Here, we report a normal-mode-based refinement of the SIV gp120 structure that was originally determined to 4.0 Å resolution. One feature of this structure determination is the rather low data-to-parameter ratio: the number of unique reflections is less than twice the number of atoms in the structure. In order to limit the number of parameters in refinement in the original structure determination, the TLS method as implemented in REFMAC
5 was employed in order to derive a better B
-factor model. One TLS group equivalent to 20 independent refinement parameters was used during the refinement (Chen et al.
). In the normal-mode-based refinement reported here, we found that the use of the first four lowest-frequency normal modes, which is equivalent to ten independent refinement parameters, in addition to the total of 20 independent refinement parameters for one TLS group and one parameter for the TLS scaling factor (see §
2 for more details), was able to produce the best B
-factor model. The normal-mode-derived B
-factor model revealed much higher structural flexibility in the inner domain than in the outer domain (Fig. 4
). This structural flexibility is also reflected in the elongated shape of the ellipsoids for the inner domain, which are in marked contrast to the more spherical ellipsoids for the outer domain (Fig. 4
). These observations agree well with two lines of evidence from previous studies that suggested substantial flexibility of the inner domain. One was from a comparison of the unliganded SIV gp120 structure with that of a previously solved HIV gp120–CD4 complex, which revealed a relocation as large as 28 Å of the inner domain upon CD4 binding (Chen et al.
; Kwong et al.
). The other was from the inability to locate a loop connecting the V1–V2 stem and the β5–β7–β25 sheet in the SIV gp120 core structure (Fig.1). Thus, these results reflect the power of normal-mode-based refinement in modeling structural deformations, especially functionally important ones.
On applying TLS refinement to the final normal-mode model of the gp120 core, we found that TLS did yield lower R factors compared with the published values, but did not bring them below those of final normal-mode model (Table 1). However, it is noteworthy that in our experience the use of normal-mode refinement can sometimes improve the subsequent application of TLS refinement (F. Ni, B. K. Poon, M. Lu, Q. Wang and J. Ma, unpublished data). Thus, in real applications it is always worth exploring whether the combined use of normal-mode and TLS refinements would yield improved refinement.
In this study, we noticed that the composite OMIT map based on normal-mode refinement is quite different from that of the TLS model in some regions, particularly regions with high B
factors. These differences were significant enough to allow gradual model improvements based on gradually improved electron-density maps, eventually leading to an overall improvement of the final normal-mode model of the gp120 core. We hope that the better map is a consequence of a more physically relevant representation of atomic motion by normal modes, which provide more accurate phase information than TLS. We compared the phase-angle shifts caused by one round of one-group TLS and normal-mode-based refinement on the original gp120 model (see §
3.1). Since the original model had isotropic B
factors, the inclusion of anisotropic B
factors by TLS or normal-mode methods followed by subsequent positional refinement using REFMAC
5 offers the best comparison of anisotropic B
-factor models generated by these two methods. The R
factors after one round of anisotropic B
-factor refinement and REFMAC
5 refinement were 38.8% for R
and 39.1% for R
for TLS refinement, compared with 38.7% for R
and 39.5% for R
for normal-mode refinement. Although the two anisotropic B
-factor refinement methods yielded comparable R
factors, the normal-mode method caused almost twice as large a shift compared with the TLS method in all resolution shells (the average phase-angle shifts were 10.3° and 17.3° for the TLS and normal-mode methods, respectively; Fig. 5). Thus, it is likely that the significantly larger phase-angle shifts caused by normal-mode-based refinement contribute to the substantial improvement of the electron-density maps and the consequent structural models.
Figure 5 Phase-angle shifts of one-round refinement by the normal-mode method (black bars) and the TLS method (gray bars) in relation to the original model as a function of resolution. The normal-mode method causes significantly larger phase-angle shifts in all (more ...)
In summary, the application of our normal-mode-based method to the structural refinement of SIV gp120 demonstrates the method’s potential in improving models of both protein and nonprotein components in X-ray structures and in revealing functionally important structural flexibility, even for rather low-resolution structures. In fact, low- to intermediate-resolution structures particularly need methods like this that can efficiently model B-factor distributions using an exceptionally small number of independent parameters.