We live in a macroscopic three-dimensional world, but our best description of the structure of matter is at the atomic and molecular scale. Understanding the relationship between the two scales requires that we bridge from the molecular world to the macroscopic world. Connecting these two domains with atomic precision is a central goal of the natural sciences, but it requires high spatial control of the 3D structure of matter.1 The simplest practical route to producing precisely designed 3D macroscopic objects is to form a crystalline arrangement by self-assembly, because such a periodic array has only conceptually simple requirements: [1] A motif whose 3D structure is robust, [2] dominant affinity interactions between parts of the motif when it self-associates, and [3] a predictable structures for these affinity interactions. Fulfilling all these criteria to produce a 3D periodic system is not easy, but it should readily be achieved by well-structured branched DNA motifs tailed by sticky ends.2 Complementary sticky ends associate with each other preferentially and assume the well-known B-DNA structure when they do so;3 the helically repeating nature of DNA facilitates the construction of a periodic array. It is key that the directions of propagation associated with the sticky ends not share the same plane, but extend to form a 3D arrangement of matter. Here, we report the crystal structure at 4 Å resolution of a designed, self-assembled, 3D crystal based on the DNA tensegrity triangle.4 The data demonstrate clearly that it is possible to design and self-assemble a well-ordered macromolecular 3D crystalline lattice with precise control.

Prostaglandin H synthase-1 and -2 (PGHS-1 and -2) catalyze the committed step in prostaglandin synthesis and are targets for nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin. We have determined the structure of PGHS-1 at 3 angstrom resolution with arachidonic acid (AA) bound in a chemically productive conformation. The fatty acid adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstraction by tyrosine-385, the likely radical donor. A space also exists for oxygen addition on the antarafacial surface of the carbon in the 11-position (C-11). While this conformation allows endoperoxide formation between C-11 and C-9, it also implies that a subsequent conformational rearrangement must occur to allow formation of the C-8/C-12 bond and to position C-15 for attack by a second molecule of oxygen.

Bacterial survival requires an intact peptidoglycan layer, a 3-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by d,d-transpeptidases, enzymes that are inhibited by the β-lactams which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of β-lactams is distinctly effective not only because they inhibit d,d-transpeptidases and are poor substrates for β-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the l,d-transpeptidases, that generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of l,d-transpeptidases of M. tuberculosis and a range of bacteria, including ESKAPE pathogens, and utilized this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5-0.6 A wavelength) with fluxes up to 8-18 x 10(12) photons s(-1) (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-maximum) at the sample position can be varied from 2.2 mm x 1.0 mm (horizontal x vertical, unfocused) to 0.083 mm x 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a kappa-geometry goniometer and beamline control software allows optimal strategies to be adopted in protein crystallographic experiments, thus maximizing the chances of their success. A large-area mosaic 3 x 3 CCD detector allows high-quality diffraction data to be measured rapidly to the crystal diffraction limits. The beamline layout and the X-ray optical and endstation components are described in detail, and the results of representative crystallographic experiments are presented.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-␥-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall Lshaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 Å. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V max /K m for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the -half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40-and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.

During diffraction experiments even cryo-cooled protein crystals can be significantly damaged due to chemical and physical changes induced by absorbed X-ray photons. The character and scale of the observed effects depend strongly on the temperature and the composition of crystals. The absorption of radiation energy results in incremental regular changes to the crystal structure, making its impact on the process of solving the structure strongly correlated with other experimental variables. An understanding of all the dependencies is still limited and does not allow for a precise prediction of the outcome of a particular diffraction experiment. Results are presented of diffraction experiments performed under different experimental conditions. The influence of temperature and crystal composition on different characteristics of radiation damage is analyzed. The observed effects are discussed in terms of their impact on data processing and phasing procedures.

Aldehyde reductase, a member of the aldo-keto reductase superfamily, catalyzes the NADPH-dependent reduction of a variety of aldehydes to their corresponding alcohols. The structure of porcine aldehyde reductase-NADPH binary complex has been determined by x-ray diffraction methods and refined to a crystallographic R-factor of 0.20 at 2.4 A resolution. The tertiary structure of aldehyde reductase is similar to that of aldose reductase and consists of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. Unlike aldose reductase, the N epsilon 2 of the imidazole ring of His 113 in aldehyde reductase interacts, through a hydrogen bond, with the amide group of the nicotinamide ring of NADPH.

As a growing number of clinical isolates of are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of are synthesized by nonclassical transpeptidases, namely, the l,d-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of β-lactams determines their activity against Here, we studied the interactions of β-lactams with two l,d-transpeptidases in, namely, Ldt and Ldt, and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of β-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with β-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible and clinical isolates that are resistant to most antibiotics, which suggests that dual-β-lactam therapy has potential for the treatment of Finally, we solved the first crystal structure of an l,d-transpeptidase, Ldt, and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and β-lactams, which provide an insight into the molecular basis for the relative efficacy of different β-lactams against .