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- PDB-9jiu: Ferritin mutant R63MeHis -

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Basic information

Entry
Database: PDB / ID: 9jiu
TitleFerritin mutant R63MeHis
ComponentsFerritin heavy chain
KeywordsMETAL BINDING PROTEIN / 3-methyl-histidine / apo-form / human heavy chain ferritin
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.28 Å
AuthorsTsou, J.-C. / Huang, K.-F. / Ko, T.-P. / Wang, Y.-S.
Funding support Taiwan, 4items
OrganizationGrant numberCountry
Academia Sinica (Taiwan) Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 107-2113-M-001-025-MY3 Taiwan
National Science Council (NSC, Taiwan)112-2113-M-001-015- 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 12, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,24037
Polymers267,84912
Non-polymers2,39025
Water36,0842003
1
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
hetero molecules

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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)540,47974
Polymers535,69924
Non-polymers4,78050
Water43224
TypeNameSymmetry operationNumber
point symmetry operation1
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)219.169, 219.169, 147.906
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number94
Space group name H-MP42212
Space group name HallP4n2n
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/2
#3: y+1/2,-x+1/2,z+1/2
#4: x+1/2,-y+1/2,-z+1/2
#5: -x+1/2,y+1/2,-z+1/2
#6: -x,-y,z
#7: y,x,-z
#8: -y,-x,-z
Components on special symmetry positions
IDModelComponents
11E-339-

HOH

21E-445-

HOH

31E-462-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1(chain "E" and (resid 4 through 176 or resid 200))
d_6ens_1(chain "F" and (resid 4 through 176 or resid 200))
d_7ens_1(chain "G" and (resid 4 through 176 or resid 200))
d_8ens_1(chain "H" and (resid 4 through 176 or resid 200))
d_9ens_1(chain "I" and (resid 4 through 176 or resid 200))
d_10ens_1(chain "J" and (resid 4 through 176 or resid 200))
d_11ens_1(chain "K" and (resid 4 through 176 or resid 200))
d_12ens_1(chain "L" and (resid 4 through 176 or resid 200))

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: SER / Beg label comp-ID: SER / End auth comp-ID: GLY / End label comp-ID: GLY / Auth seq-ID: 4 - 176 / Label seq-ID: 5 - 177

Dom-IDAuth asym-IDLabel asym-ID
d_1AA
d_2BB
d_3CC
d_4DD
d_5EE
d_6FF
d_7GG
d_8HH
d_9II
d_10JJ
d_11KK
d_12LL

NCS oper:
IDCodeMatrixVector
1given(0.00330615228707, 0.999992876238, -0.00182121661181), (-0.999994477997, 0.00330675973283, 0.000330628311549), (0.000336648281988, 0.0018201134475, 0.999998286926)0.132840642715, 114.332590927, -0.136036897915
2given(-0.00314356944262, -0.99999203527, -0.00245913957668), (0.999989974132, -0.00315138618234, 0.00318125773002), (-0.00318898209064, -0.00244911441709, 0.999991916083)114.709086154, -0.162671286814, 0.292523565537
3given(-0.999981655656, 0.00466172760369, -0.00386738250927), (-0.00466908466243, -0.999987303297, 0.00189549288035), (-0.00385849713478, 0.00191351524513, 0.999990725187)114.605200448, 114.44380712, 0.0829405934391
4given(-0.501749485671, -0.498979884, -0.706587948519), (0.50110018199, 0.498161764976, -0.707625228158), (0.705085853807, -0.70912194387, 8.65013240883E-5)166.744797363, 52.2953467985, 74.0684411111
5given(0.501217254221, 0.500983119821, 0.705547431238), (0.498169373409, 0.499625028638, -0.70866219467), (-0.707536952723, 0.706675841113, 0.000846238104145)-52.2867111596, 52.3842802004, 73.9669936331
6given(-0.50058154651, -0.502811689773, -0.704697466947), (-0.500100348643, -0.496494400522, 0.70950190383), (-0.706624197563, 0.707583009179, -0.0029203665106)166.781324997, 62.0331881686, 74.2171198699
7given(0.500729484625, 0.503189254929, 0.704322764754), (-0.497566575724, -0.498496373834, 0.709879474274), (0.708306067989, -0.705905049562, 0.000758322158925)-52.3060214694, 62.0011940461, 73.6710662796
8given(-0.498137680334, 0.501965122089, 0.707028901557), (0.50238583905, -0.497511074172, 0.707171266243), (0.706730019281, 0.707469962106, -0.00435115660578)4.97049617181, 4.68091865428, -6.89419384088
9given(-0.501373663003, 0.49898707971, -0.706849591023), (-0.495445744925, 0.504183307367, 0.707342001021), (0.709336284059, 0.704848272222, -0.0055632062246)109.705699979, 4.68773541779, -6.73862913869
10given(0.497928197379, -0.502588124737, 0.706733816319), (0.497581390059, -0.501881937153, -0.707479527213), (0.710267745658, 0.703931600393, 0.000177329469597)5.15679598064, 109.53598966, -7.13150829725
11given(0.497060946794, -0.504521313099, -0.705966472151), (-0.497506118911, 0.500879687636, -0.708242331522), (0.710927617144, 0.703262243507, -0.00203470862098)109.713234292, 109.456142802, -6.97131715813

