Identification of new, well-populated amino-acid sidechain rotamers involving hydroxyl-hydrogen atoms and sulfhydryl-hydrogen atoms
© Ho and Agard; licensee BioMed Central Ltd. 2008
Received: 16 April 2008
Accepted: 07 October 2008
Published: 07 October 2008
An important element in homology modeling is the use of rotamers to parameterize the sidechain conformation. Despite the many libraries of sidechain rotamers that have been developed, a number of rotamers have been overlooked, due to the fact that they involve hydrogen atoms.
We identify new, well-populated rotamers that involve the hydroxyl-hydrogen atoms of Ser, Thr and Tyr, and the sulfhydryl-hydrogen atom of Cys, using high-resolution crystal structures (<1.2 Å). Although there were refinement artifacts in these structures, comparison with the electron-density maps allowed the placement of hydrogen atoms involved in hydrogen bonds. The χ2 rotamers in Ser, Thr and Cys are consistent with tetrahedral bonding, while the χ3 rotamers in Tyr are consistent with trigonal-planar bonding. Similar rotamers are found in hydrogen atoms that were computationally placed with the Reduce program from the Richardson lab.
Knowledge of these new rotamers will improve the evaluation of hydrogen-bonding networks in protein structures.
One important conformational parameter of a protein structure is the sidechain χ torsion angle . In crystal structures, these torsion angles were found to be rotameric : they cluster around specific values, values that can be explained in terms of relatively simple stereo-chemical considerations [3, 4]. Consequently, libraries of sidechain rotamers have been compiled [5, 6]. These libraries have proven useful in parameterizing sidechain conformations for homology modeling , monte-carlo simulations , and protein design . Rotamer libraries are also used to build and verify crystallographic models . Although the sidechain rotamers have been extensively studied, there remain a number of rotamers involving hydrogen atoms that have been overlooked.
Due to the difficulty in placing hydrogen atoms in protein electron density maps, it has long been customary to omit hydrogen atoms in reporting the crystal structure of a protein. However, Richardson and co-workers showed that positions of hydrogen atoms in high-resolution crystal structures can be confidently projected from the topology of the heavy atoms . The projected hydrogen atoms, in most cases, form better van-der-Waals contacts with the neighboring atoms than do the heavy atoms themselves. The heavy atoms accommodate the packing of the hydrogen atoms, even though the hydrogen atoms cannot be seen in the crystal structure.
One of the reasons why the projection of hydrogen atoms works so well is that the positions of most of the sidechain hydrogen atoms are stereo-chemically restricted. For example, the Hβ atom of Val can only adopt one tetrahedral-bonding position off the Cβ position given that 3 other C atoms are also bound to Cβ. For other hydrogen atoms, symmetry between equivalent methyl-hydrogen atoms results in similar restrictions. For example, in Val, the three equivalent Hγ1 atoms bound to Cγ1 saturate the three available tetrahedral-bonding positions at Cγ1. Nevertheless, there exist four types of sidechain hydrogen atoms in which there is ambiguity in projecting their positions. For instance, in Ser, there are three different ways to place the Hγ atom onto the tetrahedral-bonding positions of the Oγ atom. This freedom is also found in the hydroxyl-hydrogen atoms of Thr and Tyr, and in the sulfhydryl-hydrogen in Cys.
As the positions of most of the sidechain hydrogen atoms are so restricted, little attention has been paid to their conformation in crystal structures. However, given the growing number of structures containing hydrogen atoms in the data bank, it has become practical to revisit the question of sidechain hydrogen rotamers for the four classes of ambiguous sidechain hydrogen atoms. The positions of these sidechain hydrogen atoms should be parameterized by χ torsion angles, and we would like to know if these angles display rotameric preferences. Here, we study the distributions of these χ torsion angles in three data-sets: (1) high-resolution X-ray structures that contains explicit hydrogen atoms, (2) neutron diffraction structures and (3) structures with computationally-placed hydrogen positions.
The rotamers in high-resolution X-ray structures with hydrogen atoms
For the first part of the analysis, we use high-resolution X-ray structures that have explicit hydrogen atoms in the Ser, Thr, Tyr and Cys residues. As such, the data-set consists of structures found in the RCSB.ORG website  with resolution < 1.2 Å, where hydrogen atoms are found in the structure. The hydrogen atoms are filtered for residues with no alternate conformations and where the neighboring heavy atom has a B-factor < 40. The structures were further selected depending on the availability of the electron density maps in the Electron Density Server . This results in 27 structures: 1AHO, 1DY5, 1JM1, 1M40, 1RW1, 2AXW, 2FDN, 1BXO, 1EUW, 1KQP, 1MUW, 1TT8, 2BF9, 2FFY, 1C75, 1F94, 1L9L, 1O7J, 1UCS, 2CAL, 2H5C, 1CEX, 1GQV, 1LS1, 1RB9, 2AWK, 2ERL.
