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- EMDB-43167: A DARPin displayed on a designed tetrahedral protein scaffold -

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Basic information

Entry
Database: EMDB / ID: EMD-43167
TitleA DARPin displayed on a designed tetrahedral protein scaffold
Map data
Sample
  • Complex: DARPin with a helical connection to a chain of a designed protein scaffold
    • Protein or peptide: DARPin protein scaffold
Keywordsscaffold / tetrahedral / darpin / symmetrical / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsSuder DS / Gonen S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM142797 United States
CitationJournal: Int J Mol Sci / Year: 2024
Title: Mitigating the Blurring Effect of CryoEM Averaging on a Flexible and Highly Symmetric Protein Complex through Sub-Particle Reconstruction.
Authors: Diana S Suder / Shane Gonen /
Abstract: Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution ...Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution reconstruction as signals from thousands of particles are averaged together. This "blurring" effect can be difficult to overcome and is possibly more pronounced when averaging highly symmetric complexes. Approaches to mitigating flexibility during CryoEM processing are becoming increasingly critical as the technique advances and is applied to more dynamic proteins and complexes. Here, we detail the use of sub-particle averaging and signal subtraction techniques to precisely target and resolve flexible DARPin protein attachments on a designed tetrahedrally symmetric protein scaffold called DARP14. Particles are first aligned as full complexes, and then the symmetry is reduced by alignment and focused refinement of the constituent subunits. The final reconstructions we obtained were vastly improved over the fully symmetric reconstructions, with observable secondary structure and side-chain placement. Additionally, we were also able to reconstruct the core region of the scaffold to 2.7 Å. The data processing protocol outlined here is applicable to other dynamic and symmetric protein complexes, and our improved maps could allow for new structure-guided variant designs of DARP14.
History
DepositionDec 18, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43167.png

Downloads & links

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Map

FileDownload / File: emd_43167.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0175
Minimum - Maximum-0.041910056 - 0.06977328
Average (Standard dev.)-0.00010579692 (±0.0016967498)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 314.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43167_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43167_half_map_2.map
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 with a helical connection to a chain of a designed protein...

EntireName: DARPin with a helical connection to a chain of a designed protein scaffold
Components
  • Complex: DARPin with a helical connection to a chain of a designed protein scaffold
    • Protein or peptide: DARPin protein scaffold

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Supramolecule #1: DARPin with a helical connection to a chain of a designed protein...

SupramoleculeName: DARPin with a helical connection to a chain of a designed protein scaffold
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: DARPin protein scaffold

MacromoleculeName: DARPin protein scaffold / type: protein_or_peptide / ID: 1
Details: with a helical connection to a chain of a designed protein scaffold
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 32.208723 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: KDSPIIEANG TLDELTSFIG EAKHYVDEEM KGILEEIQND IYKIMGEIGS KGKIEGISEE RIAWLLKLIL RYMEMVNLKS FVLPGGTLE SAKLDVCRTI ARRALRKVLT VTREFGIGAE AAAYLLALSD LLFLLARVIE IELGKKLLEA ARAGQDDEVR I LMANGADV ...String:
KDSPIIEANG TLDELTSFIG EAKHYVDEEM KGILEEIQND IYKIMGEIGS KGKIEGISEE RIAWLLKLIL RYMEMVNLKS FVLPGGTLE SAKLDVCRTI ARRALRKVLT VTREFGIGAE AAAYLLALSD LLFLLARVIE IELGKKLLEA ARAGQDDEVR I LMANGADV NAHDDQGSTP LHLAAWIGHP EIVEVLLKHG ADVNARDTDG WTPLHLAADN GHLEIVEVLL KYGADVNAQD AY GLTPLHL AADRGHLEIV EVLLKHGADV NAQDKFGKTA FDISIDNGNE DLAEILQ

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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: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6c9k

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF
Details: resolution ranges drastically across the map, though.
Number images used: 287323
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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