Biology subjects

McMullan, G.

Publications and source records attributed to McMullan, G..

3 recordsLinked to original sources

bioRxiv2025

Measurement of atomic scattering factors by cryo-electron microscopy

Determination of specimen structure from cryo-electron microscopy (cryo-EM) experiments relies on an accurate model of the electrostatic potential of the specimen. For biological macromolecules, the potential is strongly influenced by the presence of chemical bonds between atoms, a fact unaccounted for by models of electron scattering that are currently standard in the field. We propose a Bayesian approach to the estimation of atomic scattering factors which incorporates the effect of the molecular environment while remaining fast, interpretable and transferable between molecules. Our algorithm infers atomic scattering factors directly from maps of the electrostatic potential determined by cryo-EM single particle analysis, bypassing the need for computationally-intensive theoretical calculations. The algorithm is used to infer empirical scattering factors from high-resolution reconstructions of catalase enzymes. To illustrate its broad applicability, the algorithm is also applied to a training set of publicly-available cryo-EM data. The empirical scattering factors show improved agreement with a test set of cryo-EM reconstructions. The predictions are further validated by comparison with magnetic susceptibility values of organic compounds, as well as by application to the refinement of atomic models. Significance StatementUnderstanding the structure of biomolecules is key to explaining their function. Cryo-electron microscopy is a method for reconstructing the electrostatic potential distribution of a biological macro-molecule, a quantity which contains information about atomic positions and the redistribution of charge due to chemical bonding. These factors should be modelled when inferring the structure of the molecule from its electrostatic potential. We develop an improved model of the potential that takes into account chemical bonding while remaining computationally tractable. The parameters of the model are inferred from a selection of cryo-electron microscopy datasets using Bayesian methods.

biophysics↗

bioRxiv2020

Defocus-dependent Thon-ring fading

The brightness of modern Schottky field-emission guns can produce electron beams that have very high spatial coherence, especially for the weak-illumination conditions that are used for single-particle electron cryo-microscopy in structural biology. Even so, many users have observed defocus-dependent Thon-ring fading that has led them to restrict their data collection strategy to imaging with relatively small defocus values. In this paper, we reproduce the observation of defocus-dependent Thon-ring fading and produce a quantitative analysis and clear explanation of its causes. We demonstrate that a major cause is the delocalization of high-resolution Fourier components outside the field of view of the camera. We also show that it is important to make a correction for linear magnification anisotropy, even if it is quite small, before circular averaging of the Thon rings, as is also true before merging data from particles in many orientations. Under the conditions used in this paper, which are typical of those used in single-particle electron cryomicroscopy, fading of the Thon rings due to source coherence is negligible. The principal conclusion is that much higher values of defocus can be used than is currently thought to be possible. This increased understanding should give electron microscopists the confidence to use higher amounts of defocus to allow, for example, better visibility of their particles and Ewald sphere correction.

biophysics↗

bioRxiv2020

Single-particle cryo-EM at atomic resolution

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 [A] resolution cryo-EM reconstruction for a prototypical human membrane protein, the {beta}3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 [A] resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

biophysics↗