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The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals.

ABSTRACT: In recent years, nano and microcrystals have emerged as a valuable source of high-resolution structural information owing to the invention of serial femtosecond crystallography (SFX) with X-ray free electron lasers and microcrystal electron diffraction (MicroED) using electron cryomicroscopes. Once considered useless for structure determination, nano/microcrystals now confer significant advantages for static and time-resolved structure determination from a wide variety of difficult-to-study targets. MicroED has been used to obtain sub-Ångstrom resolution maps in which hydrogen atoms can be clearly resolved from only a few nano/microcrystals, while SFX has been used to probe protein dynamics following reaction initiation on time scales from femtoseconds to minutes. We review these two complementary techniques and their abilities for high-resolution structure determination.

SUBMITTER: Zatsepin NA

PROVIDER: S-EPMC6778504 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals.

Zatsepin Nadia A NA Li Chufeng C Colasurd Paige P Nannenga Brent L BL

Current opinion in structural biology 20190722

In recent years, nano and microcrystals have emerged as a valuable source of high-resolution structural information owing to the invention of serial femtosecond crystallography (SFX) with X-ray free electron lasers and microcrystal electron diffraction (MicroED) using electron cryomicroscopes. Once considered useless for structure determination, nano/microcrystals now confer significant advantages for static and time-resolved structure determination from a wide variety of difficult-to-study targ ...[more]

PMID: 31345629

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Project description:The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

Project description:Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

Dataset Information

The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals.

Publications

The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals.

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