• 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • In this study the DHFR bp deletion was not


    In this study, the DHFR 19-bp THZ2 was not associated with spina bifida risk in mothers and children and did not affect DHFR expression. The DHFR 9-bp repeat had no effect on spina bifida risk, but an effect of the DHFR 6/6 genotype on DHFR expression cannot be ruled out.
    Introduction Because radiation damage hampers x-ray crystallography, which is indispensable for structural biology, cryo-cooling is routinely performed before diffraction image collection, whereby the damage to the crystal due to ionizing radiation can be delayed (1, 2). However, a concern with cryo-cooling is that the structures determined at such low temperatures may not retain information on the dynamics of protein, which is often crucial for protein function. Although the backbone structure is often very similar between protein structures determined at room temperature and cryo-temperature, the protein and unit cell volumes usually shrink, and the lattice contact increases up to 50%, depending on the crystal (see, e.g., (3, 4, 5, 6)). Fraser et al. suggested a significant, biologically relevant impact of cryo-cooling over side-chain ensembles (7, 8). In particular, a side-chain conformer, suggested to be catalytically important, was only detectable with room-temperature diffraction of cyclophilin A (7). Comparison between NMR and crystal structures of dihydrofolate reductase (DHFR) suggests that the room-temperature crystal structure is crucial for inferring protein dynamics (9). In addition, to evaluate the effect of cryo-cooling in a more systematic way, crystal structures of DHFR determined at room temperature and cryo-temperature, determined THZ2 by two independent groups, were compared, and it was found that the cryo-cooling affects crystal structures “idiosyncratically yet reproducibly” (10). Another study also suggested that the difference observed between the two temperatures is not likely due to the greater sensitivity to ionizing radiation at room temperature (11). Considering the crucial role played by cryo-cooling on the structure and dynamics of proteins, as evidenced in numerous earlier studies, molecular dynamics (MD) simulation of protein crystal at low temperature should provide insights regarding the cryo-cooling effect. MD simulation has been used in many studies to obtain atomistic details of protein conformation and dynamics that are not accessible by experiments. Recent development of hardware and software has been enabling MD simulations of large systems in longer timescales (12, 13). However, as per the current literature, exhaustive MD simulations of the crystal environment at such low temperatures are scarce, presumably because of the difficulty of sampling at low temperatures and technical complexity of the MD simulation of the crystal environment. Classical MD simulation often suffers from insufficient sampling, particularly for large systems. The sampling issue can be alleviated by the replica-exchange MD (REMD) method (14, 15, 16, 17, 18), which has been successfully applied to Trp-cage (19) to study cold denaturation (20). Simulations of the crystal environment, in which a unit cell is built and periodic boundary conditions are applied in accordance with translational symmetry of the crystal concerned, have been reported (21, 22, 23, 24, 25, 26, 27, 28, 29). It is suggested that because the crystal packing may shift a docking pose, a hydrogen bond that is not stable in solution may have been overly emphasized for ligand specificity (26), demonstrating the importance of consideration of lattice packing for interpretation of crystal structures.