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- PDB-9j48: GFP bound to 24-mer DARPin-apoferritin model 6c -

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Basic information

Entry
Database: PDB / ID: 9j48
TitleGFP bound to 24-mer DARPin-apoferritin model 6c
Components
  • Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
  • Green fluorescent protein
KeywordsMETAL BINDING PROTEIN/LUMINESCENT PROTEIN / GFP / DARPin / apoferritin / scaffold / METAL BINDING PROTEIN-LUMINESCENT PROTEIN complex
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 / bioluminescence / generation of precursor metabolites and energy / 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 / Green fluorescent protein, GFP ...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 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Ferritin-like superfamily
Similarity search - Domain/homology
Green fluorescent protein / Ferritin heavy chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsLu, X. / Yan, M. / Zhang, H.M. / Hao, Q.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2019YFA0906004 China
CitationJournal: IUCrJ / Year: 2025
Title: A large, general and modular DARPin-apoferritin scaffold enables the visualization of small proteins by cryo-EM.
Authors: Xin Lu / Ming Yan / Yang Cai / Xi Song / Huan Chen / Mengtan Du / Zhenyi Wang / Jia'an Li / Liwen Niu / Fuxing Zeng / Quan Hao / Hongmin Zhang /
Abstract: Single-particle cryo-electron microscopy (cryo-EM) has emerged as an indispensable technique in structural biology that is pivotal for deciphering protein architectures. However, the medium-sized ...Single-particle cryo-electron microscopy (cryo-EM) has emerged as an indispensable technique in structural biology that is pivotal for deciphering protein architectures. However, the medium-sized proteins (30-40 kDa) that are prevalent in both eukaryotic and prokaryotic organisms often elude the resolving capabilities of contemporary cryo-EM methods. To address this challenge, we engineered a scaffold strategy that securely anchors proteins of interest to a robust, symmetric base via a selective adapter. Our most efficacious constructs, namely models 4 and 6c, feature a designed ankyrin-repeat protein (DARPin) rigidly linked to an octahedral human apoferritin via a helical linker. By utilizing these large, highly symmetric scaffolds (∼1 MDa), we achieved near-atomic-resolution cryo-EM structures of green fluorescent protein (GFP) and maltose-binding protein (MBP), revealing nearly all side-chain densities of GFP and the distinct structural features of MBP. The modular design of our scaffold allows the adaptation of new DARPins through minor amino-acid-sequence modifications, enabling the binding and visualization of a diverse array of proteins. The high symmetry and near-spherical shape of the scaffold not only mitigates the prevalent challenge of preferred particle orientation in cryo-EM but also significantly reduces the demands of image collection and data processing. This approach presents a versatile solution, breaking through the size constraints that have traditionally limited single-particle cryo-EM.
History
DepositionAug 9, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
a: Green fluorescent protein
B: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
b: Green fluorescent protein
C: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
c: Green fluorescent protein
D: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
d: Green fluorescent protein
E: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
e: Green fluorescent protein
F: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
f: Green fluorescent protein
G: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
g: Green fluorescent protein
H: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
h: Green fluorescent protein
I: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
i: Green fluorescent protein
J: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
j: Green fluorescent protein
K: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
k: Green fluorescent protein
L: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
l: Green fluorescent protein
M: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
m: Green fluorescent protein
N: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
n: Green fluorescent protein
O: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
o: Green fluorescent protein
P: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
p: Green fluorescent protein
Q: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
q: Green fluorescent protein
R: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
r: Green fluorescent protein
S: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
s: Green fluorescent protein
T: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
t: Green fluorescent protein
V: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
v: Green fluorescent protein
W: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
w: Green fluorescent protein
X: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
x: Green fluorescent protein
Y: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
y: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)1,677,42048
Polymers1,677,42048
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed


Mass: 43268.602 Da / Num. of mol.: 24
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
#2: Protein ...
Green fluorescent protein


Mass: 26623.918 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: gfp / Production host: Escherichia coli (E. coli) / References: UniProt: A0A059PIQ0
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: GFP bound with the 24-mer DARPin-apoferritin scaffold / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris1
250 mMsodium chlorideNacl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.28 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 39

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Processing

EM software
IDNameVersionCategory
1cryoSPARC9.01particle selection
2SerialEMimage acquisition
4cryoSPARC9.01CTF correction
7PHENIX1.16model fitting
9PHENIX1.16model refinement
10Coot0.9model refinement
11cryoSPARC9.01initial Euler assignment
12cryoSPARC9.01final Euler assignment
13cryoSPARC9.01classification
14cryoSPARC9.013D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 750123
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17397 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3AJO

3ajo


Accession code: 3AJO / Source name: PDB / Type: experimental model

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