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- PDB-9uol: Cryo-EM structure of pyrene-modified TIP60 double mutant (G12C/S5... -

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Entry
Database: PDB / ID: 9uol
TitleCryo-EM structure of pyrene-modified TIP60 double mutant (G12C/S50C) with addition of Nile Red
ComponentsTIP60 double mutant (G12C/S50C)
KeywordsDE NOVO PROTEIN / Artificial designed protein complex / Fusion protein / Protein nanocage / Protein nanoparticle / Pyrene modification
Function / homology:
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsYamashita, M. / Kawakami, N. / Arai, R. / Ikeda, A. / Moriya, T. / Senda, T. / Miyamoto, K.
Funding support Japan, 8items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP24ama121001 Japan
Japan Society for the Promotion of Science (JSPS)JP18K05324 Japan
Japan Society for the Promotion of Science (JSPS)JP17KK0104 Japan
Japan Society for the Promotion of Science (JSPS)JP19H02522 Japan
Japan Society for the Promotion of Science (JSPS)JP24K01267 Japan
Japan Society for the Promotion of Science (JSPS)JP24KJ1946 Japan
Japan Science and TechnologyJPMJSP2123 Japan
Other privateJapan Association for Chemical Innovation (JACI) 8th Research Encouragement Award Japan
Citation
Journal: Biomater Sci / Year: 2025
Title: misteINK: a protein nanocage-based ink with reversible, stimuli-responsive color shifts.
Authors: Maika Yamashita / Norifumi Kawakami / Ryoichi Arai / Akihito Ikeda / Toshio Moriya / Toshiya Senda / Kenji Miyamoto /
Abstract: Dyes exhibiting polarity-dependent color changes, known as solvatochromism, have great potential for creating sensors, smart materials, and responsive coatings. However, full-range color shifts ...Dyes exhibiting polarity-dependent color changes, known as solvatochromism, have great potential for creating sensors, smart materials, and responsive coatings. However, full-range color shifts require a technique to disperse dyes across a wide range of solvent polarities, which remains a persistent challenge. For example, hydrophobic dyes often aggregate in water, preventing effective color shifts. Although surfactants can assist in dye dispersion, they can also prevent solvent molecules from accessing the dye. To address this, we used a 60-mer protein nanocage, TIP60, with a densely pyrene-modified interior surface. The modification did not induce protein denaturation, as monitored by small-angle X-ray scattering, and greatly increased the aqueous solubility of a hydrophobic solvatochromic dye, Nile Red (NR), while preserving its fluorescence. The NR-loaded solution appeared blue, reflecting the polar environment surrounding NR. Cryogenic electron microscopy suggested that the pyrenes interacted with each other to form a binding site for NR. This interaction also contributed to thermostability of TIP60 (65 °C to 86 °C) and stability against sodium dodecyl sulfate, as observed by electrophoresis experiments. When brushed onto plain copy paper, the NR-loaded nanocage appeared bluish-purple and shifted reversibly to purplish red upon heating, returning on cooling-presumably nanocage dissociation and reassembly. The color change was also sensitive to humidity. We term this material "misteINK", a protein-based ink with reversible temperature- and humidity-dependent color changes. These findings demonstrate that a single-step interior modification enables the rational design of protein materials for tuning dye photophysics, providing a powerful strategy for designing protein-based functional materials.
#1: Journal: Chempluschem / Year: 2023
Title: Hydrophobization of a TIP60 Protein Nanocage for the Encapsulation of Hydrophobic Compounds.
Authors: Maika Yamashita / Norifumi Kawakami / Kenji Miyamoto /
Abstract: Encapsulation of hydrophobic molecules in protein-based nanocages is a promising approach for dispersing these molecules in water. Here, we report a chemical modification approach to produce a ...Encapsulation of hydrophobic molecules in protein-based nanocages is a promising approach for dispersing these molecules in water. Here, we report a chemical modification approach to produce a protein nanocage with a hydrophobic interior surface based on our previously developed nanocage, TIP60. The large pores of TIP60 act as tunnels for small molecules, allowing modification of the interior surface by hydrophobic compounds without nanocage disassembly. We used four different hydrophobic compounds for modification. The largest modification group tested, pyrene, resulted in a modified TIP60 that could encapsulate aromatic photosensitizer zinc phthalocyanine (ZnPC) more efficiently than the other modification compounds. The encapsulated ZnPC generated singlet oxygen upon light activation in the aqueous phase, whereas ZnPC alone formed inert aggregates under the same experimental conditions. Given that chemical modification allows a wider diversity of modifications than mutagenesis, this approach could be used to develop more suitable nanocages for encapsulating hydrophobic molecules of interest.
