2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor

Abstract

Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β-galactosidase and the cell-permeant inhibitor phenylethyl β-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.

Fig. 1.. Cryo-EM density map of β-galactosidase-PETG complex at 2.2 Å resolution.

(A) Surface representation of the density map of one of the four protomers in the tetrameric complex. (B to D) Visualization of selected map regions showing delineation of secondary structural elements, amino acid densities and carbonyl moieties (indicated by *). The density for Phe 627 is thinned out in the center of the aromatic ring, revealing the presence of a “hole” in the ring, a feature typically observed in structures determined by x-ray crystallography at resolutions of ~2 Å.

Fig. 2.. Visualization of tightly bound water molecules in the structure of the β-galactosidase-PETG complex.

(A to F) Selected examples of densities for water molecules (highlighted in yellow) hydrogen-bonded in pearl-string-like chains (A), connected to the polypeptide backbone and multiple amino acid side chains (B to E), or interacting with the Mg²⁺ ion in the active site (F).

Fig. 3.. Active site structure in PETG-liganded E. coli β-galactosidase.

(A) Uncorrected cryo-EM density map showing density for PETG, an associated water molecule and five of the amino acids that line the binding pocket. The density for the PETG ligand appears to be at lower resolution than the surrounding amino acids, presumably because of some wobble in the binding pocket. (B) Plot of distances of various parts of PETG to residues in the vicinity of (β-galactosidase from E. coli determined using LIGPLOT (www.ebi.ac.uk/thornton-srv/software/LIGPLOT/). (C) Superposition of the ligand binding pocket structures in (β-galactosidase from E. coli (cyan, determined by cryo-EM at 2.2 Å resolution) and T. reesei (green, determined by x-ray crystallography at 1.4 Å resolution) illustrating the differences in protein and ligand structures, and comparison between the corresponding configurations of PETG (inset). (D) Plot of distances of various parts of PETG to amino acids in the vicinity of β-galactosidase from T. reesei determined using LIGPLOT.

Fig. 4.. Illustration of map quality at the level of amino acids.

(A) Visualization of map density for examples of each of the 20 standard amino acids, which are grouped into neutral (nonpolar and polar), basic and acidic categories. (B and C) Illustration of contours of densities for multiple lie residues (B), and front, tilted and edge views for Tyr552 (C). In each case, the density contours are consistent with the 2.2 Å resolution we report.