Stephan L. Ginell

From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal

We live in a macroscopic three-dimensional (3D) 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 a 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. 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: a motif that has a robust 3D structure, dominant affinity interactions between parts of the motif when it self-associates, and predictable structures for these affinity interactions. Fulfilling these three criteria to produce a 3D periodic system is not easy, but should readily be achieved with well-structured branched DNA motifs tailed by sticky ends. Complementary sticky ends associate with each other preferentially and assume the well-known B-DNA structure when they do so; the helically repeating nature of DNA facilitates the construction of a periodic array. It is essential that the directions of propagation associated with the sticky ends do 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. The data demonstrate clearly that it is possible to design and self-assemble a well-ordered macromolecular 3D crystalline lattice with precise control.

The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results

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.

The many faces of radiation-induced changes

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.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Å, 100K; 0.80 Å, 15K; 1.75 Å, 293K), neutron Laue data (2.2 Å, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy.

The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.

Non-classical transpeptidases yield insight into new antibacterials

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.

X-ray-induced deterioration of disulfide bridges at atomic resolution.

Overall and site-specific X-ray-induced damage to porcine pancreatic elastase was studied at atomic resolution at temperatures of 100 and 15 K. The experiments confirmed that irradiation causes small movements of protein domains and bound water molecules in protein crystals. These structural changes occur not only at 100 K but also at temperatures as low as 15 K. An investigation of the deterioration of disulfide bridges demonstrated the following. (i) A decrease in the occupancy of S(γ) atoms and the appearance of new cysteine rotamers occur simultaneously. (ii) The occupancy decrease is observed for all S(γ) atoms, while new rotamers arise for some of the cysteine residues; the appearance of new conformations correlates with the accessibility to solvent. (iii) The sum of the occupancies of the initial and new conformations of a cysteine residue is approximately equal to the occupancy of the second cysteine residue in the bridge. (iv) The most pronounced changes occur at doses below 1.4 × 10(7) Gy, with only small changes occurring at higher doses. Comparison of the radiation-induced changes in an elastase crystal at 100 and 15 K suggested that the dose needed to induce a similar level of deterioration of the disulfide bonds and atomic displacements at 15 K to those seen at 100 K is more than two times higher.

Temperature and cryoprotectant influence secondary quinone binding position in bacterial reaction centers

We have determined the first de novo position of the secondary quinone QB in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, QB occupies primarily the proximal binding site. In contrast, our room temperature structure showed that QB is largely in the distal position. Both data sets were collected in dark‐adapted conditions. We estimate that the occupancy of the QB site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far‐reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.

X-Ray-Radiation-Induced Cooperative Atomic Movements in Protein

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is. We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to approximately 100 K and diffracting to 1 A. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.

The absence of tertiary interactions in a self-assembled DNA crystal structure

DNA is a highly effective molecule for controlling nanometer‐scale structure. The convenience of using DNA lies in the programmability of Watson–Crick base‐paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky‐ended cohesion. Recently, the tensegrity triangle motif has been used to self‐assemble three‐dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine–PO4 tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation. Copyright