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- EMDB-28615: MicroED structure of an Aeropyrum pernix protoglobin mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-28615
TitleMicroED structure of an 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: FE (III) ION
  • Ligand: water
KeywordsMicroED / protoglobin / directed evolution / 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 / Resolution: 2.1 Å
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 18, 2022-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28615.png

Downloads & links

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Map

FileDownload / File: emd_28615.map.gz / Format: CCP4 / Size: 25.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.51 Å/pix.
x 190 pix.
= 97.608 Å		0.49 Å/pix.
x 179 pix.
= 86.918 Å		0.5 Å/pix.
x 196 pix.
= 98.899 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.51372 Å / Y: 0.48558 Å / Z: 0.50459 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.41858
Minimum - Maximum-3.4921148 - 9.948743
Average (Standard dev.)-0.0057642898 (±0.9483574)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-119-134-90
Dimensions179196190
Spacing190179196
CellA: 97.60764 Å / B: 86.918076 Å / C: 98.899025 Å
α: 104.095 ° / β: 98.5833 ° / γ: 90.109 °

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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: FE (III) ION
  • 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: 22.187307 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
IPGYDYGRVE KSPITDLEFD LLKKTVMLGE KDVMYLKKAG DVLKDQVDEI LDLLVGWRAS NEHLIYYFSN PDTGEPIKEY LERVRARFG AWILDTTSRD YNREWLDYQY EVGLRHHRSK KGVTDGVRTV PHIPLRYLIA QIYPLTATIK PFLAKKGGSP E DIEGMYNA WFKSVVLQVA IWSHPYTKEN DW

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

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Macromolecule #3: IMIDAZOLE

MacromoleculeName: IMIDAZOLE / type: ligand / ID: 3 / Number of copies: 15 / Formula: IMD
Molecular weightTheoretical: 69.085 Da
Chemical component information

ChemComp-IMD:
IMIDAZOLE

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Macromolecule #4: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 308 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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 / Mesh: 200 / 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
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
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX (ver. 1.20.1-4487-000)
Crystallography statisticsNumber intensities measured: 33826 / Number structure factors: 33826 / Fourier space coverage: 72 / R merge: 0.43 / Overall phase residual: 0 / High resolution: 2.1 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.1 Å / Shell - Low resolution: 24.27 Å / Shell - Number structure factors: 33826 / Shell - Phase residual: 21.11 / Shell - Fourier space coverage: 73.1 / Shell - Multiplicity: 4.1

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 26.51 / Target criteria: maximum likelihood
Output model

PDB-8eum:
MicroED structure of an Aeropyrum pernix protoglobin mutant

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