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- EMDB-61728: Cryo-EM structure of ferritin variant R63MeH/R67MeH -

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Basic information

Entry
Database: EMDB / ID: EMD-61728
TitleCryo-EM structure of ferritin variant R63MeH/R67MeH
Map data
Sample
  • Complex: 24-mer complex of ferritin variant R63MeH/R67MeH
    • Protein or peptide: Ferritin heavy chain
  • Ligand: FE (III) ION
  • Ligand: water
KeywordsCryo-EM / ferritin variant / non-canonical amino acid / H-N-3-Methyl-L-histidine(MeH) / METAL BINDING PROTEIN
Function / homology
Function and homology information


iron ion sequestering activity / ferritin complex / negative regulation of ferroptosis / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / negative regulation of fibroblast proliferation / ferric iron binding ...iron ion sequestering activity / ferritin complex / negative regulation of ferroptosis / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / negative regulation of fibroblast proliferation / ferric iron binding / autophagosome / Iron uptake and transport / ferrous iron binding / tertiary granule lumen / iron ion transport / ficolin-1-rich granule lumen / intracellular iron ion homeostasis / immune response / iron ion binding / negative regulation of cell population proliferation / Neutrophil degranulation / extracellular exosome / extracellular region / identical protein binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Ferritin heavy chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.81 Å
AuthorsWang CH / Wang YS
Funding support Taiwan, 3 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan) Taiwan
Ministry of Science and Technology (MoST, Taiwan)107-2113-M-001-025-MY3 Taiwan
National Science Council (NSC, Taiwan)113-2113-M-001 -006 - Taiwan
CitationJournal: J Am Chem Soc / Year: 2024
Title: Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme.
Authors: Jo-Chu Tsou / Chun-Ju Tsou / Chun-Hsiung Wang / An-Li A Ko / Yi-Hui Wang / Huan-Hsuan Liang / Jia-Cheng Sun / Kai-Fa Huang / Tzu-Ping Ko / Shu-Yu Lin / Yane-Shih Wang /
Abstract: Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine's moderately nucleophilic imidazole side chain. ...Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine's moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole's electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin's B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out .
History
DepositionSep 27, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61728.png

Downloads & links

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Map

FileDownload / File: emd_61728.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.67 Å/pix.
x 420 pix.
= 280.56 Å		0.67 Å/pix.
x 420 pix.
= 280.56 Å		0.67 Å/pix.
x 420 pix.
= 280.56 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.668 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-1.3786767 - 2.2617073
Average (Standard dev.)0.0006852655 (±0.08018499)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 280.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_61728_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_61728_half_map_2.map
Projections & Slices
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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : 24-mer complex of ferritin variant R63MeH/R67MeH

EntireName: 24-mer complex of ferritin variant R63MeH/R67MeH
Components
  • Complex: 24-mer complex of ferritin variant R63MeH/R67MeH
    • Protein or peptide: Ferritin heavy chain
  • Ligand: FE (III) ION
  • Ligand: water

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Supramolecule #1: 24-mer complex of ferritin variant R63MeH/R67MeH

SupramoleculeName: 24-mer complex of ferritin variant R63MeH/R67MeH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ferritin heavy chain

MacromoleculeName: Ferritin heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.27168 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTTASTSQVR QNYHQDSEAA INRQINLELY ASYVYLSMSY YFDRDDVALK NFAKYFLHQS HEE(MHS)EHA(MHS)KL MK LQNQRGG RIFLQDIKKP DCDDWESGLN AMECALHLEK NVNQSLLELH KLATDKNDPH LCDFIETHYL NEQVKAIKEL GDH VTNLRK MGAPESGLAE YLFDKHTLGD SDNES

UniProtKB: Ferritin heavy chain

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Macromolecule #2: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 2 / Number of copies: 48 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 3096 / 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
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.999 µm / Nominal defocus min: 0.202 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.81 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1566061
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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