#2: Journal: Chem Commun (Camb) / Year: 2021
Title: Icosahedral 60-meric porous structure of designed supramolecular protein nanoparticle TIP60.
Authors: Junya Obata / Norifumi Kawakami / Akihisa Tsutsumi / Erika Nasu / Kenji Miyamoto / Masahide Kikkawa / Ryoichi Arai /
Abstract: Supramolecular protein nanoparticles and nanocages have potential in a broad range of applications. Recently, we developed a uniform supramolecular protein nanoparticle, TIP60, symmmetrically self- ...Supramolecular protein nanoparticles and nanocages have potential in a broad range of applications. Recently, we developed a uniform supramolecular protein nanoparticle, TIP60, symmmetrically self-assembled from fusion proteins of a pentameric Sm-like protein and a dimeric MyoX-coil domain. Herein, we report the icosahedral 60-meric structure of TIP60 solved using single-particle cryo-electron microscopy. Interestingly, the structure revealed 20 regular-triangle-like pores on the surface. TIP60 and its mutants have many modifiable sites on their exterior and interior surfaces. The TIP60 architecture will be useful in the development of biomedical and biochemical nanoparticles/nanocages for future applications.
#3: Journal: Angew Chem Int Ed Engl / Year: 2018
Title: Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces.
Authors: Norifumi Kawakami / Hiroki Kondo / Yuki Matsuzawa / Kaoru Hayasaka / Erika Nasu / Kenji Sasahara / Ryoichi Arai / Kenji Miyamoto /
Abstract: Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously ...Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.
History
DepositionApr 26, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release
Revision 1.1Nov 26, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TIP60 double mutant (G12C/S50C)
B: TIP60 double mutant (G12C/S50C)
C: TIP60 double mutant (G12C/S50C)
D: TIP60 double mutant (G12C/S50C)
E: TIP60 double mutant (G12C/S50C)
F: TIP60 double mutant (G12C/S50C)
G: TIP60 double mutant (G12C/S50C)
H: TIP60 double mutant (G12C/S50C)
I: TIP60 double mutant (G12C/S50C)
J: TIP60 double mutant (G12C/S50C)
K: TIP60 double mutant (G12C/S50C)
L: TIP60 double mutant (G12C/S50C)
M: TIP60 double mutant (G12C/S50C)
N: TIP60 double mutant (G12C/S50C)
O: TIP60 double mutant (G12C/S50C)
P: TIP60 double mutant (G12C/S50C)
Q: TIP60 double mutant (G12C/S50C)
R: TIP60 double mutant (G12C/S50C)
S: TIP60 double mutant (G12C/S50C)
T: TIP60 double mutant (G12C/S50C)
V: TIP60 double mutant (G12C/S50C)
W: TIP60 double mutant (G12C/S50C)
X: TIP60 double mutant (G12C/S50C)
Y: TIP60 double mutant (G12C/S50C)
Z: TIP60 double mutant (G12C/S50C)
AA: TIP60 double mutant (G12C/S50C)
BA: TIP60 double mutant (G12C/S50C)
CA: TIP60 double mutant (G12C/S50C)
DA: TIP60 double mutant (G12C/S50C)
EA: TIP60 double mutant (G12C/S50C)
FA: TIP60 double mutant (G12C/S50C)
GA: TIP60 double mutant (G12C/S50C)
HA: TIP60 double mutant (G12C/S50C)
IA: TIP60 double mutant (G12C/S50C)
JA: TIP60 double mutant (G12C/S50C)
KA: TIP60 double mutant (G12C/S50C)
LA: TIP60 double mutant (G12C/S50C)
MA: TIP60 double mutant (G12C/S50C)
NA: TIP60 double mutant (G12C/S50C)
OA: TIP60 double mutant (G12C/S50C)
PA: TIP60 double mutant (G12C/S50C)
QA: TIP60 double mutant (G12C/S50C)
RA: TIP60 double mutant (G12C/S50C)
SA: TIP60 double mutant (G12C/S50C)
TA: TIP60 double mutant (G12C/S50C)
UA: TIP60 double mutant (G12C/S50C)
VA: TIP60 double mutant (G12C/S50C)
WA: TIP60 double mutant (G12C/S50C)
XA: TIP60 double mutant (G12C/S50C)
YA: TIP60 double mutant (G12C/S50C)
ZA: TIP60 double mutant (G12C/S50C)
AB: TIP60 double mutant (G12C/S50C)
BB: TIP60 double mutant (G12C/S50C)
CB: TIP60 double mutant (G12C/S50C)
DB: TIP60 double mutant (G12C/S50C)
EB: TIP60 double mutant (G12C/S50C)
FB: TIP60 double mutant (G12C/S50C)
GB: TIP60 double mutant (G12C/S50C)
HB: TIP60 double mutant (G12C/S50C)
IB: TIP60 double mutant (G12C/S50C)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,107,060180
Polymers1,071,38360
Non-polymers35,677120
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, light scattering, The molecular weight of 1133 kDa determined by SEC-MALS is consistent with that of 60-mer.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
TIP60 double mutant (G12C/S50C)


