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- EMDB-60822: MultiBody Refinement of dimeric DARPin and its bound GFP on a sym... -

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Basic information

Entry
Database: EMDB / ID: EMD-60822
TitleMultiBody Refinement of dimeric DARPin and its bound GFP on a symmetric scaffold
Map dataBody output from Relion Multibody refinement.
Sample
  • Complex: DARPin and its bound GFP on a symmetric scaffold
    • Protein or peptide: DARPin
    • Protein or peptide: Green fluorescent protein
KeywordsDARPin / dimeric / GFP / scaffold / BIOSYNTHETIC PROTEIN
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / Green fluorescent protein
Function and homology information
Biological speciessynthetic construct (others) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 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 16, 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_60822.png

Downloads & links

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Map

FileDownload / File: emd_60822.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBody output from Relion Multibody refinement.
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0193
Minimum - Maximum-0.062162917 - 0.08491932
Average (Standard dev.)-0.0000053455387 (±0.002734565)
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_60822_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Sharpened map, using the Relion local resolution implementation....

Fileemd_60822_additional_1.map
AnnotationSharpened map, using the Relion local resolution implementation. Model was primarily refined against this map. This map is submitted at the same time and the temporary EMDB code is EMD-61130.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

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Half map: Body half-map 2 from Relion Multibody refinement

Fileemd_60822_half_map_1.map
AnnotationBody half-map 2 from Relion Multibody refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Body half-map 1 from Relion Multibody refinement

Fileemd_60822_half_map_2.map
AnnotationBody half-map 1 from Relion Multibody refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DARPin and its bound GFP on a symmetric scaffold

EntireName: DARPin and its bound GFP on a symmetric scaffold
Components
  • Complex: DARPin and its bound GFP on a symmetric scaffold
    • Protein or peptide: DARPin
    • Protein or peptide: Green fluorescent protein

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Supramolecule #1: DARPin and its bound GFP on a symmetric scaffold

SupramoleculeName: DARPin and its bound GFP on a symmetric scaffold / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: DARPin

MacromoleculeName: DARPin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
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

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Macromolecule #2: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / 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 state3D array

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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: OTHER / 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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 113239
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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