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Abstract
X-ray crystallography and single-particle analysis cryogenic electron microscopy are essential techniques for uncovering the three-dimensional structures of biological macromolecules. Both techniques rely on the Fourier transform to calculate experimental maps. However, one of the crucial parameters, resolution, is rather broadly defined. Here, the methods to determine the resolution in X-ray crystallography and single-particle analysis are summarized. In X-ray crystallography, it is becoming increasingly more common to include reflections discarded previously by traditionally used standards, allowing for the inclusion of incomplete and anisotropic reflections into the refinement process. In general, the resolution is the smallest lattice spacing given by Bragg’s law for a particular set of X-ray diffraction intensities; however, typically the resolution is truncated by the user during the data processing based on certain parameters and later it is used during refinement. However, at which resolution to perform such a truncation is not always clear and this makes it very confusing for the novices entering the structural biology field. Furthermore, it is argued that the effective resolution should be also reported as it is a more descriptive measure accounting for anisotropy and incompleteness of the data. In single particle cryo-EM, the situation is not much better, as multiple ways exist to determine the resolution, such as Fourier shell correlation, spectral signal-to-noise ratio and the Fourier neighbor correlation. The most widely accepted is the Fourier shell correlation using a threshold of 0.143 to define the resolution (so-called “gold-standard”), although it is still debated whether this is the correct threshold. Besides, the resolution obtained from the Fourier shell correlation is an estimate of varying resolution across the density map. In reality, the interpretability of the map is more important than the numerical value of the resolution.
Highlights
Introduction“Seeing is believing” is at the heart of structural biology
Faculty of Science and Engineering, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Groningen Biomolecular Sciences & Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
For crystallography [1] and near atomic resolution of 1.54 Å achieved by single particle analysis [2] and
Summary
“Seeing is believing” is at the heart of structural biology Both X-ray crystallography and single-particle cryogenic electron microscopy (cryo-EM) have become essential for uncovering the three-dimensional (3D) structures of biological macromolecules. In the light microscopy field, the resolution was first defined by Lord Rayleigh as the smallest distance at which two point sources can be still distinguished [6]. The definition of Lord Rayleigh is not applicable in X-ray crystallography and cryo-EM, because both techniques make use of Fourier space to determine the resolution of data. The recent technological progress in the structural biology and the enormous effort of software developers to make their software user-friendly have brought many new users sometimes without deep understanding of techniques The aim of this mini-review is to give an introductory overview on how the resolution of data is determined in X-ray crystallography and single particle cryo-EM
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