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- EMDB-43158: DARP14 EM map -

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Basic information

Entry
Database: EMDB / ID: EMD-43158
TitleDARP14 EM map
Map data
Sample
  • Complex: A12B12 tetrahedral complex of a designed protein scaffold called DARP14
    • Complex: Subunit A in a designed protein scaffold called DARP14
    • Complex: Subunit B in a designed protein scaffold called DARP14
Keywordsscaffold / tetrahedral / darpin / symmetrical / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 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_43158.png

Downloads & links

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Map

FileDownload / File: emd_43158.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0007
Minimum - Maximum-0.020596996 - 0.048554625
Average (Standard dev.)0.000040863066 (±0.0026026133)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 251.51999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43158_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_43158_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 : A12B12 tetrahedral complex of a designed protein scaffold called ...

EntireName: A12B12 tetrahedral complex of a designed protein scaffold called DARP14
Components
  • Complex: A12B12 tetrahedral complex of a designed protein scaffold called DARP14
    • Complex: Subunit A in a designed protein scaffold called DARP14
    • Complex: Subunit B in a designed protein scaffold called DARP14

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Supramolecule #1: A12B12 tetrahedral complex of a designed protein scaffold called ...

SupramoleculeName: A12B12 tetrahedral complex of a designed protein scaffold called DARP14
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: synthetic construct (others)

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Supramolecule #2: Subunit A in a designed protein scaffold called DARP14

SupramoleculeName: Subunit A in a designed protein scaffold called DARP14
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: synthetic construct (others)

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Supramolecule #3: Subunit B in a designed protein scaffold called DARP14

SupramoleculeName: Subunit B in a designed protein scaffold called DARP14
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.5 mg/mL
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: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 309973
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION / Details: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION / Details: RELION

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