Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.

Crystallographic Fragment Screening on the Shigella Type III Secretion System Chaperone IpgC.

The Shigella pathogenicity factor IpgC belongs to the class II of type III secretion system chaperones, whose members are characterized by a tetratricopeptide repeat (TPR) domain consisting of three and a half TPR motifs. Since IpgC is essential for Shigella virulence, we determined a high-resolution crystal structure of this chaperone to facilitate its use as a target for the structure-based design of anti-shigellosis compounds. The crystal structure revealed two possible homodimer assemblies, which strongly differ from the homodimer architectures so far known for IpgC and orthologues thereof. Through crystallographic fragment screening, we identified 10 small molecules that bind to IpgC and, therefore, are available for expansion to generate larger, more potent binders. A follow-up compound, based on one of our fragment hits, binds to a strictly conserved site, which overlaps with the binding site of the chaperone's substrates, IpaB and IpaC. Therefore, it constitutes a promising starting point for the design of functional IpgC inhibitors.

TriMem: A parallelized hybrid Monte Carlo software for efficient simulations of lipid membranes.

Lipid membranes are integral building blocks of living cells and perform a multitude of biological functions. Currently, molecular simulations of cellular-scale membrane remodeling processes at atomic resolution are extremely difficult, due to their size, complexity, and the large times-scales on which these processes occur. Instead, elastic membrane models are used to simulate membrane shapes and transitions between them and to infer their properties and functions. Unfortunately, an efficiently parallelized open-source simulation code to do so has been lacking. Here, we present TriMem, a parallel hybrid Monte Carlo simulation engine for triangulated lipid membranes. The kernels are efficiently coded in C++ and wrapped with Python for ease-of-use. The parallel implementation of the energy and gradient calculations and of Monte Carlo flip moves of edges in the triangulated membrane enable us to simulate large and highly curved membrane structures. For validation, we reproduce phase diagrams of vesicles with varying surface-to-volume ratios and area difference. We also compute the density of states to verify correct Boltzmann sampling. The software can be used to tackle a range of large-scale membrane remodeling processes as a step toward cell-scale simulations. Additionally, extensive documentation make the software accessible to the broad biophysics and computational cell biology communities.

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

19F-NMR Unveils the Ligand-Induced Conformation of a Catalytically Inactive Twisted Homodimer of tRNA-Guanine Transglycosylase.

Understanding the structural arrangements of protein oligomers can support the design of ligands that interfere with their function in order to develop new therapeutic concepts for disease treatment. Recent crystallographic studies have elucidated a novel twisted and functionally inactive form of the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative target in the fight against shigellosis. Active-site ligands have been identified that stimulate the rearrangement of one monomeric subunit by 130° against the other one to form an inactive twisted homodimer state. To assess whether the crystallographic observations also reflect the conformation in solution and rule out effects from crystal packing, we performed 19F-NMR spectroscopy with the introduction of 5-fluorotryptophans at four sites in TGT. The inhibitor-induced conformation of TGT in solution was assessed based on 19F-NMR chemical shift perturbations. We investigated the effect of C(4) substituted lin-benzoguanine ligands and identified a correlation between dynamic protein rearrangements and ligand-binding features in the corresponding crystal structures. These involve the destabilization of a helix next to the active site and the integrity of a flexible loop-helix motif. Ligands that either completely lack an attached C(4) substituent or use it to stabilize the geometry of the functionally competent dimer state do not indicate the presence of the twisted dimer form in the NMR spectra. The perturbation of crucial structural motifs in the inhibitors correlates with an increasing formation of the inactive twisted dimer state, suggesting these ligands are able to shift a conformational equilibrium from active C2-symmetric to inactive twisted dimer conformations. These findings suggest a novel concept for the design of drug candidates for further development.

Unraveling a Ligand-Induced Twist of a Homodimeric Enzyme by Pulsed Electron-Electron Double Resonance.

Mechanistic insights into protein-ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes. For the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative virulence target of shigellosis, ligand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one. However, crystallographic observation of both end states does neither verify the ligand-induced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approach that combines site-directed spin labeling (SDSL) with distance measurements based on pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with a pyranose-substituted ligand being the most potent one in shifting the equilibrium toward the twisted dimer. Our experiments suggest a dissociation-association mechanism for the formation of the twisted dimer upon ligand binding.

Targeting a Cryptic Pocket in a Protein-Protein Contact by Disulfide-Induced Rupture of a Homodimeric Interface.

Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.

Sensitive protein alignments at tree-of-life scale using DIAMOND.

We are at the beginning of a genomic revolution in which all known species are planned to be sequenced. Accessing such data for comparative analyses is crucial in this new age of data-driven biology. Here, we introduce an improved version of DIAMOND that greatly exceeds previous search performances and harnesses supercomputing to perform tree-of-life scale protein alignments in hours, while matching the sensitivity of the gold standard BLASTP.

