An automatic sample dispenser has been constructed to aid with protein crystallization trials. This dispenser contains a bank of Hamilton syringes driven by stepper motors under computer control which is used to set up small samples (2 μl or less) for batch crystallization. Software has been written to create a series of trials which form a two‐dimensional array of crystallization conditions. A specially designed fluoropolymer multibore microtip allows the very small volumes to be mixed and dispensed with great accuracy.
The field of Membrane Protein Structural Biology has grown significantly since its first landmark in 1985 with the first three-dimensional atomic resolution structure of a membrane protein. Nearly twenty-six years later, the crystal structure of the beta2 adrenergic receptor in complex with G protein has contributed to another landmark in the field leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane protein structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration and third-generation synchrotrons, has enhanced membrane protein structure determination rate. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins, and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies amazingly remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible. In this review we draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition we also highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.
Influenza A and B, and many unrelated viruses including rhinovirus, RSV, adenovirus, metapneumovirus and coronavirus share the same seasonality, since these viral acute respiratory tract infections (vARIs) are much more common in winter than summer. Unfortunately, early investigations that used recycled "pedigree" virus strains seem to have led microbiologists to dismiss the common folk belief that vARIs often follow chilling. Today, incontrovertible evidence shows that ambient temperature dips and host chilling increase the incidence and severity of vARIs. This review considers four possible mechanisms, M1 - 4, that can explain this link: (M1) increased crowding in winter may enhance viral transmission; (M2) lower temperatures may increase the stability of virions outside the body; (M3) chilling may increase host susceptibility; (M4) lower temperatures or host chilling may activate dormant virions. There is little evidence for M1 or M2, which are incompatible with tropical observations. Epidemiological anomalies such as the repeated simultaneous arrival of vARIs over wide geographical areas, the rapid cessation of influenza epidemics, and the low attack rate of influenza within families are compatible with M4, but not M3 (in its simple form). M4 seems to be the main driver of seasonality, but M3 may also play an important role.
Microseed matrix-screening combined with random screens (rMMS) is a significant recent breakthrough in protein crystallization. In this study, a very reproducible assay for crystal seeds was set up that allowed the following recommendations to be made: (1) the suitability of a solution for suspending seed crystals can be predicted by incubating (uncrushed) crystals in it for one day and observing crystal stability. (2) For routine rMMS, seed crystals should be suspended in the crystallization cocktail that gave the original crystals. (3) Seed crystals can be suspended in PEG or NaCl solutions to reduce the prevalence of salt crystals. (4) Protein complexes can be seeded with seed crystals suspended in PEG. If necessary, seed crystals can also be suspended in the original crystallization cocktail with any individual ingredients that destabilize the complex removed. ( 5) "Preseeding" of the protein stock should not be used if rMMS is available, because it is less effective. ( 6) Seed crystals can be harvested from microfluidic devices. (7) Heterogeneous nucleants and cross-seeding are less effective than rMMS, but they can be used if seed crystals cannot be obtained. A theoretical case and practical suggestions are also put forward for producing crystals with different space groups.
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