N~24~)di(propanoato)(2-)]iron

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 64 / 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 R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
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: 40105 / Number structure factors: 40105 / Fourier space coverage: 70 / R merge: 0.32 / Overall phase residual: 0 / High resolution: 2.5 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.5 Å / Shell - Low resolution: 24.37 Å / Shell - Number structure factors: 40105 / Shell - Phase residual: 29.27 / Shell - Fourier space coverage: 7 / Shell - Multiplicity: 4.3
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: 20.59 / Target criteria: maximum likelihood
Output model

PDB-8eun:
MicroED structure of an Aeropyrum pernix protoglobin metallo-carbene complex

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