The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein-Protein Interface of Homodimeric tRNA-Guanine Transglycosylase.

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.

Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.

Efficient Ensemble Refinement by Reweighting.

Ensemble refinement produces structural ensembles of flexible and dynamic biomolecules by integrating experimental data and molecular simulations. Here we present two efficient numerical methods to solve the computationally challenging maximum-entropy problem arising from a Bayesian formulation of ensemble refinement. Recasting the resulting constrained weight optimization problem into an unconstrained form enables the use of gradient-based algorithms. In two complementary formulations that differ in their dimensionality, we optimize either the log-weights directly or the generalized forces appearing in the explicit analytical form of the solution. We first demonstrate the robustness, accuracy, and efficiency of the two methods using synthetic data. We then use NMR J-couplings to reweight an all-atom molecular dynamics simulation ensemble of the disordered peptide Ala-5 simulated with the AMBER99SB*-ildn-q force field. After reweighting, we find a consistent increase in the population of the polyproline-II conformations and a decrease of α-helical-like conformations. Ensemble refinement makes it possible to infer detailed structural models for biomolecules exhibiting significant dynamics, such as intrinsically disordered proteins, by combining input from experiment and simulation in a balanced manner.

Swapping Interface Contacts in the Homodimeric tRNA-Guanine Transglycosylase: An Option for Functional Regulation.

The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, recognizes tRNA only as a homodimer and performs full nucleobase exchange at the wobble position. Active-site inhibitors block the enzyme function by competitively replacing tRNA. In solution, the wild-type homodimer dissociates only marginally, whereas mutated variants show substantial monomerization in solution. Surprisingly, one inhibitor transforms the protein into a twisted state, whereby one monomer unit rotates by approximately 130°. In this altered geometry, the enzyme is no longer capable of binding and processing tRNA. Three sugar-type inhibitors have been designed and synthesized, which bind to the protein in either the functionally competent or twisted inactive state. They crystallize with the enzyme side-by-side under identical conditions from the same crystallization well. Possibly, the twisted inactive form corresponds to a resting state of the enzyme, important for its functional regulation.

Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase.

The bacterial enzyme tRNA-guanine transglycosylase (TGT) is involved in the biosynthesis of queuosine, a modified nucleoside present in the anticodon wobble position of tRNAHis, tRNATyr, tRNAAsp, and tRNAAsn. Although it forms a stable homodimer endowed with two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time. In contrast, its mammalian counterpart constitutes a heterodimer consisting of a catalytic and a noncatalytic subunit, termed QTRT1 and QTRT2, respectively. Both subunits are homologous to the bacterial enzyme, yet only QTRT1 possesses all the residues required for substrate binding and catalysis. In mice, genetic inactivation of the TGT results in the uncontrolled oxidation of tetrahydrobiopterin and, accordingly, phenylketonuria-like symptoms. For this reason and because of the recent finding that mammalian TGT may be utilized for the treatment of multiple sclerosis, this enzyme is of potential medical relevance, rendering detailed knowledge of its biochemistry and structural architecture highly desirable. In this study, we performed the kinetic characterization of the murine enzyme, investigated potential quaternary structures of QTRT1 and QTRT2 via noncovalent mass spectrometry, and, finally, determined the crystal structure of the murine noncatalytic TGT subunit, QTRT2. In the crystal, QTRT2 is clearly present as a homodimer that is strikingly similar to that formed by bacterial TGT. In particular, a cluster of four aromatic residues within the interface of the bacterial TGT, which constitutes a "hot spot" for dimer stability, is present in a similar constellation in QTRT2.

Soaking suggests "alternative facts": Only co-crystallization discloses major ligand-induced interface rearrangements of a homodimeric tRNA-binding protein indicating a novel mode-of-inhibition.

For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy. Since bacterial TGT is only functional as homodimer, its activity can be inhibited either by blocking its active site or by preventing dimerization. Recently, we discovered that in some crystal structures obtained by soaking the full conformational adaptation most likely induced in solution upon ligand binding is not displayed. Thus, soaked structures may be misleading and suggest irrelevant binding modes. Accordingly, we re-investigated these complexes by co-crystallization. The obtained structures revealed large conformational rearrangements not visible in the soaked complexes. They result from spatial perturbations in the ribose-34/phosphate-35 recognition pocket and, consequently, an extended loop-helix motif required to prevent access of water molecules into the dimer interface loses its geometric integrity. Thermodynamic profiles of ligand binding in solution indicate favorable entropic contributions to complex formation when large conformational adaptations in the dimer interface are involved. Native MS titration experiments reveal the extent to which the homodimer is destabilized in the presence of each inhibitor. Unexpectedly, one ligand causes a complete rearrangement of subunit packing within the homodimer, never observed in any other TGT crystal structure before. Likely, this novel twisted dimer is catalytically inactive and, therefore, suggests that stabilizing this non-productive subunit arrangement may be used as a further strategy for TGT inhibition.

