Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel
(Impact Factor: 12.88).
11/2013; 7(12). DOI: 10.1021/nn402515q
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these 'difficult to crystallize' proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is setup separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop the crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.
Available from: Raimund Fromme
[Show abstract] [Hide abstract]
ABSTRACT: Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.
Philosophical Transactions of The Royal Society B Biological Sciences 07/2014; 369(1647). DOI:10.1098/rstb.2013.0316 · 7.06 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The maximum thickness permissible within the single-scattering approximation for the determination of the structure of perfectly ordered protein microcrystals by transmission electron diffraction is estimated for tetragonal hen-egg lysozyme protein crystals using several approaches. Multislice simulations are performed for many diffraction conditions and beam energies to determine the validity domain of the required single-scattering approximation and hence the limit on crystal thickness. The effects of erroneous experimental structure factor amplitudes on the charge density map for lysozyme are noted and their threshold limits calculated. The maximum thickness of lysozyme permissible under the single-scattering approximation is also estimated using R-factor analysis. Successful reconstruction of density maps is found to result mainly from the use of the phase information provided by modeling based on the protein data base through molecular replacement (MR), which dominates the effect of poor quality electron diffraction data at thicknesses larger than about 200 Å. For perfectly ordered protein nanocrystals, a maximum thickness of about 1000 Å is predicted at 200 keV if MR can be used, using R-factor analysis performed over a subset of the simulated diffracted beams. The effects of crystal bending, mosaicity (which has recently been directly imaged by cryo-EM) and secondary scattering are discussed. Structure-independent tests for single-scattering and new microfluidic methods for growing and sorting nanocrystals by size are reviewed.
Ultramicroscopy 08/2014; 148(S3). DOI:10.1017/S1431927614007946 · 2.44 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The use of X-ray crystallography for the structure determination of biological macromolecules has experienced a steady expansion over the last 20 years with the Protein Data Bank growing from <1000 deposited structures in 1992 to >100 000 in 2014. The large number of structures determined each year not only reflects the impact of X-ray crystallography on many disciplines in the biological and medical fields but also its accessibility to non-expert laboratories. Thus protein crystallography is now largely a mainstream research technique and is routinely integrated in high-throughput pipelines such as structural genomics projects and structure-based drug design. Yet, significant frontiers remain that continuously require methodological developments. In particular, membrane proteins, large assemblies, and proteins from scarce natural sources still represent challenging targets for which obtaining the large diffracting crystals required for classical crystallography is often difficult. These limitations have fostered the emergence of microcrystallography, novel approaches in structural biology that collectively aim at determining structures from the smallest crystals. Here, we review the state of the art of macromolecular microcrystallography and recent progress achieved in this field.
Australian Journal of Chemistry 10/2014; 67(12):1793-1806. DOI:10.1071/CH14455 · 1.56 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.