Elution Profiles Of Proteins Research Articles

A batch screening technique for the calculation of chromatographic separability

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures

In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.

Native Size-Exclusion Chromatography–Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery—suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these—the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex—as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Global landscape of protein complexes in postprandial-state livers from diet-induced obese and lean mice

Obesity is associated with a spectrum of nonalcoholic fatty liver disease (NAFLD) which is characterized by steatosis. Prolonged fat deposition aggravates liver dysfunctions leading to an advanced form of NAFLD such as steatohepatitis and cirrhosis. As liver function in the postprandial state is critical for macronutrient metabolism and energy homeostasis, we sought to determine the differences in protein complex profiles in lean and fatty livers in the postprandial state. Protein complex profiling is of interest as proteins often do not function alone and the information on the interactions may reveal novel etiology of NAFLD, which is currently limited compared with proteome profiles or RNA-sequencing profiles. To this end, we fractionated liver lysates using size-exclusion chromatography (SEC) and analyzed each fraction using untargeted LC-MS/MS. We identified 1172 proteins that were discovered in lean and fatty livers, and their elution profiles were compared. We found that the majority of liver proteins were present as putative complexes. Also, the fatty liver protein elution profile showed great conservations as lean liver despite the metabolic disease state. Yet, we discovered a few proteins that showed different elution patterns in the fatty liver, including Acadm, Aldh1a7, Aldh1a1, Akr1a1, Eif3l, Fkbp2, G6pdx, Gm20441, Hao1, Pcna, Pkm, Ppif, Prdx4, Stmn1, Tagln, Tubb4b, Ubqln2, and Usp14, which may be involved in high fat diet-induced alterations of protein oligomerization and hepatic functions. Overall, our protein complex profiling could expand our understanding of hepatic abnormalities that cannot be uncovered by simple quantitation of protein expression.

A co-fractionation mass spectrometry-based prediction of protein complex assemblies in the developing rice aleurone-subaleurone.

Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and system-level control. This is of great biological importance and practical significance in heterotrophic rice (Oryza sativa) endosperm and aleurone-subaleurone tissues, which are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this study was to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable large-scale multimerization state measurements and protein complex predictions. The predicted complexes had predicted functions across diverse functional categories, including novel heteromeric RNA binding protein complexes that may influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about system-level posttranslational control during the early stages of rice seed development.

Prediction of the elution profiles of proteins in mixed salt systems in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is often used for purifying proteins. A mathematical model to describe the complex effects of salts on the adsorption equilibria in HIC has recently been introduced by our group. It describes not only the influence of single salts, but also salt mixtures, in which cooperative effects may occur. The influence of the salts is thereby modeled with a Taylor series expansion in the individual ion molarities. In the present study, the model of the adsorption equilibrium is coupled with a lumped kinetic model of the adsorption kinetics to obtain a model of the elution of proteins in HIC adsorption columns. The column model is tested using experimental data on the adsorption of bovine serum albumin (BSA) and lysozyme (LYS) on the mildly hydrophobic resin Toyopearl PPG-600M at pH 7. The studied salts are ammonium chloride, sodium chloride, ammonium sulfate, and sodium sulfate as well as binary and ternary mixtures of them. The parameters of the lumped kinetic model are protein-specific and were fitted to the elution profiles of the single proteins in presence of single salts. The model was then used to predict the elution profiles of BSA and LYS solutions containing both proteins, for single salts as well as for binary and ternary salt mixtures. Both isocratic and gradient elution were studied. Furthermore, the model was applied to identify the optimal overall ionic strength for the separation of the two proteins by isocratic elution and the optimal linear gradient of the salt concentration in a multicriteria approach where the conflicting goals are high separation yield and low elution volume.

Abstract 391: Flow Field-flow Fractionation: a Powerful Underused Size Fractionation Technique for Composition Analysis of Lipoprotein Particles

Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques for composition analysis of lipoproteins are limited by traditional preparative fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work a highly effective but underused size fractionation technique, asymmetric flow field-flow fractionation (AFFFF) was applied. We compared AFFFF with traditional techniques, including gel electrophoresis (GE), size exclusion chromatography (SEC), and gradient ultracentrifugation (GUC). Direct comparison of the techniques was performed by analysis of the same set of samples using sudan black staining. The AFFFF size separation allowed size resolution most similar to GE techniques, either tube gel or gradient gel. In addition, AFFFF and SEC was compared by collection of fractions where the average size in each fraction was measured by dynamic light scattering, and all major lipids and apolipoproteins were quantified by liquid chromatography tandem mass spectrometry methods (LC-MS/MS) in each fraction. The size resolution by AFFFF vs. SEC was compared based on the elution profiles of endogenous plasma proteins. On the hydrodynamic size scale, the half peak width of individual size species were 0.8-1 nm by AFFFF vs. 3-8 nm by SEC, showing the superior size resolution capability of the AFFFF technique. The gentle nature of the AFFFF separation relative to GUC was demonstrated by AFFFF separation of HDL sub-fractions with and without pre-fractionation by GUC, showing the significant loss of apoA-IV and apoCs, as well as a general shift of all lipid and protein profiles to smaller hydrodynamic size as a result of intense shearing forces and high salt conditions during GUC. Our results demonstrate that AFFFF coupled with LC-MS/MS deserves to be among the gold standard techniques for size and composition analysis of HDL, LDL and VLDL sub-classes.

