Crystal Cryocooling Distorts Conformational Heterogeneity in a Model Michaelis Complex of DHFR

Abstract

Summary Most macromolecular X-ray structures are determined from cryocooled crystals, but it is unclear whether cryocooling distorts functionally relevant flexibility. Here we compare independently acquired pairs of high-resolution datasets of a model Michaelis complex of dihydrofolate reductase (DHFR), collected by separate groups at both room and cryogenic temperatures. These datasets allow us to isolate the differences between experimental procedures and between temperatures. Our analyses of multiconformer models and time-averaged ensembles suggest that cryocooling suppresses and otherwise modifies sidechain and mainchain conformational heterogeneity, quenching dynamic contact networks. Despite some idiosyncratic differences, most changes from room temperature to cryogenic temperature are conserved, and likely reflect temperature-dependent solvent remodeling. Both cryogenic datasets point to additional conformations not evident in the corresponding room-temperature datasets, suggesting that cryocooling does not merely trap pre-existing conformational heterogeneity. Our results demonstrate that crystal cryocooling consistently distorts the energy landscape of DHFR, a paragon for understanding functional protein dynamics.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

“…Consequently, electron density at the catalytic site of an NC structure is derived from the mixture of both substrate and product, making interpretation of data complicated and unreliable. Third, cryogenic manipulations for reducing radiation damages in SRX have also been focused as a factor that changes the population of amino acid residues (33, 34) and enzyme-substrate complexes (35). Crystallographic (36), computational (37), and spectroscopic (38-40) studies actually show that binding modes of NO 2 − and NO in CuNiR crystal structures can differ from those in physiological environments.…”

Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca2+-calmodulin

“…Moreover, the degree of overall atomic disorder between the two models is also similar, with an average ADP for all protein atoms of 17.4 Å 2 and an average protein ADP anisotropy of 0.388 for Ca 2+ -SeMet CaM, which is comparable to the average protein ADP of 16.4 Å 2 and protein ADP anisotropy of 0.353 previously reported for 1exr. The consistencies between 1exr and our new model suggest functional conformational disorder, rather then idiosyncrasies resulting from cryocooling (Keedy et al, 2014). Therefore, the increased number of modeled disordered residues in Ca 2+ -SeMet CaM represents an improved model of conformational disorder compared with the conservative approach taken with 1exr.…”

“…The manually built Ca 2+ -SeMet CaM model contains $39% of the residues in alternate conformations, while the automated disorder-modeling approach implemented in qFit2.0 indicates that 93% of the residues sample multiple conformations. Considered in the light of recent results from diverse proteins (Deis et al, 2014;Fraser et al, 2009Fraser et al, , 2011Keedy et al, 2014;Lang et al, 2010Lang et al, , 2014, there is compelling evidence that crystalline proteins are more extensively disordered than previously thought and that traditional criteria for modeling disorder are probably too conservative. Being free of model bias, experimental phase information could aid in the development of new criteria for the detection and modeling of protein conformational disorder.…”

Expanding the Range of Protein Function at the Far End of the Order-Structure Continuum

“…4E-BP2 is phosphorylated initially on residues Thr 37 and Thr 46 and later on residues Ser 65 and Thr 70 (49). The initial phosphorylation on Thr 37 and Thr 46 greatly weakens the interaction between 4E-BP2 and eIFE4, and subsequent phosphorylation of residues Ser 65 , Thr 70 , and Ser 83 further lowers the affinity between 4E-BP2 and eIFE4 (41,49)). Accompanying studies suggest that 4E-BP2 forms a four-strand ␤-structure upon phosphorylation of residues Thr 37 and Thr 46 , and that this structure is further stabilized by the phosphorylation of Ser 65 , Thr 70 , and Thr 83 (41).…”

“…The initial phosphorylation on Thr 37 and Thr 46 greatly weakens the interaction between 4E-BP2 and eIFE4, and subsequent phosphorylation of residues Ser 65 , Thr 70 , and Ser 83 further lowers the affinity between 4E-BP2 and eIFE4 (41,49)). Accompanying studies suggest that 4E-BP2 forms a four-strand ␤-structure upon phosphorylation of residues Thr 37 and Thr 46 , and that this structure is further stabilized by the phosphorylation of Ser 65 , Thr 70 , and Thr 83 (41). Residues 54 -56, which form part of a helix when bound to eIFE4, are incorporated in one of the ␤-strands, and residues 58 -60 form a disordered loop (Fig.…”