The three-dimensional crystal structure of an arginine kinase transition-state analogue complex has been re®ned at 1.2 A Ê resolution, with an overall R factor of 12.3%. The current model provides a unique opportunity to analyze the structure of a bimolecular (phosphagen kinase) enzyme in its transition state. This atomic resolution structure con®rms in-line transfer of the phosphoryl group and the catalytic importance of the precise alignment of the substrates. The structure is consistent with a concerted proton transfer that has been proposed for an unrelated kinase. Re®nement of anisotropic temperature factors and translation±libration±screw (TLS) analyses led to the identi®cation of four rigid groups and their prevalent modes of motion in the transition state. The relative magnitudes of the mobility of rigid groups are consistent with their proposed roles in catalysis.

Dynamic macromolecular assemblies, such as ribosomes, viruses, and muscle protein complexes, are often more amenable to visualization by electron microscopy than by high-resolution X-ray crystallography or NMR. When high-resolution structures of component structures are available, it is possible to build an atomic model that gives information about the molecular interactions at greater detail than the experimental resolution, due to constraints of modeling placed upon the interpretation. There are now several competing computational methods to search systematically for orientations and positions of components that match the experimental image density, and continuing developments will be reviewed. Attention is now also moving toward the related task of optimization, with flexible and/or multifragment models and sometimes with stereochemically restrained refinement methods. This paper will review the various approaches and describe advances in the authors' methods and applications of real-space refinement.

In E. coli, the SecM nascent polypeptide causes elongation arrest, while interacting with 23S RNA bases A2058 and A749-753 in the exit tunnel of the large ribosomal subunit. We compared atomic models fitted by real-space refinement into cryo-electron microscopy reconstructions of a pretranslocational and SecM-stalled E. coli ribosome complex. A cascade of RNA rearrangements propagates from the exit tunnel throughout the large subunit, affecting intersubunit bridges and tRNA positions, which in turn reorient small subunit RNA elements. Elongation arrest could result from the inhibition of mRNA.(tRNAs) translocation, E site tRNA egress, and perhaps translation factor activation at the GTPase-associated center. Our study suggests that the specific secondary and tertiary arrangement of ribosomal RNA provides the basis for internal signal transduction within the ribosome. Thus, the ribosome may itself have the ability to regulate its progression through translation by modulating its structure and consequently its receptivity to activation by cofactors.

A new semi-empirical force field has been developed to describe hydrogen-bonding interactions with a directional component. The hydrogen bond potential supports two alternative target angles, motivated by the observation that carbonyl hydrogen bond acceptor angles have a bimodal distribution. It has been implemented as a module for a macromolecular refinement package to be combined with other force field terms in the stereochemically restrained refinement of macromolecules. The parameters for the hydrogen bond potential were optimized to best fit crystallographic data from a number of protein structures. Refinement of medium-resolution structures with this additional restraint leads to improved structure, reducing both the free R-factor and over-fitting. However, the improvement is seen only when stringent hydrogen bond selection criteria are used. These findings highlight common misconceptions about hydrogen bonding in proteins, and provide explanations for why the explicit hydrogen bonding terms of some popular force field sets are often best switched off.Keywords: Hydrogen bonds; crystallography; force field; hydrogen bond restraint Force fields are critical to molecular simulation in many aspects of life sciences research. Understanding, analyzing, and predicting three-dimensional structural models of molecular systems-including their conformations, binding affinities, and related properties-all depend on accurate atomic force fields. For this reason, there has been a great deal of effort devoted to the development and improvement of potential energy functions and their parameterization.The force fields commonly used for determining atom positions within a molecule use a combination of valence (or bonded) and nonbonded energy terms (Weiner and Kollman 1981;Brooks et al. 1983;Karplus 1987;Dinur and Hagler 1991). The overall potential energy of a molecular system may be written as E Empirical ‫ס‬ E bond + E angle + E dihedral + E vdw + E elec + E hb where the different energy terms are given by empirical formulae or harmonic functions penalizing deviations from ideal values. These values are determined from small-molecule crystallographic or spectroscopic data or from calibration to quantum mechanics calculations (Lifson and Stern 1982;Brooks et al. 1983;Nemethy et al. 1983;Hermans et al. 1984;Weiner et al. 1984Weiner et al. , 1986Nilsson and Karplus 1986;Dinur and Hagler 1991;Engh and Huber 1991). For macromolecular refinement with data from Xray crystallography, an additional term is added, E X-ray , which restrains the model against the diffraction data. The total potential energy is then E total ‫ס‬ E Empirical + w a E X-ray where w a is a weighting factor.Energy minimizations and dynamics simulations are often limited by inadequate description of the various force

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Current methods of free R factor cross-validation assume that the structure factors of the test and working sets are independent of one another. This assumption is only an approximation when the modeled structure occupies anything less than the full asymmetric unit. Through progressive elimination of reflections from the working set, starting with those expected to be most correlated to the test set, small biases in free R can be measured, presumably because of over-sampling of the Fourier transform owing to bulk solvent in the crystal. This level of bias may be of little practical importance, but it rises to significant levels with increasing non-crystallographic symmetry owing to wider correlations between structure factors than hitherto appreciated. In the presence of 15-fold non-crystallographic symmetry, with resolutions commonly attainable in macromolecular crystallography, it may not be possible to calculate an unbiased free R factor. Methods are developed for the calculation of reduced-bias free R factors through elimination of the strongest correlations between test and working sets. With 180-fold non-crystallographic symmetry they may not be an accurate indicator of absolute quality, but they do yield the correct optimal weighting for stereochemical restraints.

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