Search

We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events. The authors introduce the spitrobot, a crystal plunger, enabling cryo-trapping with millisecond time-resolution via the liquid application method (LAMA). Ligand binding and reaction intermediates are demonstrated in three different enzymes. [ABSTRACT FROM AUTHOR]

Fixed‐target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X‐ray free‐electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high‐throughput experiments, which depend on high‐quality protein microcrystals. The batch crystallization procedures that are typically applied require time‐ and sample‐intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor‐diffusion conditions to in‐chip crystallization. Based on conventional hanging‐drop vapor‐diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high‐quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively unexplored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in‐chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal‐containing cells. These proof‐of‐principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens. [ABSTRACT FROM AUTHOR]

ID30A‐3 (or MASSIF‐3) is a mini‐focus (beam size 18 µm × 14 µm) highly intense (2.0 × 1013 photons s−1), fixed‐energy (12.81 keV) beamline for macromolecular crystallography (MX) experiments at the European Synchrotron Radiation Facility (ESRF). MASSIF‐3 is one of two fixed‐energy beamlines sited on the first branch of the canted undulator setup on the ESRF ID30 port and is equipped with a MD2 micro‐diffractometer, a Flex HCD sample changer, and an Eiger X 4M fast hybrid photon‐counting detector. MASSIF‐3 is recommended for collecting diffraction data from single small crystals (≤15 µm in one dimension) or for experiments using serial methods. The end‐station has been in full user operation since December 2014, and here its current characteristics and capabilities are described. [ABSTRACT FROM AUTHOR]

Since the first crystal structures were determined by the Braggs just over 100 years ago, crystallography has rapidly evolved in parallel with developments in photon, neutron, and electron sources to become the “go-to” technique for structure-function studies. It can provide atomic resolution structural information on an entire three-dimensional molecular or crystalline structure and is used from the study of bulk phase materials through to individual protein molecules [1, 2]. [ABSTRACT FROM PUBLISHER]

The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for `spectroscopy'). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed. [ABSTRACT FROM AUTHOR]