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- PDB-7eq9: Cryo-EM structure of designed protein nanoparticle TIP60 (Truncat... -

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Basic information

Entry
Database: PDB / ID: 7eq9
TitleCryo-EM structure of designed protein nanoparticle TIP60 (Truncated Icosahedral Protein composed of 60-mer fusion proteins)
ComponentsTIP60
KeywordsDE NOVO PROTEIN / Artificial designed protein complex / VIRUS LIKE PARTICLE
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsObata, J. / Kawakami, N. / Tsutsumi, A. / Miyamoto, K. / Kikkawa, M. / Arai, R.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19H02522 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)JP16K05841 Japan
Japan Agency for Medical Research and Development (AMED)JP20am0101115 Japan
Citation
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.
#1: 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 30, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed
Revision 1.2Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)1,067,65460
Polymers1,067,65460
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, Molecular weight estimated by SAXS is 1063 kDa corresponding to molecular weight of 60-meric complex (1066 kDa). This SAXS data has been deposited to SASBDB as accession code SASDDZ8. ...Evidence: SAXS, Molecular weight estimated by SAXS is 1063 kDa corresponding to molecular weight of 60-meric complex (1066 kDa). This SAXS data has been deposited to SASBDB as accession code SASDDZ8., light scattering, Molecular weight determined by SEC-MALS is 1096 kDa corresponding to molecular weight of 60-meric complex (1066 kDa).
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
TIP60


Mass: 17794.227 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Details: Designed fusion protein of pentameric Sm-like protein (3BY7) and dimeric MyoX-coil domain (2LW9)
Source: (gene. exp.) synthetic construct (others) / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star

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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: 60-meric complex of designed fusion proteins TIP60 / Type: COMPLEX
Details: 60-meric complex of designed fusion proteins of a pentameric Sm-like protein (3BY7) and a dimeric MyoX-coil domain (2LW9)
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.066 MDa / Experimental value: YES
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pETDuet-1
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethaneTris1
2300 mMsodium chlorideNaClSodium chloride1
31 mMethylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1755
Image scansWidth: 3838 / Height: 3710

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 175419
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115596 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 205.47 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDPdb chain residue range
13BY7

3by7

3BY712-86
22LW9

2lw9

2LW921-47
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 205.47 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002263000
ELECTRON MICROSCOPYf_angle_d0.469384540
ELECTRON MICROSCOPYf_chiral_restr0.041710080
ELECTRON MICROSCOPYf_plane_restr0.003110740
ELECTRON MICROSCOPYf_dihedral_angle_d3.34528100

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