Search

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as beta-D-2'-O,4'-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The beta-D-2'-O,4'-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability. A 7-mer helix derived from the terminal part of an aptamer that was targeted against ricin was chosen. The ricin aptamer originally consisted of natural RNA building blocks and showed high affinity in ricin binding. For future stabilization of the aptamer, the terminal helix has been constructed as an ;all-locked' LNA and was successfully crystallized in order to investigate its structural properties. Optimization of crystal growth succeeded by the use of different metal salts as additives, such as CuCl(2), MgCl(2), MnCl(2), CaCl(2), CoCl(2) and ZnSO(4). Preliminary X-ray diffraction data were collected and processed to 2.8 A resolution. The LNA crystallized in space group P6(5), with unit-cell parameters a = 50.11, b = 50.11, c = 40.72 A. The crystals contained one LNA helix per asymmetric unit with a Matthews coefficient of 3.17 A(3) Da(-1), which implies a solvent content of 70.15%.

The aminoacylation of tRNA is a crucial step in cellular protein biosynthesis. Recognition of the cognate tRNA by the correct aminoacyl-tRNA synthetase is ensured by tRNA identity elements. In tRNA(Arg), the identity elements consist of the anticodon, parts of the D-loop and the discriminator base. The minor groove of the aminoacyl stem interacts with the arginyl-tRNA synthetase. As a consequence of the redundancy of the genetic code, six tRNA(Arg) isoacceptors exist. In the present work, three different Escherichia coli tRNA(Arg) acceptor-stem helices were crystallized. Two of them, the tRNA(Arg) microhelices RR-1660 and RR-1662, were examined by X-ray diffraction analysis and diffracted to 1.7 and 1.8 A resolution, respectively. The tRNA(Arg) RR-1660 helix crystallized in space group P1, with unit-cell parameters a = 26.28, b = 28.92, c = 29.00 A, alpha = 105.74, beta = 99.01, gamma = 97.44 degrees , whereas the tRNA(Arg) RR-1662 helix crystallized in space group C2, with unit-cell parameters a = 33.18, b = 46.16, c = 26.04 A, beta = 101.50 degrees .

Interest has been focused on comparative X-ray structure analyses of different tRNA(Gly) acceptor-stem helices. tRNA(Gly)/glycyl-tRNA synthetase belongs to the so-called class II system, in which the tRNA identity elements consist of simple and unique determinants that are located in the tRNA acceptor stem and the discriminator base. Comparative structure investigations of tRNA(Gly) microhelices provide insight into the role of tRNA identity elements. Predominant differences in the structures of glycyl-tRNA synthetases and in the tRNA identity elements between prokaryotes and eukaryotes point to divergence during the evolutionary process. Here, the crystallization and high-resolution X-ray diffraction analysis of a human tRNA(Gly) acceptor-stem microhelix with sequence 5'-G(1)C(2)A(3)U(4)U(5)G(6)G(7)-3', 5'-C(66)C(67)A(68)A(69)U(70)G(71)C(72)-3' is reported. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 37.32, b = 37.61, c = 30.47 A, beta = 112.60 degrees and one molecule per asymmetric unit. A data set was collected using synchrotron radiation and data were processed within the resolution range 50.0-1.18 A. The structure was solved by molecular replacement.

Chemically synthesized RNAs with the unnatural L-configuration possess enhanced in vivo stability and nuclease resistance, which is a highly desirable property for pharmacological applications. For a structural comparison, both L- and D-RNA oligonucleotides of a shortened Thermus flavus 5S rRNA A-helix were chemically synthesized. The enantiomeric RNA duplexes were stochiometrically cocrystallized as a racemate, which enabled analysis of the D- and L-RNA enantiomers in the same crystals. In addition to a biochemical investigation, diffraction data were collected to 3.0 A resolution using synchrotron radiation. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 35.59, c = 135.30 A, gamma = 120 degrees and two molecules per asymmetric unit.

The digestive fluid of the marine crab Cancer pagurus (Decapoda, Brachyura) contains highly stable proteases which display enhanced activity in aqueous mixtures of organic solvents. Three trypsins were isolated from the gastric fluid and two of them, C.p.TryII and C.p.TryIII, were purified to homogeneity by anion-exchange chromatography and crystallized by hanging-drop vapour diffusion. Diffraction data were collected at a synchrotron to 0.97 and 3.2 A resolution, respectively. The crystal of C.p.TryII belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 52.06, b = 62.00, c = 71.66 A. Based on the Matthews coefficient, one protein molecule per asymmetric unit is suggested. In contrast, crystals of C.p.TryIII, which belong to the cubic space group P2(1)3 with unit-cell parameters a = b = c = 215.4 A, are assumed to contain 12 molecules per asymmetric unit.

The tRNA(Gly) and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNA(Gly). To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNA(Gly) acceptor-stem helix was crystallized. Data were collected to 2.0 A resolution using synchrotron radiation. Crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 35.35, c = 130.82 A, alpha = beta = 90, gamma = 120 degrees and two molecules in the asymmetric unit.

