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- EMDB-60931: 24-mer DARPin-apoferritin scaffold in complex with the maltose bi... -

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Basic information

Entry
Database: EMDB / ID: EMD-60931
Title24-mer DARPin-apoferritin scaffold in complex with the maltose binding protein
Map data
Sample
  • Complex: 24-mer DARPin-apoferritin in complex with the maltose binding protein
    • Protein or peptide: DARPin,Ferritin heavy chain, N-terminally processed
    • Protein or peptide: Maltodextrin-binding protein
KeywordsDARPin / apoferritin / scaffold / maltose binding protein / BIOSYNTHETIC PROTEIN
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 / carbohydrate transmembrane transporter activity / maltose binding ...iron ion sequestering activity / ferritin complex / negative regulation of ferroptosis / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / negative regulation of fibroblast proliferation / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ferric iron binding / autophagosome / Iron uptake and transport / ferrous iron binding / tertiary granule lumen / iron ion transport / outer membrane-bounded periplasmic space / 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 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain ...Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Bacterial extracellular solute-binding protein / Ferritin-like / Bacterial extracellular solute-binding protein / Ferritin-like superfamily
Similarity search - Domain/homology
Maltodextrin-binding protein / Ferritin heavy chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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
DepositionJul 24, 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_60931.png

Downloads & links

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Map

FileDownload / File: emd_60931.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.3538298 - 1.127321
Average (Standard dev.)0.00996923 (±0.06538334)
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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Half map: #2

Fileemd_60931_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_60931_half_map_2.map
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Sample components

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Entire : 24-mer DARPin-apoferritin in complex with the maltose binding protein

EntireName: 24-mer DARPin-apoferritin in complex with the maltose binding protein
Components
  • Complex: 24-mer DARPin-apoferritin in complex with the maltose binding protein
    • Protein or peptide: DARPin,Ferritin heavy chain, N-terminally processed
    • Protein or peptide: Maltodextrin-binding protein

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Supramolecule #1: 24-mer DARPin-apoferritin in complex with the maltose binding protein

SupramoleculeName: 24-mer DARPin-apoferritin in complex with the maltose binding protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DARPin,Ferritin heavy chain, N-terminally processed

MacromoleculeName: DARPin,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: 41.047145 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGP GSAKEEILEE IKKAKQEIAG GGGGSELGKE LLEAARAGQD DEVRILMARG AEVNAADNTG TTPLHLAAYS GHLEIVEVL LKYGAEVNAA DVFGYTPLHL AAYWGHLEIV EVLLKNGADV NARDSDGMTP LHLAAKWGHL EIVEVLLRYG A DVEAQDKF ...String:
MGHHHHHHGP GSAKEEILEE IKKAKQEIAG GGGGSELGKE LLEAARAGQD DEVRILMARG AEVNAADNTG TTPLHLAAYS GHLEIVEVL LKYGAEVNAA DVFGYTPLHL AAYWGHLEIV EVLLKNGADV NARDSDGMTP LHLAAKWGHL EIVEVLLRYG A DVEAQDKF GKTPFDLAID NGNEDIAEVL QALLAINRQI NLELYASYVY LSMSYYFDRD DVALKNFAKY FLHQSHEERE HA EKLMKLQ NQRGGAISLQ DIKKPDCDDW ESGLNAMECA LHLEKNVNQS LLELHKLATD CNDPHLCDFI ETHYLNEQVK AIK ELGDHV TNLRKMGAPE SGLAEYLFDK HTLGSGSGAE IEQAKKEIAY LIKK

UniProtKB: Ferritin heavy chain

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Macromolecule #2: Maltodextrin-binding protein

MacromoleculeName: Maltodextrin-binding protein / type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 45.694176 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE L KAKGKSAL ...String:
MGSSHHHHHH SSGLVPRGSH MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE L KAKGKSAL MFNLQEPYFT WPLIAADGGY AFKYENGKYD IKDVGVDNAG AKAGLTFLVD LIKNKHMNAD TDYSIAEAAF NK GETAMTI NGPWAWSNID TSKVNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT DEGLEAVNKD KPL GAVALK SYEEELAKDP RIAATMENAQ KGEIMPNIPQ MSAFWYAVRT AVINAASGRQ TVDEALKDAQ TNSSSNNNNN NNNN NLGIE GRENLYFQGG S

UniProtKB: Maltodextrin-binding 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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.28 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ajo

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91926
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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