Microseed Matrix Screening Research Articles

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

Structural and Functional Characterization of Three Novel Fungal Amylases with Enhanced Stability and pH Tolerance.

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.

Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68.

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

Human carbonic anhydrase IX (CA IX) is a multi-domain membrane protein that is therefore difficult to express or crystalize. To prepare crystals that are suitable for neutron studies, we are using only the catalytic domain of CA IX with six surface mutations, named surface variant (SV). The crystallization of CA IX SV, and also partly deuterated CA IX SV, was enabled by the use of microseed matrix screening (MMS). Only three drops with crystals were obtained after initial sparse matrix screening, and these were used as seeds in subsequent crystallization trials. Application of MMS, commercial screens, and refinement resulted in consistent crystallization and diffraction-quality crystals. The crystallization protocols and strategies that resulted in consistent crystallization are presented. These results demonstrate not only the use of MMS in the growth of large single crystals for neutron studies with defined conditions, but also that MMS enabled re-screening to find new conditions and consistent crystallization success.

Combining crystallogenesis methods to produce diffraction-quality crystals of a psychrophilic tRNA-maturation enzyme.

The determination of conditions for the reproducible growth of well diffracting crystals is a critical step in every biocrystallographic study. On the occasion of a new structural biology project, several advanced crystallogenesis approaches were tested in order to increase the success rate of crystallization. These methods included screening by microseed matrix screening, optimization by counter-diffusion and crystal detection by trace fluorescent labeling, and are easily accessible to any laboratory. Their combination proved to be particularly efficient in the case of the target, a 48 kDa CCA-adding enzyme from the psychrophilic bacterium Planococcus halocryophilus. A workflow summarizes the overall strategy, which led to the production of crystals that diffracted to better than 2 Å resolution and may be of general interest for a variety of applications.

Microseed matrix screening for crystallization: theory, practice and a new technique

Microseed matrix screening for crystallization: theory, practice and a new technique

A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

Microseed matrix screening for optimization in protein crystallization: what have we learned?

Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans.

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å(3) Da(-1) corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans.

"How to use ""random"" microseeding before you get your first crystals"

Random Microseed Matrix-Screening (rMMS), where seed crystals are added automatically to random crystallization screens, is a significant recent breakthrough in protein crystallization [1]. One industrial group used the method to solve 38 out of 70 structures generated in a three year period, finding particular success with antibody complexes [2]. rMMS not only produces more hits, it also generates better-diffracting crystals - because crystals are more likely to grow in the metastable zone. This assumes, however, that you have some initial crystal hits to make a seed stock from. This presentation will look at unusual methods of nucleation, including cross-seeding [3], heterogeneous nucleation, and nucleation with precipitants [4].

Protein crystallization with microseed matrix screening: application to human germline antibody Fabs.

The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Crystallization of a challenging antigen–antibody complex: TLR3 ECD with three noncompeting Fabs

The mechanism of action of therapeutic antibodies can be elucidated from the three-dimensional crystal structures of their complexes with antigens, but crystallization remains the primary bottleneck to structure determination. Methods that resulted in the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 with the three Fab fragments combined with microseed matrix screening and additive screening. Fine purification entailed the application of a very shallow gradient in anion-exchange chromatography, resulting in the resolution of two separate complex peaks which had different crystallizabilities. Subsequent structure determination defined the epitopes of the respective antibodies and revealed a mechanistic hypothesis that is currently under investigation. The results also showed that cocrystallization with multiple noncompeting Fab fragments can be a viable path when an antigen complex with a single Fab proves to be recalcitrant to crystallization.

Random microseeding: a theoretical and practical exploration of the microseed matrix-screening (MMS) method, with new recommendations for achieving crystallization success

Douglas Instruments has completed theoretical and practical studies of Microseed Matrix Screening (MMS), a powerful approach to protein crystallization, introduced by Ireton and Stoddard in 2004 [1]. The method was automated by D'Arcy et al. [2], who first used seeding with random screening kits. Experience shows that MMS with random screens generates more hits or better-diffracting crystals in about 75% of cases where at least one crystal can be obtained.

Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes

Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP(C)-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb_PrP_01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb_PrP_01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 Å, β = 101.96° and diffracted to 2.1 Å resolution using synchrotron radiation. Human PrP (90-231)/Nb_PrP_01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 Å, β = 96.23° and diffracted to 1.5 Å resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes.

Practical variations on the microseed matrix screening (MMS) method in protein crystallisation

Practical variations on the microseed matrix screening (MMS) method in protein crystallisation

Promoting crystallization of antibody–antigen complexesviamicroseed matrix screening

The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate.

Microseed matrix screening: a modified version

Microseed matrix screening: a modified version