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- EMDB-61130: GFP bound to 24-mer DARPin-apoferritin model 6c -

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Basic information

Entry
Database: EMDB / ID: EMD-61130
TitleGFP bound to 24-mer DARPin-apoferritin model 6c
Map data
Sample
  • Complex: GFP bound with the 24-mer DARPin-apoferritin scaffold
    • Protein or peptide: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
    • Protein or peptide: Green fluorescent protein
KeywordsGFP / DARPin / apoferritin / scaffold / METAL BINDING PROTEIN/LUMINESCENT PROTEIN / METAL BINDING PROTEIN-LUMINESCENT PROTEIN complex
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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsLu X / Yan M / Zhang HM / Hao Q
Funding support China, 1 items
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, 2024-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61130.png

Downloads & links

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Map

FileDownload / File: emd_61130.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 344.32 Å		1.08 Å/pix.
x 320 pix.
= 344.32 Å		1.08 Å/pix.
x 320 pix.
= 344.32 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.62385 - 3.5405865
Average (Standard dev.)0.013023531 (±0.14319329)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 344.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61130_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_61130_half_map_1.map
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Half map: #1

Fileemd_61130_half_map_2.map
Projections & Slices
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Sample components

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Entire : GFP bound with the 24-mer DARPin-apoferritin scaffold

EntireName: GFP bound with the 24-mer DARPin-apoferritin scaffold
Components
  • Complex: GFP bound with the 24-mer DARPin-apoferritin scaffold
    • Protein or peptide: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
    • Protein or peptide: Green fluorescent protein

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Supramolecule #1: GFP bound with the 24-mer DARPin-apoferritin scaffold

SupramoleculeName: GFP bound with the 24-mer DARPin-apoferritin scaffold / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminal...

MacromoleculeName: Designed ankyrin repeat proteins,Ferritin heavy chain, N-terminally processed
type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.268602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGP GSRMKQLEDK VEELLSKNYH LENEVARLKK LVGGSGGSGG SELGKELLEA ARAGQDDEVA VLMARGAEVN AADDVGVTP LHLAAQRGHL AIVSVLLAFG ASVNAADLWG QTPLHLAATA GHLEIVEVLL RSGASVNARD NIGHTPLHLA A WAGHLEIV ...String:
MGHHHHHHGP GSRMKQLEDK VEELLSKNYH LENEVARLKK LVGGSGGSGG SELGKELLEA ARAGQDDEVA VLMARGAEVN AADDVGVTP LHLAAQRGHL AIVSVLLAFG ASVNAADLWG QTPLHLAATA GHLEIVEVLL RSGASVNARD NIGHTPLHLA A WAGHLEIV EVLLAYGADV FAQDKFGKTP FDLAIDNGNE DIAEVLQRLL ECRRDAEAAI NYQINLELYA SYVYLSMSYY FD RDDVALK NFAKYFLHQS HEEREHAEKL MKLQNQRGGE ISLQSISSPD SDDWESGLNA MESALHLEKA VNASLLRLHK LAT DCNDPH LCDFIETHYL NEQVKAIKEL GDHVTNLRKM GAPESGLAEY LFDKHTLGSG SGAEIEQAKK EIAYLIKK

UniProtKB: Ferritin heavy chain

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Macromolecule #2: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 26.623918 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN ...String:
MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HHHHHH

UniProtKB: Green fluorescent protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
50.0 mMNaclsodium chloride
GridModel: Homemade / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.28 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 750123
CTF correctionSoftware - Name: cryoSPARC (ver. 9.01) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ajo

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 9.01) / Number images used: 17397
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 9.01)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 9.01)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 9.01)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3ajo


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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