Controlled dehydration of a ruthenium complex-DNA crystal induces reversible DNA kinking

Hydration-dependent DNA deformation has been known since Rosalind Franklin recognised that the relative humidity of the sample had to be maintained to observe a single conformation in DNA fibre diffraction. We now report for the first time the crystal structure, at the atomic level, of a dehydrated form of a DNA duplex and demonstrate the reversible interconversion to the hydrated form at room temperature. This system, containing d(TCGGCGCCGA) in the presence of Λ-[Ru(TAP)2(dppz)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyridophenazine), undergoes a partial transition from an A/B hybrid to the A-DNA conformation, at 84-79% relative humidity. This is accompanied by an increase in kink at the central step from 22° to 51°, with a large movement of the terminal bases forming the intercalation site. This transition is reversible on rehydration. Seven datasets, collected from one crystal at room temperature, show the consequences of dehydration at near-atomic resolution. This result highlights that crystals, traditionally thought of as static systems, are still dynamic and therefore can be the subject of further experimentation

X-ray scattering imaging of intervertebral discs under load

Background An improved understanding of intervertebral disc (IVD) structure and function is required for treatment development. Loading induces micro-fractures at the interface between the nucleus pulposus (NP) and the annulus fibrosus (AF), which is hypothesized to induce a cascade of cellular changes leading to degeneration. However, there is limited understanding of the structural relationship between the NP and AF at this interface and particularly response to load. Here, X-ray scattering is utilised to provide hierarchical morphometric information of collagen structure across the IVD, especially the interface region under load. Methodology IVDs were imaged using the I22 SAXS/WAXS beamline at Diamond Light Source. Peaks associated with the D-banded structure of collagen fibrils were fitted to quantify their azimuthal distribution, as well the magnitude and direction of internal strains under static and applied strain (0–20%). Results IVD tissue regions exhibited structural “AF-like” and “NP-like” fingerprints. Demonstrating high internal strains on collagen fibres particularly within the NP region of the disc. AF and NP regions showed distinct collagen orientation and internal strains with an apparent lack of bracing structure seen at the interface between the differential mechanical tissues. X-ray scattering under tensile strain provided structural information at high resolution, with clear differences observed between normal and degenerate discs under load. Conclusion X ray scattering has been utilised to develop an improved understanding of collagen structure across the intervertebral disc which can be utilised to gain an increased understanding of load induced propagation of micro fissures and disc degeneration

Example data set from Diamond Light Source VMXi beamline (Eiger 4M data, NeXus format)

<p>Data set recorded from Thermolysin crystal record <em>in situ</em> with Eiger 4M detector, to demonstrate file format used for this instrument at Diamond Light Source. Processing results using xia2 / DIALS:</p> <p> </p> <pre>For AUTOMATIC/DEFAULT/SAD Overall Low High High resolution limit 1.97 5.35 1.97 Low resolution limit 46.86 46.87 2.01 Completeness 59.8 73.2 6.0 Multiplicity 5.8 8.0 1.1 I/sigma 13.6 24.4 1.8 Rmerge(I) 0.072 0.050 0.308 Rmerge(I+/-) 0.067 0.048 0.000 Rmeas(I) 0.078 0.054 0.436 Rmeas(I+/-) 0.076 0.054 0.000 Rpim(I) 0.028 0.018 0.308 Rpim(I+/-) 0.035 0.023 0.000 CC half 0.997 0.998 0.450 Wilson B factor 13.401 Anomalous completeness 51.3 78.8 0.8 Anomalous multiplicity 3.3 4.8 1.0 Anomalous correlation 0.039 -0.006 0.000 Anomalous slope 0.987 dF/F 0.103 dI/s(dI) 1.086 Total observations 85069 8229 82 Total unique 14747 1031 73 Assuming spacegroup: P 6 2 2 Other likely alternatives are: P 61 2 2 P 65 2 2 P 62 2 2 P 64 2 2 P 63 2 2 Unit cell (with estimated std devs): 93.7184(3) 93.7184(3) 130.864(2) 90.0 90.0 120.0 </pre> <p> </p

Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering

Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D1/2 = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures

Room-Temperature Macromolecular Crystallography Using a Micro-Patterned Silicon Chip with Minimal Background Scattering

Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D1/2 = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures

Tropocollagen springs allow collagen fibrils to stretch elastically

The mechanical properties of connective tissues are tailored to their specific function, and changes can lead to dysfunction and pathology. In most mammalian tissues the mechanical environment is governed by the micro- and nano-scale structure of collagen and its interaction with other tissue components, however these hierarchical properties remain poorly understood. In this study we use the human cornea as a model system to characterise and quantify the dominant deformation mechanisms of connective tissue in response to cyclic loads of physiological magnitude. Synchronised biomechanical testing, x-ray scattering and 3D digital image correlation revealed the presence of two dominant mechanisms: collagen fibril elongation due to a largely elastic, spring-like straightening of tropocollagen supramolecular twist, and a more viscous straightening of fibril crimp that gradually increased over successive loading cycles. The distinct mechanical properties of the two mechanisms suggest they have separate roles in vivo. The elastic, spring-like mechanism is fast-acting and likely responds to stresses associated with the cardiac cycle, while the more viscous crimp mechanism will respond to slower processes, such as postural stresses. It is anticipated that these findings will have broad applicability to understanding the normal and pathological functioning of other connective tissues such as skin and blood vessels that exhibit both helical structures and crimp

Effects of mineralization on the hierarchical organization of collagen—a synchrotron X-ray scattering and polarized second harmonic generation study

The process of mineralization fundamentally alters collagenous tissue biomechanics. While the structure and organization of mineral particles have been widely studied, the impact of mineralization on collagen matrix structure, particularly at the molecular scale, requires further investigation. In this study, synchrotron X-ray scattering (XRD) and polarization-resolved second harmonic generation microscopy (pSHG) were used to study normally mineralizing turkey leg tendon in tissue zones representing different stages of mineralization. XRD data demonstrated statistically significant differences in collagen D-period, intermolecular spacing, fibril and molecular dispersion and relative supramolecular twists between non-mineralizing, early mineralizing and late mineralizing zones. pSHG analysis of the same tendon zones showed the degree of collagen fibril organization was significantly greater in early and late mineralizing zones compared to non-mineralizing zones. The combination of XRD and pSHG data provide new insights into hierarchical collagen–mineral interactions, notably concerning possible cleavage of intra- or interfibrillar bonds, occlusion and reorganization of collagen by mineral with time. The complementary application of XRD and fast, label-free and non-destructive pSHG optical measurements presents a pathway for future investigations into the dynamics of molecular scale changes in collagen in the presence of increasing mineral deposition