Abstract
In 2020, cryo-EM single-particle analysis achieved true atomic resolution thanks to technological developments in hardware and software. The number of high-resolution reconstructions continues to grow, increasing the importance of the accurate determination of atomic coordinates. Here, a new Python package and program called Servalcat is presented that is designed to facilitate atomic model refinement. Servalcat implements a refinement pipeline using the program REFMAC5 from the CCP4 package. After the refinement, Servalcat calculates a weighted Fo - Fc difference map, which is derived from Bayesian statistics. This map helps manual and automatic model building in real space, as is common practice in crystallography. The Fo - Fc map helps in the visualization of weak features including hydrogen densities. Although hydrogen densities are weak, they are stronger than in the electron-density maps produced by X-ray crystallography, and some H atoms are even visible at ∼1.8 Å resolution. Servalcat also facilitates atomic model refinement under symmetry constraints. If point-group symmetry has been applied to the map during reconstruction, the asymmetric unit model is refined with the appropriate symmetry constraints.
Highlights
Atomic model refinement is the optimization of the model’s parameters against the observed data
The optimal sharpening will differ depending on the region, but here we use an overall isotropic B value estimated by comparing |Fo| with |from the atomic model (Fc)| calculated from a copy of the initial model with all atomic displacement parameters (ADPs) set to zero
The case of Electron Microscopy Data Bank (EMDB) entry EMD-8123 (Supplementary Fig. S2), atoms in apoferritin and GABAAR maps by cryo-EM single-particle analysis (SPA) at sharpening by the overall B value obtained by line fitting gave 1.2 and 1.7 Aresolution, respectively
Summary
FT: Fourier transform of unknown true map (complex values). Fn: Fourier transform of noise in the observed map (complex values). Fo1, Fo2: Fourier transforms of the two unweighted and unsharpened half maps from independent reconstructions (complex values). Fo: Fourier transform of the observed full map, (Fo1 + Fo2)/2. Fc: Fourier transform of calculated map from atomic coordinates (complex values). K: resolution-dependent scale factor between Fo and FT. D: resolution-dependent scale factor between Fo and Fc. T2 : variance of signal, var(FT). S: column vector of position in reciprocal space. ST: row vector of position in reciprocal space. X: column vector of position in real space. B: displacement parameter of an atom, or blurring parameter for a local or global region of a map. All quantities in Fourier space are dependent on s
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