What Glues a Homodimer Together: Systematic Analysis of the Stabilizing Effect of an Aromatic Hot Spot in the Protein-Protein Interface of the tRNA-Modifying Enzyme Tgt.

Shigella bacteria constitute the causative agent of bacillary dysentery, an acute inflammatory disease causing the death of more than one million humans per year. A null mutation in the tgt gene encoding the tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) was found to drastically decrease the pathogenicity of Shigella bacteria, suggesting the use of Tgt as putative target for selective antibiotics. The enzyme is only functionally active as a homodimer; thus, interference with the formation of its protein-protein interface is an attractive opportunity for therapeutic intervention. To better understand the driving forces responsible for the assembly, stability, and formation of the homodimer, we studied the properties of the residues that establish the dimer interface in detail. We performed site-directed mutagenesis and controlled shifts in the monomer/dimer equilibrium ratio in solution in a concentration-dependent manner by native mass spectrometry and used crystal structure analysis to elucidate the geometrical modulations resulting from mutational variations. The wild-type enzyme exhibits nearly exclusive dimer geometry. A patch of four aromatic amino acids, embedded into a ring of hydrophobic residues and further stabilized by a network of H-bonds, is essential for the stability of the dimer's contact. Accordingly, any perturbance in the constitution of this aromatic patch by nonaromatic residues reduces dimer stability significantly, with some of these exchanges resulting in a nearly exclusively monomeric state. Apart from the aromatic hot spot, the interface comprises an extended loop-helix motif that exhibits remarkable flexibility. In the destabilized mutated variants, the loop-helix motif adopts deviating conformations in the interface region, and a number of water molecules, penetrating into the interface, are observed.

Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization.

High resolution crystal structure of Clostridium propionicum ß-alanyl-CoA:ammonia lyase, a new member of the "hot dog fold" protein superfamily.

Clostridium propionicum is the only organism known to ferment ß-alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo-acyl carrier protein. The first step in the fermentation is a CoA-transfer to ß-alanine. Subsequently, the resulting ß-alanyl-CoA is deaminated by the enzyme ß-alanyl-CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl-CoA. We have determined the crystal structure of Acl in its apo-form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called "hot dog fold" which is characterized by a five-stranded antiparallel ß-sheet with a long α-helix packed against it. The functional unit of all "hot dog fold" proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA-like acyl-CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking.

Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn) by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering) by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

Launching spiking ligands into a protein-protein interface: a promising strategy to destabilize and break interface formation in a tRNA modifying enzyme.

Apart from competitive active-site inhibition of protein function, perturbance of protein-protein interactions by small molecules in oligodomain enzymes opens new perspectives for innovative therapeutics. tRNA-guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode. A special feature of this enzyme is the short distance between active site and rim of the dimer interface. This suggests design of expanded active-site inhibitors decorated with rigid, needle-type substituents to spike into potential hot spots of the interaction interface. Ligands with attached ethinyl-type substituents have been synthesized and characterized by Kd measurements, crystallography, noncovalent mass spectrometry, and computer simulations. In contrast to previously determined crystal structures with nonextended active-site inhibitors, a well-defined loop-helix motif, involved in several contacts across the dimer interface, falls apart and suggests enhanced flexibility once the spiking ligands are bound. Mass spectrometry indicates significant destabilization but not full disruption of the complexed TGT homodimer in solution. As directed interactions of the loop-helix motif obviously do not determine dimer stability, a structurally conserved hydrophobic patch composed of several aromatic amino acids is suggested as interaction hot spot. The residues of this patch reside on a structurally highly conserved helix-turn-helix motif, which remains unaffected by the bound spiking ligands. Nevertheless, it is shielded from solvent access by the loop-helix motif that becomes perturbed upon binding of the spiking ligands, which serves as a possible explanation for reduced interface stability.

A crystallization screen based on alternative polymeric precipitants.

Most commercially available crystallization screens are sparse-matrix screens with a predominance of inorganic salts and polyethylene glycols (PEGs) as precipitants. It was noted that commercially available screens are largely unsatisfactory for the purpose of the crystallization of multimeric protein and protein-nucleic acid complexes. This was reasoned to be a consequence of the redundancy in screening crystallization parameter space by the predominance of PEG as a precipitant in standard screens and it was suggested that this limitation could be overcome by introducing a variety of other organic polymers. Here, a set of 288 crystallization conditions was devised based on alternative polymeric precipitants and tested against a set of 20 different proteins/complexes; finally, a screen comprising the 96 most promising conditions designed to complement PEG- and salt-based commercial screens was proposed.