REFINED STRUCTURE

A similar problem arises with present cross-validated measures of fit [92], because they also are applied to the final clean list of restraints. Residual dipolar couplings offer an entirely different and, owing to their long-range nature, very powerful way of validating structures against experimental data [93]. Similar to cross-validation, a set of residual dipolar couplings can be excluded from the refinement, and the deviations from this set are evaluated in the refined structures. 

Wlodawer, A., Deisenhofer, J., Huber, R. Comparison of two highly refined structures of bovine pancreatic trypsin inhibitor. /. Mol. Biol. 193 145-156, 1987. 

Refined structure of an intact IgG2a monoclonal antibody. Biochemistry 36 1581-1597, 1997. 

In refined structures at high resolution (around 2 A) there are usually no major errors in the orientation of individual residues, and the estimated errors in atomic positions are around 0.1-0.2 A provided the amino acid sequence is known. Hydrogen bonds both within the protein and to bound ligands can be identified with a high degree of confidence. 

X-ray structures are determined at different levels of resolution. At low resolution only the shape of the molecule is obtained, whereas at high resolution most atomic positions can be determined to a high degree of accuracy. At medium resolution the fold of the polypeptide chain is usually correctly revealed as well as the approximate positions of the side chains, including those at the active site. The quality of the final three-dimensional model of the protein depends on the resolution of the x-ray data and on the degree of refinement. In a highly refined structure, with an R value less than 0.20 at a resolution around 2.0 A, the estimated errors in atomic positions are around 0.1 A to 0.2 A, provided the amino acid sequence is known. 

It can be concluded that MD is a very powerful tool to refine structures of proteins and polypeptides in solution, based on 2D NMR data. This combination of techniques emerges as an important means to determine the 3 D structure of macromolecules up to a molecular weight of 20,000 in solution or in micelles or membrane fragments. 

These structures show that RpII essentially consists of the small core of four-stranded )9-sheet and three relatively large loops. Residues 6-16, 23-30, and 35-40 form loops 1, 2, and 3, respectively, and the chain reversals are accomplished by tight turns involving residues D8-D11, E28-E31, and V36-P39. Segments involved in )5-sheet strands and loops alternate in the primary sequence of Rp toxins. As mentioned above, these structures indicate that the )5-sheet is highly twisted in order to form the correct disulfide bonds. Comparison of different refined structures of RpII indicate that the structure of loop 1 in RpII is less well defined than... 

Fig. 9.8 Comparison of experimentally determined and back-calculated one-bond RDCs for the crystal structure (A), the ROE-derived structure (B) and the (RDC-hROE)-refined structure (C) of cyclosporin A. (D) Evaluation of the resulting models for the...

X-ray single crystal analysis Data collection. Siemens SMART IK CCD. Diffractometer. Data collection by SMART (Siemens, 1995) cell refinement SMART data reduction SAINT (Siemens, 1995) program(s) used to solve structure SHELXS97 (Sheldrick, 1997) program(s) used to refine structure SHELXL97 (Sheldrick, 1997) molecular graphics XP in SHELXTL (Sheldrick,... 

As at room temperature Bragg reflections contain both nuclear and magnetic structure factors, the nuclear structure was refined from a combination of polarized and unpolarized neutron data. Contrary to the ideal structure where only three atomic sites are present, it has been shown [11, 12] that some Y atoms were substituted by pairs of cobalt. These pairs, parallel to the c-axis are responsible for a structure deformation which shrinks the cobalt hexagons surrounding the substitutions. The amount of these substituted Y was refined to be 0.046 0.008. Furthermore, the thermal vibration parameter of Coi site appeared to be very anisotropic. The nuclear structure factors Fn were calculated from this refined structure and were introduced in the polarized neutron data to get the magnetic structure factors Fu. 

Other exciting applications involved using parallel tempering in connection with available experimental data. For example, Falcioni and Deem [57] used X-ray data to refine structures of zeolites, and Haliloglu et al. [58] refined NMR structural data for proteins (in particular using residual dipolar coupling constraints). 

M. Ghosh, C. Anthony, K. Harlos, M.G. Goodwin, and C. Blake, The refined structure of the quino-protein methanol dehydrogenase from Methylobacterium extorquens at 1.94A. Structure 3, 177—187 (1995). 

Refined structures of substrate-bound and phosphate-bound thymidylate synthase from lactobacillus casei, J. Mol. Biol. 232 1101 (1993). 

Fig. 6. Schematic diagram of the peptide-protein interaction mode as seen in the crystallo-graphically refined structured of the Lck SH2 domain-peptide complex, Protein Databank entry code 1 LKK.PDB. The residues directly engaged in intramolecular hydrogen bonds (dotted lines) are labeled explicitly... 

Steinbacher, S., Miller, S., Baxa, U., Budisa, N., Weintraub, A., Seckler, R., and Huber, R. (1997). Phage P22 tailspike protein Crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage. J. Mol. Biol. 267, 865-880. 

Fig. 4. The NMR-refined structure of interstrand cross-linked cis- Pt(NH3)2 2 -d(CATAGCTATG)2. The deoxycytosine residues opposite to the platinated deoxygua-nines have become extrahelical (structure reference PDBID, 1DDP). Adapted from (53). 

As reported by Hyde and Andersson (1989) the well-refined structure of mineral crysoberil may be considered the reference for the structures of a number of... 

D. M. E. Szebenyi and K. Moffat, The refined structure of vitamin D-dependent calciumbinding protein from bovine intestine, J. Biol. Chem. 261, 8761-8777 (1986). 

Clearly, precision must be a small fraction of resolution and any structural model must fit column position within a few picometer as shown in Figures 3 and 6. Deviations from this rule indicate the need to refine structural models, the presence of systematic errors, or an over interpretations of data. Unfortunately, this basic rule is sometimes disregarded by ignoring mismatches of up to 1 A = 100 pm . Systematic errors often relate to the presence of scanning noise, sample tilt, or to unfavorable specimen geometry for an exit wave reconstruction... 

By far the best way to refine structures using electron diffraction data is to use multislice calculations. These will be discussed in the next chapter. However, some useful information can be obtained by regular crystallographic least squares with the assumption of kinematic data. 

Figure 9 Refined structure for both modifications of FAPPO distances of hydrogen bonding are indicated as dotted lines.

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