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A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.

ABSTRACT: Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.

SUBMITTER: de Wijn R

PROVIDER: S-EPMC6503916 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.

de Wijn Raphaël R Hennig Oliver O Roche Jennifer J Engilberge Sylvain S Rollet Kevin K Fernandez-Millan Pablo P Brillet Karl K Betat Heike H Mörl Mario M Roussel Alain A Girard Eric E Mueller-Dieckmann Christoph C Fox Gavin C GC Olieric Vincent V Gavira José A JA Lorber Bernard B Sauter Claude C

IUCrJ 20190419 Pt 3

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard te ...[more]

PMID: 31098026

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Project description: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.

Dataset Information

A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.

Publications

A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.

Similar Datasets

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