Mass: 17856.383 Da / Num. of mol.: 60 / Mutation: G12C, S50C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pACYC-Duet 1 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical...
ChemComp-A1L9F / N-(1-pyrenyl)maleimide / 1-pyren-1-ylpyrrole-2,5-dione


Mass: 297.307 Da / Num. of mol.: 120 / Source method: obtained synthetically / Formula: C20H11NO2 / Feature type: SUBJECT OF INVESTIGATION
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: Pyrene-modified TIP60 double mutant (G12C/S50C) with addition of Nile Red
Type: COMPLEX
Details: Pyrene-modified TIP60 (Truncated Icosahedral Protein composed of 60-mer fusion proteins) double mutant (G12C/S50C) with addition of Nile Red
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.108 MDa / Experimental value: YES
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pACYCDuet-1
Buffer solutionpH: 7.4 / Details: 20 mM Tris-HCl, 1 mM EDTA
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClC4H12ClNO31
21 mMEDTAC10H16N2O81
SpecimenConc.: 12.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: PIB-10 (Vacuum Device Inc.) was used for glow discharge.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 5 seconds (blot force 15)

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 77 K
Image recordingAverage exposure time: 6.02 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2110
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1.14CTF correction
7Cootmodel fitting
9PHENIX1.21.2_5419model refinement
10RELION4.0.2initial Euler assignment
11RELION4.0.2final Euler assignment
12RELION4.0.2classification
13RELION4.0.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 235783 / Details: Topaz auto-picking
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22050 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 7EQ9

7eq9


Accession code: 7EQ9 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 182.97 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002264140
ELECTRON MICROSCOPYf_angle_d0.640586820
ELECTRON MICROSCOPYf_chiral_restr0.04399900
ELECTRON MICROSCOPYf_plane_restr0.002110620
ELECTRON MICROSCOPYf_dihedral_angle_d3.57097920

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