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- PDB-8ve7: A DARPin displayed on a designed tetrahedral protein scaffold -

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Basic information

Entry
Database: PDB / ID: 8ve7
TitleA DARPin displayed on a designed tetrahedral protein scaffold
ComponentsDARPin protein scaffold
KeywordsDE NOVO PROTEIN / scaffold / tetrahedral / darpin / symmetrical
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsSuder, D.S. / Gonen, S.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DARPin protein scaffold


Theoretical massNumber of molelcules
Total (without water)32,2091
Polymers32,2091
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DARPin protein scaffold


Mass: 32208.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: with a helical connection to a chain of a designed protein scaffold
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)

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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: DARPin with a helical connection to a chain of a designed protein scaffold
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 287323
Details: resolution ranges drastically across the map, though.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034489
ELECTRON MICROSCOPYf_angle_d0.5036049
ELECTRON MICROSCOPYf_dihedral_angle_d3.699606
ELECTRON MICROSCOPYf_chiral_restr0.037715
ELECTRON MICROSCOPYf_plane_restr0.003768

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