National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35-GM142797
United States
Citation
Journal: 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.
Entire : Trimeric component of a tetrahedral complex of a designed protein...
Entire
Name: Trimeric component of a tetrahedral complex of a designed protein scaffold called DARP14
Components
Complex: Trimeric component of a tetrahedral complex of a designed protein scaffold called DARP14
Complex: Subunit A with DARPin in a designed protein scaffold called DARP14
Complex: Subunit B in a designed protein scaffold called DARP14
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Supramolecule #1: Trimeric component of a tetrahedral complex of a designed protein...
Supramolecule
Name: Trimeric component of a 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)
-
Supramolecule #2: Subunit A with DARPin in a designed protein scaffold called DARP14
Supramolecule
Name: Subunit A with DARPin in a designed protein scaffold called DARP14 type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)
Organism: synthetic construct (others)
-
Supramolecule #3: Subunit B in a designed protein scaffold called DARP14
Supramolecule
Name: 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)
-
Experimental details
-
Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
0.5 mg/mL
Buffer
pH: 8
Vitrification
Cryogen name: ETHANE
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm
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