Figure 4. Identification of the rotameric conformation of amino acid side chains and resolving of ordered water molecules in the T20S cryoEM reconstruction at 2.8 Å resolution.

(A) Different rotameric conformations adopted by the Met-14 side chain (β-subunit) between a crystal structure of the T20S determined at 3.4 Å resolution (left, PDB 1PMA) and the EM density (right). (B) Unambiguous establishment of the rotameric conformations of two different isoleucine residues: Ile70 (left, α-subunit) and Ile37 (right, β-subunit). (C) The additional density accounting for the hydroxyl group of tyrosine side chains (left, Tyr132, α-subunit) is prominent when compared to phenylalanine side chains (right, Phe91, α-subunit). (D) Numerous water molecules are resolved in the T20S cryoEM map. Left: a water molecule hydrogen-bonded to the carbonyl group of Val87 (α-subunit). Right: a water molecule hydrogen-bonded to the side chain hydroxyl of Thr102 (α-subunit). The cross-validation of the assignment of these water molecules using gold-standard half maps can be found in Figure 4—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.06380.006

Figure 4—figure supplement 1. Cross-validation of water molecule assignment through comparison of gold standard half maps.

(A) Water molecule hydrogen-bonded to Val87. Left: The EM density reconstructed using all particles (49,954). Middle and right: gold standard half-maps. (B) Water molecule hydrogen-bonded to Thr102. Left: EM density reconstructed using all particles. Middle and right: gold standard half-maps. In each case, the density attributed to water is present in both half maps confirming that it is not due to random noise. Furthermore the position of these water molecules matches those observed in a crystal structure of the proteasome determined to 1.9 Å resolution (PDB 1YAR).

DOI: http://dx.doi.org/10.7554/eLife.06380.007