Dominik Oberthür

Serial crystallography on in vivo grown microcrystals using synchrotron radiation

The structure solution of T. brucei cathepsin B from 80 in vivo grown crystals with an average volume of 9 µm3 obtained by serial synchrotron crystallography at a microfocus beamline is reported.

Room-temperature macromolecular serial crystallography using synchrotron radiation

The room-temperature structure of lysozyme is determined using 40000 individual diffraction patterns from micro-crystals flowing in liquid suspension across a synchrotron microfocus beamline.

Macromolecular diffractive imaging using imperfect crystals

The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.

Crystal structure of a mirror-image L -RNA aptamer (Spiegelmer) in complex with the natural L- protein target CCL2

We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokines activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide·L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamers intramolecular contacts and permits a detailed analysis of its structure–function relationship. Furthermore, the analysis of the intermolecular drug–target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.

Modeling of oxidized PTH (oxPTH) and non-oxidized PTH (n-oxPTH) receptor binding and relationship of oxidized to non-oxidized PTH in children with chronic renal failure, adult patients on hemodialysis and kidney transplant recipients

Background: The biological properties of oxidized and non-oxidized PTH are substantially different. Oxidized PTH (oxPTH) loses its PTH receptor-stimulating properties, whereas non-oxidized PTH (n-oxPTH) is a full agonist of the receptor. This was described in more than 20 well published studies in the 1970s and 80s. However, PTH oxidation has been ignored during the development of PTH assays for clinical use so far. Even the nowadays used third generation assay systems do not consider oxidation of PTH We recently developed an assay to differentiate between oxPTH and n-oxPTH. In the current study we established normal values for this assay system. Furthermore, we compare the ratio of oxPTH to n-oxPTH in different population with chronic renal failure: 620 children with renal failure stage 2-4 of the 4C study, 342 adult patients on dialysis, and 602 kidney transplant recipients. In addition, we performed modeling of the interaction of either oxPTH or n-oxPTH with the PTH receptor using biophysical structure approaches. Results: The children had the highest mean as well as maximum n-oxPTH concentrations as compared to adult patients (both patients on dialysis as well as kidney transplant recipients). The relationship between oxPTH and n-oxPTH of individual patients varied substantially in all three populations with renal impairment. The analysis of n-oxPTH in 89 healthy control subjects revealed that n-oxPTH concentrations in patient with renal failure were higher as compared to healthy adult controls (2.25-fold in children with renal failure, 1.53-fold in adult patients on dialysis, and 1.56-fold in kidney transplant recipients, respectively). Computer assisted biophysical structure modeling demonstrated, however, minor sterical- and/or electrostatic changes in oxPTH and n-oxPTH. This indicated that PTH oxidation may induce refolding of PTH and hence alters PTH-PTH receptor interaction via oxidation induced three-dimensional structure alteration of PTH. Conclusion: A huge proportion of circulating PTH measured by current state-of-the-art assay systems is oxidized and thus not biologically active. The relationship between oxPTH and n-oxPTH of individual patients varied substantially. Non-oxidized PTH concentrations are 1.5 - 2.25 fold higher in patients with renal failure as compared to health controls. Measurements of n-oxPTH may reflect the hormone status more precise. The iPTH measures describes most likely oxidative stress in patients with renal failure rather than the PTH hormone status. This, however, needs to be demonstrated in further clinical studies.

Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm

It is well known that most proteins and many other biomolecules fluoresce when illuminated with UV radiation, but it is also commonly accepted that utilizing this property to detect protein crystals in crystallization setups is limited by the opacity of the materials used to contain and seal them. For proteins, this fluorescence property arises primarily from the presence of tryptophan residues in the sequence. Studies of protein crystallization results in a variety of setup configurations show that the opacity of the containment hardware can be overcome at longer excitation wavelengths, where typical hardware materials are more transparent in the UV, by the use of a powerful UV-light source that is effective in excitation even though not at the maximum of the excitation response. The results show that under these circumstances UV evaluation of crystallization trials and detection of biomolecular crystals in them is not limited by the hardware used. It is similarly true that a deficiency in tryptophan or another fluorescent component that limits the use of UV light for these purposes can be effectively overcome by the addition of fluorescent prostheses that bind to the biomolecule under study. The measurements for these studies were made with a device consisting of a potent UV-light source and a detection system specially adapted (i) to be tunable via a motorized and software-controlled absorption-filter system and (ii) to convey the excitation light to the droplet or capillary hosting the crystallization experiment by quartz-fibre light guides.

Structural enzymology using X-ray free electron lasers

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Design and application of a microfluidic device for protein crystallization using an evaporation-based crystallization technique

A new crystallization system is described, which makes it possible to use an evaporation-based microfluidic crystallization technique for protein crystallization. The gas and water permeability of the used polydimethylsiloxane (PDMS) material enables evaporation of the protein solution in the microfluidic device. The rates of evaporation are controlled by the relative humidity conditions, which are adjusted in a precise and stable way by using saturated solutions of different reagents. The protein crystals could nucleate and grow under different relative humidity conditions. Using this method, crystal growth could be improved so that approximately 1 mm-sized lysozyme crystals were obtained more successfully than using standard methods. The largest lysozyme crystal obtained reached 1.57 mm in size. The disadvantage of the good gas permeability in PDMS microfluidic devices becomes an advantage for protein crystallization. The radius distributions of aggregrates in the solutions inside the described microfluidic devices were derived from in situ dynamic light scattering measurements. The experiments showed that the environment inside of the microfluidic device is more stable than that of conventional crystallization techniques. However, the morphological results showed that the protein crystals grown in the microfluidic device could lose their morphological stability. Air bubbles in microfluidic devices play an important role in the evaporation progress. A model was constructed to analyze the relationship of the rates of evaporation and the growth of air bubbles to the relative humidity.

Effects of self-seeding and crystal post-selection on the quality of Monte Carlo-integrated SFX data

Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected in this way are then combined in a scheme called Monte Carlo integration, which provides the full diffraction intensities. However, apart from having to perform this merging, the Monte Carlo integration must also average out all variations in crystal quality, crystal size, X-ray beam properties and other factors, necessitating data collection from thousands of crystals. Because the pulses provided by FELs running in the typical self-amplified spontaneous emission (SASE) mode of operation have very irregular, spiky spectra that vary strongly from pulse to pulse, it has been suggested that this is an important source of variation contributing to inaccuracies in the intensities, and that, by using monochromatic pulses produced through a process called self-seeding, fewer images might be needed for Monte Carlo integration to converge, resulting in more accurate data. This paper reports the results of two experiments performed at the Linac Coherent Light Source in which data collected in both SASE and self-seeded mode were compared. Importantly, no improvement attributable to the use of self-seeding was detected. In addition, other possible sources of variation that affect SFX data quality were investigated, such as crystal-to-crystal variations reflected in the unit-cell parameters; however, these factors were found to have no influence on data quality either. Possibly, there is another source of variation as yet undetected that affects SFX data quality much more than any of the factors investigated here.

Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse

Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.