Given the absence of agreement with the other distributions (below), and incompatibilities with tetrahedral-bonding and trigonal-planar bonding, it must be concluded that in Ser, the two rotamers at χ2 = -120° (Figure 2A) and χ2 = 0° (Figure 2B) are artifacts especially since these rotamers pack the hydrogen atom against a carbon atom. Similarly, in Tyr, the off-planar rotamer at χ3 = 60° (Figure 2C) is an artifact.
Manually-placed rotamers in X-ray structures using hydrogen-bonds
Rotamers from hydrogen-bonded hydrogen atoms fitted from electron density in high-resolution structures.
Type of χ Angle
Ser χ2: 37 counts
-79 ± 18°
69 ± 24°
189 ± 16°
Thr χ2: 60 counts
-74 ± 26°
80 ± 18°
176 ± 35°
Tyr χ3: 39 counts
5 ± 9°
174 ± 12°
Comparison with neutron structures
We can compare the high-resolution manually-placed distributions to distributions from neutron structures. While the neutron structures are generally of a lower resolution, hydrogen is a strong and negative neutron scatterer, which should allow reasonably accurate positioning of the hydrogen atoms. We found 16 neutron structures in the PDB with hydrogen atoms, containing a total of 215 Ser, 154 Thr and 85 Tyr residues. There were no Cys hydrogen atoms. In keeping with the lower resolution, the distributions in the neutron structures (Figure 1C) are more diffuse than those of the high-resolution X-ray structures (Figure 1B). In the neutron structures, the Ser distribution has a peak near 180°. In Thr, the distribution has one delineated peak at 60°. In Tyr, the distribution is diffuse with weak peaks at 0° and 180°. Qualitatively, there is agreement with distributions derived from the manually-placed hydrogen atoms of the high-resolution crystal structures (Figure 1B).
Comparison with computationally-placed hydrogen atoms
We can also compare the high-resolution manually-placed distributions to distributions derived from computationally-placed hydrogen atoms. This allows us to evaluate algorithms that project hydrogen positions from the coordinates of heavy atoms. We use a representative non-homologous set of high resolution structures (< 1.8 Å), provided by the Richardson lab , where missing hydrogen atoms have been computationally-placed using the program Reduce . These hydrogen atoms were computationally placed by optimizing hydrogen bonds and steric contacts with neighboring atoms.
In the structures with Reduce-placed hydrogen atoms, we found an artifact in the surface hydrogen atoms. As there are few neighboring contacts on the surface to help determine the position of hydrogen atoms, many surface hydrogen atoms remain at the default value, resulting in a pronounced peak at 180° (data not shown). This peak can be removed if we eliminate surface residues. Furthermore, as the Reduce algorithm uses steric contacts to optimize hydrogen positions, we need to use well-packed hydrogen atoms. Consequently, we only consider buried interior hydrogen atoms, defined as atoms with > 8 neighboring atoms, where a neighboring atom is defined if it is within 3.5 Å of another atom. We also filter out residues with alternate conformations and atoms where the B-factor > 40. There were hydrogen atoms from 5768 Ser, 5932 Thr, 3645 Tyr and 660 Cys residues in 480 structures. The large size of this data set gives the most reliable statistics.
Rotamers from computationally-placed hydrogen atoms in the Richardson set of structures.
Type of χ Angle
Ser χ2: 5768 counts
-75 ± 25°
81 ± 22°
180 ± 26°
Thr χ2: 5932 counts
-70 ± 24°
80 ± 20°
178 ± 24°
Tyr χ3: 3645 counts
2 ± 27°
180 ± 23°
Cys χ2: 660 counts
-66 ± 32°
72 ± 27°
179 ± 24°
Given the robust performance of Reduce, we investigated the position of sulfhydryl-hydrogen atoms in the set of structures provided by the Richardson lab. The hydrogen atoms are considered only if there are no other Cys residues within 4.5 Å of the SG atom of the Cys in order to avoid disulfide-bonded Cystines. The position of the sulfhydryl-hydrogen in Cys is determined by the χ2 = Cα-Cβ-Sγ-Hγ angle. The Cys χ2 distribution show a dominant rotamer at χ2 = 181° (Figure 1), which places the hydrogen atom furthest away from the backbone, in between the two Hβ atoms.
Data was collected from PDB structures using in-house Python scripts. The distributions in Figure 1 were binned using a rough guideline of ~4 bins for a peak. For the X-ray structures, 360 bins were used for the distribution from the raw coordinates, and 30 bins for the distributions from the manually-placed hydrogen atoms. For neutron structures, we used 30 bins. For the computationally-placed distributions, we used 50 bins. To calculate the means and distributions for the rotamers, we divided up the χ range into 3 equal partitions in Ser, Thr and Cys, and 2 partitions for Tyr.
Based on experimental data, we find that certain sidechain torsion angles involving hydrogen atoms have strongly preferred orientations and should thus be considered rotameric. Although there were serious artifacts found in the reported coordinates of high-resolution X-ray structures, reliable hydrogen atom positions could be directly derived from the electron-density maps of hydrogen-bonded hydrogen atoms. The χ-angle distributions of these hydrogen-bonded hydrogen atoms match the distribution of hydrogen atoms that were computationally placed by the program Reduce .
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