Cheng, Robert K.Y.
The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.
Hennig Michael, Rufer Arne
Chapter 7 in: Structural Biology in Drug Discovery: Methods, Techniques and Practices, p. 143-164, 2020
Along the entire value creation chain of drug discovery, biophysical methods are used to reveal details on the molecular properties of a target protein and its complexes with endogenous and pharmaceutical ligands. The ultimate goal of biophysical characterization of proteins with respect to drug design is their crystallization and the subsequent elucidation of the three-dimensional structure of the target/drug complex at atomic resolution. This chapter provides the example of the monotopic membrane protein carnitine palmitoyl transferase 2 from rat (CPT-2), which was used in the course of a drug discovery program to implement methods for the characterization of protein/detergent complexes. Continuous advancement of dedicated analysis software makes analytical ultracentrifugation a promising method for the purpose of characterizing protein-protein interactions. The chapter also presents a case study on minimizing macro-inhomogeneity.
In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A
Robert Cheng, Chia-Ying Huang, Michael Hennig, Herbert Nar and Gisela Schnapp
The in meso in situ serial X-ray crystallization method (Huang et al., (2015) Acta Crystallogr D Biol Crystallogr 71, 1238) combines lipid cubic phase crystallization, direct freezing of the crystallization droplet without handling of the crystals, and data collection in situ. Recently, this method was used to overcome the mechanical fragility of crystals which enabled the X-ray structure determination of chemokine receptor 2A (Apel et al., (2019) Structure 27, 427) at 2.7 Å resolution. The CCR2 structure provides the structural basis for ligand selectivity of CCR2 against chemokine receptor 5 and provides insights into the residence time of MK-0812 analogs based on molecular dynamics simulations. These findings offer new opportunities for drug discovery targeting chemokine receptors.
Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists
We determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å. An N- and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E2917.39 in high-affinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H1213.33, resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.
Interview of leadXpro's CEO Michael Hennig by the journal EuropeanCEO
Our CEO was interviewed by the journal “European CEO“. In the Summer edition of 2018 a number of emerging CEOs from Europe based companies are introduced. Michael Hennig’s interview “Taking the lead” emphasizes facts about the core business of leadXpro, the impact of membrane proteins as drug targets and the key advantages leadXpro’s technologies provide to drug discovery and the creation of differentiating medicines.
Read the article here
Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons
T. Weinert, N. Olieric, R. Cheng, S. Brünle et al.
Nature Communications 8:542 (2017)
The authors show that serial millisecondcrystallography (SMX) at synchrotrons allows fast, straightforward structure determination at room-temperature for large soluble macromolecular complexes as well as membrane proteins. Using SMX, a much larger dose can be distributed over many crystals, resulting in higher resolution structures with less-radiation damage compared with classical room-temperature methods. Modern fast frame rate detectors produce results of excellent quality, making even native SAD phasing possible in less than a single 8-h synchrotron shift. Using ultra high frame rate detectors and next-generation diffraction-limited sources, time-resolved measurements in the micro- and perhaps nanosecond range may become possible at synchrotrons. Given the comparatively simple sample preparation, data collection, data processing and its great potential for automation, the authors believe that SMX is the method of choice for room-temperature structure determination and fragment screening approaches.
X-ray Free Electron Laser: Opportunities for drug discovery
Cheng R., Abela R., Hennig M.
The authors demonstrate that XFEL structure determination has matured to reality and point out the many advantages of the technology: Unmet brilliance and focus, femtosecond pulses and low/no radiation damage and measurement at room temperature allow structure determinations of challenging systems like membrane proteins, investigation of time-resolved ligand binding, new structural insights due to room temperature and full automation of crystal diffraction experiments.
Structural biology: doors open at the European XFEL
A technology feature by Nature Methods’ technology editor Vivien Marx about free electron lasers to comment on the opening of the European X-ray Free Electron Laser (EuXFEL) in Hamburg, Germany. One paragraph of the article refers to the SwissFEL (opened in December 2016) and the applied research at this instrument that will be facilitated by leadXpro AG.