Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive.

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physio-logical temperatures would be a great benefit, is impaired.

C-phycocyanin as a highly attractive model system in protein crystallography: unique crystallization properties and packing-diversity screening.

The unique crystallization properties of the antenna protein C-phycocyanin (C-PC) from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed. C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives. Remarkably, the crystal dimensions vary from a few micrometres, as used in serial crystallography, to several hundred micrometres, with a very diverse crystal morphology.

Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology.

Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful.

Ultracompact 3D microfluidics for time-resolved structural biology.

To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s, which allows for the use of megahertz XFEL repetition rates.

Author Correction: Membrane protein megahertz crystallography at the European XFEL.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evaluation of serial crystallographic structure determination within megahertz pulse trains.

The new European X-ray Free-Electron Laser (European XFEL) is the first X-ray free-electron laser capable of delivering intense X-ray pulses with a megahertz interpulse spacing in a wavelength range suitable for atomic resolution structure determination. An outstanding but crucial question is whether the use of a pulse repetition rate nearly four orders of magnitude higher than previously possible results in unwanted structural changes due to either radiation damage or systematic effects on data quality. Here, separate structures from the first and subsequent pulses in the European XFEL pulse train were determined, showing that there is essentially no difference between structures determined from different pulses under currently available operating conditions at the European XFEL.

Membrane protein megahertz crystallography at the European XFEL.

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.

Mix-and-diffuse serial synchrotron crystallography.

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported.

Structure of a symmetric photosynthetic reaction center-photosystem.

Reaction centers are pigment-protein complexes that drive photosynthesis by converting light into chemical energy. It is believed that they arose once from a homodimeric protein. The symmetry of a homodimer is broken in heterodimeric reaction-center structures, such as those reported previously.

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.

Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from .

The gene coding for the aminoglycoside adenylyltransferase () from a clinical isolate of was cloned and expressed in strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits.

Purification and biochemical characterization of the ATP synthase from Heliobacterium modesticaldum.

Heliobacterium modesticaldum is an anaerobic photosynthetic bacterium that grows optimally at pH 6-7 and 52°C and is the only phototrophic member of the Firmicutes phylum family (gram-positive bacteria with low GC content). The ATP synthase of H. modesticaldum was isolated and characterized at the biochemical and biophysical levels.

Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center.

Heliobacteria contain a very simple photosynthetic apparatus, consisting of a homodimeric type I reaction center (RC) without a peripheral antenna system and using the unique pigment bacteriochlorophyll (BChl) g. They are thought to use a light-driven cyclic electron transport pathway to pump protons, and thereby phosphorylate ADP, although some of the details of this cycle are yet to be worked out. We previously reported that the fluorescence emission from the heliobacterial RC in vivo was increased by exposure to actinic light, although this variable fluorescence phenomenon exhibited very different characteristics to that in oxygenic phototrophs (Collins et al.

De novo design of an artificial bis[4Fe-4S] binding protein.

In nature, protein subunits containing multiple iron-sulfur clusters often mediate the delivery of reducing equivalents from metabolic pathways to the active site of redox proteins. The de novo design of redox active proteins should include the engineering of a conduit for the delivery of electrons to and from the active site, in which multiple redox active centers are arranged in a controlled manner. Here, we describe a designed three-helix protein, DSD-bis[4Fe-4S], that coordinates two iron-sulfur clusters within its hydrophobic core.

Purification of the photosynthetic reaction center from Heliobacterium modesticaldum.

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC-MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al.

Efficient light harvesting in a dark, hot, acidic environment: the structure and function of PSI-LHCI from Galdieria sulphuraria.

Photosystem I-light harvesting complex I (PSI-LHCI) was isolated from the thermoacidophilic red alga Galdieria sulphuraria, and its structure, composition, and light-harvesting function were characterized by electron microscopy, mass spectrometry, and ultrafast optical spectroscopy. The results show that Galdieria PSI is a monomer with core features similar to those of PSI from green algae, but with significant differences in shape and size. A comparison with the crystal structure of higher plant (pea) PSI-LHCI indicates that Galdieria PSI binds seven to nine light-harvesting proteins.