Dataset Information

Determination of Rigidity of Protein Bound Au(144) Clusters by Electron Cryomicroscopy.

ABSTRACT: A method for estimating the positional displacement of protein bound gold nanoparticles is presented and used to estimate the rigidity of linkage of Au(144) nanoparticles bound to a tetrameric model protein. We observe a distribution of displacement values where most Au(144) clusters are immobilized to within 3Å relative to the protein center of mass. The shape of the distribution suggests two physical processes of thermal motion and protein deformation. The application of this and similar rigid gold nanoparticle/protein conjugates in high resolution single particle electron cryo-microscopy is discussed.

SUBMITTER: Sexton JZ

PROVIDER: S-EPMC2992337 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Publications

Determination of Rigidity of Protein Bound Au(144) Clusters by Electron Cryomicroscopy.

Sexton Jonathan Z JZ Ackerson Christopher J CJ

The journal of physical chemistry. C, Nanomaterials and interfaces 20100901 38

A method for estimating the positional displacement of protein bound gold nanoparticles is presented and used to estimate the rigidity of linkage of Au(144) nanoparticles bound to a tetrameric model protein. We observe a distribution of displacement values where most Au(144) clusters are immobilized to within 3Å relative to the protein center of mass. The shape of the distribution suggests two physical processes of thermal motion and protein deformation. The application of this and similar rigid ...[more]

PMID: 21116473

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Project description:Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words).

Dataset Information

Determination of Rigidity of Protein Bound Au(144) Clusters by Electron Cryomicroscopy.

Publications

Determination of Rigidity of Protein Bound Au(144) Clusters by Electron Cryomicroscopy.

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