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- PDB-9jqc: Cryo-EM structure of ferritin variant R63BrThA/E67BrThA with Cu(II) -

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Basic information

Entry
Database: PDB / ID: 9jqc
TitleCryo-EM structure of ferritin variant R63BrThA/E67BrThA with Cu(II)
ComponentsFerritin heavy chain
KeywordsMETAL BINDING PROTEIN / Cryo-EM / ferritin variant / non-canonical amino acid / metalloenzyme / copper / L-2-(5-Bromothienyl)alanine (BrThA)
Function / homology
Function and homology information


iron ion sequestering activity / ferritin complex / Scavenging by Class A Receptors / negative regulation of ferroptosis / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / negative regulation of fibroblast proliferation / ferric iron binding ...iron ion sequestering activity / ferritin complex / Scavenging by Class A Receptors / negative regulation of ferroptosis / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / negative regulation of fibroblast proliferation / ferric iron binding / autophagosome / iron ion transport / Iron uptake and transport / ferrous iron binding / tertiary granule lumen / 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 / cytoplasm / cytosol
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
COPPER (II) ION / Ferritin heavy chain
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.73 Å
AuthorsWang, C.H. / Sun, J.C. / Wang, Y.S.
Funding support Taiwan, 3items
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, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ferritin heavy chain
B: Ferritin heavy chain
C: Ferritin heavy chain
D: Ferritin heavy chain
E: Ferritin heavy chain
F: Ferritin heavy chain
G: Ferritin heavy chain
H: Ferritin heavy chain
I: Ferritin heavy chain
J: Ferritin heavy chain
K: Ferritin heavy chain
L: Ferritin heavy chain
M: Ferritin heavy chain
N: Ferritin heavy chain
O: Ferritin heavy chain
P: Ferritin heavy chain
Q: Ferritin heavy chain
R: Ferritin heavy chain
S: Ferritin heavy chain
T: Ferritin heavy chain
U: Ferritin heavy chain
V: Ferritin heavy chain
W: Ferritin heavy chain
X: Ferritin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)515,93048
Polymers514,40524
Non-polymers1,52524
Water51,8832880
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Ferritin heavy chain / Ferritin H subunit / Cell proliferation-inducing gene 15 protein


Mass: 21433.545 Da / Num. of mol.: 24 / Mutation: R63BrThA/E67BrThA
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FTH1, FTH, FTHL6, OK/SW-cl.84, PIG15 / Production host: Escherichia coli (E. coli) / References: UniProt: P02794, ferroxidase
#2: Chemical...
ChemComp-CU / COPPER (II) ION


Mass: 63.546 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2880 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 24-mer complex of ferritin variant R63BrThA/E67BrThA with Cu(II)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2880 nm / Nominal defocus min: 204 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 1.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 472193 / Symmetry type: POINT

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