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Components

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


Mass: 22320.781 Da / Num. of mol.: 12 / Mutation: R63H
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 BL21(DE3) (bacteria) / References: UniProt: P02794, ferroxidase
#2: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Na
#3: Chemical
ChemComp-P6G / HEXAETHYLENE GLYCOL / POLYETHYLENE GLYCOL PEG400


Mass: 282.331 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C12H26O7 / Comment: precipitant*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2003 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.32 Å3/Da / Density % sol: 62.92 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5 / Details: 0.1M Bicine pH 8.5, 20%(v/v) PEG 300

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: BL15A1 / Wavelength: 1 Å
DetectorType: RAYONIX MX300HE / Detector: CCD / Date: May 1, 2024
RadiationMonochromator: LN2-Cooled, Fixed-Exit Double Crystal Monochromator
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.28→30 Å / Num. obs: 159010 / % possible obs: 97.5 % / Redundancy: 7.3 % / Biso Wilson estimate: 37.41 Å2 / Rmerge(I) obs: 0.2 / Rpim(I) all: 0.08 / Rrim(I) all: 0.223 / Net I/σ(I): 8.4
Reflection shellResolution: 2.28→2.36 Å / Redundancy: 7.2 % / Rmerge(I) obs: 0.783 / Mean I/σ(I) obs: 2 / Num. unique obs: 14962 / CC1/2: 0.949 / CC star: 0.987 / Rpim(I) all: 0.305 / Rrim(I) all: 0.843 / % possible all: 93.1

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
HKL-2000data collection
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.28→29.89 Å / SU ML: 0.2781 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 39.2356
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2718 1982 1.3 %
Rwork0.2387 150838 -
obs0.2392 152820 93.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 41.14 Å2
Refinement stepCycle: LAST / Resolution: 2.28→29.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17080 0 118 2004 19202
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003117521
X-RAY DIFFRACTIONf_angle_d0.583523574
X-RAY DIFFRACTIONf_chiral_restr0.03972468
X-RAY DIFFRACTIONf_plane_restr0.00383100
X-RAY DIFFRACTIONf_dihedral_angle_d12.94316563
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAX-RAY DIFFRACTIONTorsion NCS0.433995144769
ens_1d_3AAX-RAY DIFFRACTIONTorsion NCS0.431876930565
ens_1d_4AAX-RAY DIFFRACTIONTorsion NCS0.400250417692
ens_1d_5AAX-RAY DIFFRACTIONTorsion NCS0.405303324171
ens_1d_6AAX-RAY DIFFRACTIONTorsion NCS0.477420698192
ens_1d_7AAX-RAY DIFFRACTIONTorsion NCS0.339041253189
ens_1d_8AAX-RAY DIFFRACTIONTorsion NCS0.43501202208
ens_1d_9AAX-RAY DIFFRACTIONTorsion NCS0.426176141562
ens_1d_10AAX-RAY DIFFRACTIONTorsion NCS0.466407144859
ens_1d_11AAX-RAY DIFFRACTIONTorsion NCS0.469116044628
ens_1d_12AAX-RAY DIFFRACTIONTorsion NCS0.41036919446
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.28-2.330.35411300.30239916X-RAY DIFFRACTION87.1
2.33-2.40.32711380.280910432X-RAY DIFFRACTION91.75
2.4-2.470.36451380.286310573X-RAY DIFFRACTION92.51
2.47-2.550.32671410.27810719X-RAY DIFFRACTION93.99
2.55-2.640.31791430.27310911X-RAY DIFFRACTION95.62
2.64-2.740.30411440.259511005X-RAY DIFFRACTION96.3
2.74-2.870.31251430.252210959X-RAY DIFFRACTION95.91
2.87-3.020.29641410.250810889X-RAY DIFFRACTION95.03
3.02-3.210.30051430.245910798X-RAY DIFFRACTION94.05
3.21-3.460.29581410.241410787X-RAY DIFFRACTION93.77
3.46-3.80.24431430.213910785X-RAY DIFFRACTION93.43
3.8-4.350.23761400.211210672X-RAY DIFFRACTION91.95
4.35-5.480.20961460.206311052X-RAY DIFFRACTION94.48
5.48-29.890.25661510.245611340X-RAY DIFFRACTION93.93

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