Analysis of the quality of crystallographic data and the limitations of structural models

Abstract

Crystal structures provide visual models of biological macromolecules, which are widely used to interpret data from functional studies and generate new mechanistic hypotheses. Because the quality of the collected x-ray diffraction data directly affects the reliability of the structural model, it is essential that the limitations of the models are carefully taken into account when making interpretations. Here we use the available crystal structures of members of the glutamate transporter family to illustrate the importance of inspecting the data that underlie the structural models. Crystal structures of glutamate transporters in multiple different conformations have been solved, but most structures were determined at relatively low resolution, with deposited models based on crystallographic data of moderate quality. We use these examples to demonstrate the extent to which mechanistic interpretations can be made safely.

Figure 1.

Schematic representation of the glutamate transporter transport cycle. (A) EAATs couple glutamate uptake to symport of three sodium ions and one proton and to antiport of one potassium ion. (B) The archaeal homologues Glt_Tk and Glt_Ph couple aspartate uptake only to symport of three sodium ions. Both mammalian and archaeal homologues were shown to support chloride conductance uncoupled to substrate transport. One protomer of the homotrimeric protein is depicted schematically in the membrane plane. The scaffold and transport domains are shown in yellow and blue, respectively; the position of membrane is indicated with the black lines, where “in” and “out” stand for inside and outside the cell, respectively. Sodium (magenta), proton (dark green), chloride (gray), and potassium (light green) ions are shown as circles, and substrate as a purple triangle. Possible chloride ion pathway is depicted with a dashed arrow.

Figure 2.

Structural architecture of the glutamate transporter homologues. (A) Extracellular view of the Glt_Tk homotrimer; cartoon representation. The scaffold and transport domains of one of the protomers are shown in yellow and blue, respectively. (B) Cross-section of the Glt_Tk trimer in the OFC (left) and Glt_Ph in the IFC (right); protein in surface representation, the position of membrane indicated with the black lines. (C) Substrate-binding site in Glt_Tk (residue numbering for Glt_Ph in parentheses). l-Aspartate (black) and amino acid residues involved in substrate coordination are shown as sticks and sodium ions as purple spheres. HP1 and HP2 are shown in cyan and green, respectively. (D and E) Cartoon representation of protomers in OFC (D) and IFC (E). Color scheme as in A and C. PDB codes 5E9S and 3KBC, respectively.

Figure 3.

Examples of electron densities for Glt_Tk and Glt_Ph structures. (A–C) Representation of electron densities for the conserved NMDGT motif (shown as sticks) in the following structures: (A) Glt_Tk OFC (PDB code 5DWY); (B) Glt_Ph iOFC (PDB code 3V8G); and (C) Glt_Ph with asymmetric IFC protomers (PDB code 4X2S). The 2Fo-Fc electron-density maps (shown in blue mesh) are contoured at 1σ.

Figure 4.

Absence of electron density in the Na1 site for the apo Glt_Ph structure (PDB code 4OYF). The electron-density map (2Fo-Fc) is shown as a blue mesh and contoured at 1σ. The Fo-Fc map is colored in green (3σ) and red (−3σ). See Glossary for explanation of 2Fo-Fc and Fo-Fc maps. Cartoon representation; sodium ion Na1 assigned in this structure is shown as a purple sphere, and amino acid residues supposedly involved in its coordination are shown as sticks.

Figure 5.

Representation of the electron density for the thallium ions in the suggested cation-binding site (Tl^Ct) and Na2 site (Tl2) of Glt_Ph (PDB code 4P1A). The 2Fo-Fc map is colored in blue and contoured at 3σ. The Fo-Fc map is colored in green and red (±3σ). Difference maps are used to check the fit of the model to the diffraction data (see Glossary). The Fo-Fc difference map is a tool to visualize possible misfits and errors: positive peaks (green) indicate missing parts of the model, and negative peaks (red) indicate that these parts of the model are not supported by experimental data, and hence have to be removed. Additionally, negative density peaks might indicate inappropriate refinement of occupancies/B-factors and/or severe radiation damage. Cartoon representation; thallium ions are shown as brown spheres.

Figure 6.

Absence of electron density for the benzyl group of TBOA in the Glt_Ph structure (PDB code 2NWW). Possible alternative orientation of the benzyl group of TBOA (shown with an arrow). The electron-density omit map is shown in gray mesh (1σ). The Fo-Fc map is colored in green (3σ) and red (−3σ). Cartoon representation. HP2 loop is shown in purple. TBOA (shown in black) and residues involved in its binding are presented as sticks. Omit maps are used to remove bias (largely introduced by molecular replacement, where phases are taken from the similar structure, or caused by erroneous modeling) and can be used to verify assignment of ligands in binding sites. This is achieved by excluding a part of the model from the refinement procedure followed by the calculation of a bias-free difference map.

Figure 7.

Contacts between Glt_Ph asymmetric IFS protomers related by noncrystallographic symmetry (PDB code 4X2S). Superposition of unlocked protomers B (green) and C (gray) and a locked protomer A (yellow). Chain C_sym of a symmetry molecule that forms an interface with chain B is shown in blue. Chains B and C_sym are symmetry mates, where steric clashes between the loop 4c-5 (chain B) and helix HP1b (chain C_sym) may have caused the shift of 4c-5 hairpin (shown with a dashed arrow), creating an “unlocked” conformation. Cartoon representation; amino acid residues that could cause steric clashes are shown as sticks.