A rapid and accurate approach for prediction of interactomes from co-elution data (PrInCE)

BackgroundAn organism’s protein interactome, or complete network of protein-protein interactions, defines the protein complexes that drive cellular processes. Techniques for studying protein complexes have traditionally applied targeted strategies such as yeast two-hybrid or affinity purification-mass spectrometry to assess protein interactions. However, given the vast number of protein complexes, more scalable methods are necessary to accelerate interaction discovery and to construct whole interactomes. We recently developed a complementary technique based on the use of protein correlation profiling (PCP) and stable isotope labeling in amino acids in cell culture (SILAC) to assess chromatographic co-elution as evidence of interacting proteins. Importantly, PCP-SILAC is also capable of measuring protein interactions simultaneously under multiple biological conditions, allowing the detection of treatment-specific changes to an interactome. Given the uniqueness and high dimensionality of co-elution data, new tools are needed to compare protein elution profiles, control false discovery rates, and construct an accurate interactome.ResultsHere we describe a freely available bioinformatics pipeline, PrInCE, for the analysis of co-elution data. PrInCE is a modular, open-source library that is computationally inexpensive, able to use label and label-free data, and capable of detecting tens of thousands of protein-protein interactions. Using a machine learning approach, PrInCE offers greatly reduced run time, more predicted interactions at the same stringency, prediction of protein complexes, and greater ease of use over previous bioinformatics tools for co-elution data. PrInCE is implemented in Matlab (version R2017a). Source code and standalone executable programs for Windows and Mac OSX are available at https://github.com/fosterlab/PrInCE, where usage instructions can be found. An example dataset and output are also provided for testing purposes.ConclusionsPrInCE is the first fast and easy-to-use data analysis pipeline that predicts interactomes and protein complexes from co-elution data. PrInCE allows researchers without bioinformatics expertise to analyze high-throughput co-elution datasets.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Purification and Electrophoretic Characterization of Lactate Dehydrogenase from Mammalian Blood: A Different Twist on a Classic Experiment

A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size exclusion, and affinity chromatography. Protein and enzyme activity elution profiles are determined by graphical analysis of assay data collected using rapid microplate spectrophotometric assays. Students perform quantitative assays on LDH pools and use these data to build a purification table for use in evaluating the protocol. The protocol typically generates final overall fold-purifications from 1500 to 2500 and activity recoveries of 45–60%. Electrophoretic separations in both denaturing and native-gel format are analyzed both visually and by use of commercial digital analysis software to assess the isoenzyme pattern of isolated LDH and to further evaluate the purification. Assessment of student work revealed a high level of achievement of course learning goals that include development of critical thinking skills required to (1) critically evaluate an experimental protocol and (2) draw conclusions based on a variety of forms of experimental data.

Application of spectral deconvolution and inverse mechanistic modelling as a tool for root cause investigation in protein chromatography

In chromatographic protein purification, process variations, aging of columns, or processing errors can lead to deviations of the expected elution behavior of product and contaminants and can result in a decreased pool purity or yield. A different elution behavior of all or several involved species leads to a deviating chromatogram. The causes for deviations are however hard to identify by visual inspection and complicate the correction of a problem in the next cycle or batch. To overcome this issue, a tool for root cause investigation in protein chromatography was developed. The tool combines a spectral deconvolution with inverse mechanistic modelling. Mid-UV spectral data and Partial Least Squares Regression were first applied to deconvolute peaks to obtain the individual elution profiles of co-eluting proteins. The individual elution profiles were subsequently used to identify errors in process parameters by curve fitting to a mechanistic chromatography model. The functionality of the tool for root cause investigation was successfully demonstrated in a model protein study with lysozyme, cytochrome c, and ribonuclease A. Deviating chromatograms were generated by deliberately caused errors in the process parameters flow rate and sodium-ion concentration in loading and elution buffer according to a design of experiments. The actual values of the three process parameters and, thus, the causes of the deviations were estimated with errors of less than 4.4%. Consequently, the established tool for root cause investigation is a valuable approach to rapidly identify process variations, aging of columns, or processing errors. This might help to minimize batch rejections or contribute to an increased productivity.