Modified nucleic acids are of great interest with respect to their nuclease resistance and enhanced thermostability. In therapeutical and diagnostic applications, such molecules can substitute for labile natural nucleic acids that are targeted against particular diseases or applied in gene therapy. The so-called `locked nucleic acids' contain modified sugar moieties such as 2′- O,4′- C-methylene-bridged β- d-ribofuranose and are known to be very stable nucleic acid derivatives. The structure of locked nucleic acids in single or multiple LNA-substituted natural nucleic acids and in LNA-DNA or LNA-RNA heteroduplexes has been well investigated, but the X-ray structure of an `all-locked' nucleic acid double helix has not been described to date. Here, the crystallization and X-ray diffraction data analysis of an `all-locked' nucleic acid helix, which was designed as an LNA originating from a tRNASer microhelix RNA structure, is presented. The crystals belonged to space group C2, with unit-cell parameters a = 77.91, b = 40.74, c = 30.06 Å, β = 91.02°. A high-resolution and a low-resolution data set were recorded, with the high-resolution data showing diffraction to 1.9 Å resolution. The crystals contained two double helices per asymmetric unit, with a Matthews coefficient of 2.48 Å3 Da−1 and a solvent content of 66.49% for the merged data. [ABSTRACT FROM AUTHOR]

The aminoacylation of tRNA is a crucial step in cellular protein biosynthesis. Recognition of the cognate tRNA by the correct aminoacyl-tRNA synthetase is ensured by tRNA identity elements. In tRNAArg, the identity elements consist of the anticodon, parts of the D-loop and the discriminator base. The minor groove of the aminoacyl stem interacts with the arginyl-tRNA synthetase. As a consequence of the redundancy of the genetic code, six tRNAArg isoacceptors exist. In the present work, three different Escherichia coli tRNAArg acceptor-stem helices were crystallized. Two of them, the tRNAArg microhelices RR-1660 and RR-1662, were examined by X-ray diffraction analysis and diffracted to 1.7 and 1.8 Å resolution, respectively. The tRNAArg RR-1660 helix crystallized in space group P1, with unit-cell parameters a = 26.28, b = 28.92, c = 29.00 Å, α = 105.74, β = 99.01, γ = 97.44°, whereas the tRNAArg RR-1662 helix crystallized in space group C2, with unit-cell parameters a = 33.18, b = 46.16, c = 26.04 Å, β = 101.50°. [ABSTRACT FROM AUTHOR]

The major dissimilarities between the eukaryotic/archaebacterial-type and eubacterial-type glycyl-tRNA synthetase systems (GlyRS; class II aminoacyl-tRNA synthetases) represent an intriguing example of evolutionarily divergent solutions to similar biological functions. The differences in the identity elements of the respective tRNAGly systems are located within the acceptor stem and include the discriminator base U73. In the present work, the human tRNAGly acceptor-stem microhelix was crystallized in an attempt to analyze the structural features that govern the correct recognition of tRNAGly by the eukaryotic/archaebacterial-type glycyl-tRNA synthetase. The crystals of the human tRNAGly acceptor-stem helix belong to the monoclinic space group C2, with unit-cell parameters a = 37.12, b = 37.49, c = 30.38 Å, α = γ = 90, β = 113.02°, and contain one molecule per asymmetric unit. A high-resolution data set was acquired using synchrotron radiation and the data were processed to 1.2 Å resolution. [ABSTRACT FROM AUTHOR]

The pharmacokinetic properties of an aptamer against the tumour-marker protein tenascin-C have recently been successfully improved by the introduction of locked nucleic acids (LNAs) into the terminal stem of the aptamer. Since it is believed that this post-SELEX optimization is likely to provide a more general route to enhance the in vitro and in vivo stability of aptamers, elucidation of the structural basis of this improvement was embarked upon. Here, the crystallographic and X-ray diffraction data of the isolated aptamer stem encompassed in a six-base-pair duplex both with and without the LNA modification are presented. The obtained all-LNA crystals belong to space group P41212 or P43212, with unit-cell parameters a = b = 52.80, c = 62.83 Å; the all-RNA crystals belong to space group R32, with unit-cell parameters a = b = 45.21, c = 186.97 Å, γ = 120.00°. [ABSTRACT FROM AUTHOR]

ThiM [5-(hydroxyethyl)-4-methylthiazole kinase; EC 2.7.1.50] from Staphylococcus aureus is an essential enzyme of thiamine or vitamin B1 metabolism and has been crystallized by the vapour-diffusion method. The crystals belonged to the primitive space group P1, with unit-cell parameters a = 62.06, b = 62.40, c = 107.82 Å, α = 92.25, β = 91.37, γ = 101.48° and six protomers in the unit cell, corresponding to a packing parameter VM of 2.3 Å3 Da−1. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The phase problem was solved by molecular replacement. [ABSTRACT FROM AUTHOR]

Thiaminase type II (TenA) catalyzes the deamination of aminopyrimidines, including the cleavage of thiamine to 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole in the metabolism of thiamine (vitamin B1), in Staphylococcus aureus ( Sa). SaTenA was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 2.6 Å resolution usng synchrotron radiation. The crystal is orthorhombic, belonging to space group P212121 with unit-cell parameters a = 103.5, b = 104.1, c = 109.6 Å. With four molecules in the asymmetric unit, the Matthews coefficient is 2.85 Å3 Da−1. Initial attempts to solve the structure by molecular-replacement techniques were successful. [ABSTRACT FROM AUTHOR]

The successful crystallization of proteins is important because their molecular three-dimensional structures can be obtained through X-ray diffraction when in their crystalline form. Investigation of the crystallization process is beneficial for this purpose. We have reported that protein crystallization is sensitive to audible sound, which is commonly present but is often ignored. Here we investigate the effect of audible sound parameters, especially frequency, on a protein crystallization. We show a significant facilitation of protein crystallization using 5000 Hz audible sound, possible mechanism was also tried to be clarified. Suitably controlled audible sound can be beneficial for promoting protein crystallization. Therefore, audible sound can be used as a simple tool to promote protein crystallization. In addition, the processing of other types of materials may also be affected by audible sound. [ABSTRACT FROM AUTHOR]