Ribosomal Crystallography and Heteropolytungstates

Exploring Wells-Dawson Clusters Associated With the Small Ribosomal Subunit

“…Polyoxomoetalates (POMs; Wu, 1920; Dawson, 1953; Pope, 1976; Acerete et al, 1979b) have been used for many applications including being a selective and effective inhibitor of enzymes (Stephan et al, 2013), such as ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs; Lee et al, 2015), and as an artificial proteases (Stroobants et al, 2013), as nanocages for heteroanions (Zheng et al, 2015), and effective in catalysis (Wang and Yang, 2015). POMs have also been found to facilitate X-ray structure analysis of proteins and have been used for solving the structure of proteins such as the small subunit of the ribosome (Janell et al, 2001; Bashan and Yonath, 2008; Yonath, 2009). Proteins are synthesized in an organelle referred to as the ribosome, located in the endosomal reticulum.…”

“…That is, the co-ordinates are fine-tuned by solving many crystals of interacting species, such as antibiotics, and phasing agents, including the polyoxometalates (POMs) such as the polyoxotungstates shown in red in Figure 1. Over the series of structures of ribosomal small subunit from the extremophile Thermus thermophilus (T30S) by Yonath et al (Thygesen et al, 1996; Tocilj et al, 1999; Weinstein et al, 1999; Schluenzen et al, 2000; Janell et al, 2001; Auerbach-Nevo et al, 2005; Bashan and Yonath, 2008; Yonath, 2009) the Wells-Dawson cluster, P 2 W 18 O626- (P 2 W 18 ), (Figure 2) was used extensively. This approach was accompanied by studies with complementary techniques such as electron cryomicroscopy (Cryo-EM) to yield additional details (Winkler et al, 2017; Brown and Shao, 2018).…”

“…Examining the interactions of the metal complexes with proteins is interesting but has several issues, as detailed in previous works (Janell et al, 2001; Crans et al, 2014; McLauchlan et al, 2015; Sanchez Lombardo et al, 2015). Briefly, issues relating to resolution (Weinstein et al, 1999; Rudenko et al, 2003; Dauter, 2005; Blazevic et al, 2015), phasing (Thygesen et al, 1996; Pioletti et al, 2001), and degradation (including photoreduction) (Rich et al, 1998; George et al, 2012) all become of critical importance.…”

“…Briefly, issues relating to resolution (Weinstein et al, 1999; Rudenko et al, 2003; Dauter, 2005; Blazevic et al, 2015), phasing (Thygesen et al, 1996; Pioletti et al, 2001), and degradation (including photoreduction) (Rich et al, 1998; George et al, 2012) all become of critical importance. Protein X-ray crystallography is thus very different than small molecule crystallography because the protein structure diffraction data are often collected across numerous crystals rather than a single crystal more typical of a small molecule, although techniques have been developed to alleviate this issue in certain cases (Janell et al, 2001; Heras and Martin, 2005). Seeking improvements in the structure, interacting species, such as antibiotics, and phasing agents, including the polyoxotungstates were used to improve the overall structure ( vide infra ).…”

“…Since first described by Perutz with hemoglobin (Green et al, 1954), heavy atom derivatization has been employed for many years in solving structures and is well-reviewed as a technique in the literature (Garman and Murray, 2003; Dauter, 2005). In practice, ribosomal protein crystals may be soaked with P 2 W 18 solutions and large amounts of the P 2 W 18 remain in the crystal even after washing, helping with diffraction and phasing (Janell et al, 2001). In addition, the Yonath group reported that the P 2 W 18 POM served to anchor the protein conformation and stabilize the ribosomal proteins in their preparations, although other groups did not observe similar stabilization of the ribosome in their preparations (Wimberly et al, 2000; Clemons et al, 2001).…”