the-automated-crystallography-pipelines-at-embl-htx-facility

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble

EMBL Grenoble operates the High Throughput Crystallization Laboratory (HTX Lab), a large-scale user facility offering high throughput crystallography ...

European Molecular Biology Laboratory

Construct design and sample formulation are critical in structural biology projects. Large numbers of sample variants are often produced and analyzed for a single target and significant effort is dedicated to sample characterization in order to identify at an early stage the most promising samples to help save manpower and time. Here, we present a method based on a thermal stability assay that can help estimate the likelihood of biological samples to produce crystals. This assay is rapid, inexpensive and consumes very small amounts of sample. The results can be used to prioritize certain constructs at an early stage or as an objective test to help decide when to undertake other type of approaches addressed at improving sample properties. 

Estimation of crystallization likelihood through a fluorimetric thermal stability assay.

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities. 

Automated harvesting and processing of protein crystals through laser photoablation.

In eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied.

Article	Year
Estimation of crystallization likelihood through a fluorimetric thermal stability assay. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24203333	2014
Automated harvesting and processing of protein crystals through laser photoablation. Acta crystallographica. Section D, Structural biology\| PubMed ID: 27050125	2016
Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases. Microbial cell factories\| PubMed ID: 27894344	2016

Vincent Mariaule

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