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model.

Predicting protein elution for overloaded ion exchange columns requires models capable of describing protein binding over broad ranges of protein and salt concentrations. Although approximate mechanistic models are available, they do not always have the accuracy needed for precise predictions. The aim of this work is to develop a method to predict protein chromatographic behavior from batch isotherm data without relying on a mechanistic model. The method uses a systematic empirical interpolation (EI) scheme coupled with a lumped kinetic model with rate parameters determined from HETP measurements for non-binding conditions, to numerically predict the column behavior. For two experimental systems considered in this work, predictions based on the EI scheme are in excellent agreement with experimental elution profiles under highly overloaded conditions without using any adjustable parameters. A qualitative study of the sensitivity of predicting protein elution profiles to the precision, granularity, and extent of the batch adsorption data shows that the EI scheme is relatively insensitive to the properties of the dataset used, requiring only that the experimental ranges of protein and salt concentrations overlap those under which the protein actually elutes from the column and possess a ± 10% measurement precision.

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

Modification of the Zonal Elution Method for Detection of Transient Protein–Protein Interactions Involving Ligand Exchange

A new affinity chromatography method was developed by modifying a zonal elution method. The new method targets transient protein-protein interactions, such as those encountered during direct ligand transfer between the ligand transporter and its cognate receptor. A ligand-loaded transport protein is immobilized on the solid support, and a plug containing a putative receptor is flowed through the column. Elution profiles of proteins not interacting with the immobilized transporter can be approximated with a simple Gaussian curve, while the elution profiles of cognate receptors show significant delay and exhibit complex shape. Ligand transfer from the immobilized transporter molecules to the receptors is verified by both UV absorbance measurements and mass spectrometry. The sensitivity of the method is demonstrated using retinoic acid (RA) transfer from various isoforms of cellular RA binding proteins (CRABPs) and RA receptor γ (RARγ). Although these interactions have been hypothesized long ago to proceed via direct mechanism (i.e., via transient docking of the receptor and the transporter), the existing biophysical techniques failed to detect the presence of the transporter-receptor complexes. However, the modified zonal elution method provides unequivocal evidence of direct interaction between RARγ and one of the CRABP isoforms (CRABP II) during the ligand transfer to the receptor.

Low-resolution structure of Drosophila translin

Crystals of native Drosophila melanogaster translin diffracted to 7Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size-exclusion chromatography analyses but the methylated protein displayed reduced DNA-binding activity. Crystals of the methylated protein diffracted to 4.2Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P21 with eight protomers in the asymmetric unit. Only 2% of low-resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to Rwork (Rfree) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel-filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up-down dimer as the basic structural subunit of translin-like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel-filtration columns. This study demonstrates clearly that low-resolution X-ray structure can be useful in understanding complex biological oligomers.

Effects of resin ligand density on yield and impurity clearance in preparative cation exchange chromatography. II. Process characterization

Ion exchange resins are key raw materials in biopharmaceutical manufacturing processes, and variability in ligand density has the potential to compromise process robustness if not controlled within appropriate ranges. In this study, yield and impurity clearance were evaluated for several preparative cation exchange chromatography steps using SP Sepharose™ Fast Flow resins at the high, low, and center of the commercial ligand density specification range. This was accomplished using a design of experiments (DoE) approach coupled to high-throughput screening in 96-well plate format, as well as column chromatography experiments with gradient elution. Results of the DoE study indicated that ligand density variation within the commercial specification range of SP Sepharose™ Fast Flow had no effect on yield, HCP clearance, aggregate clearance, or distribution of charge variants. However, results from the column experiments showed that ligand density has the potential to influence protein elution profiles which can lead to small changes in impurity clearance in some cases.

Thermally-modulated on/off-adsorption materials for pharmaceutical protein purification

For the development of temperature-responsive adsorption materials for pharmaceutical protein purification, poly( N-isopropylacrylamide-co- N,N-dimethylaminopropylacrylamide-co- N-tert-butylacrylamide) (P(IPAAm- co-DMAPAAm- co-tBAAm) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP). The prepared silica beads as a chromatographic stationary phase were evaluated by observing their thermo-responsive elution profiles of plasma proteins including human serum albumin (HSA) and γ-globulin. Chromatograms of two proteins indicated that negatively-charged HSA was adsorbed on the cationic copolymer brush modified silica beads at higher temperatures with low concentration of phosphate buffer (PB) (pH 7.0) as a mobile phase. The HSA adsorption was attributed to (1) an enhanced electrostatic interaction with the cationic copolymer brush at low concentration of PB and (2) an increased hydrophobic interaction from the dehydrated copolymer at high temperature. Step-temperature gradient enabled HSA and γ-globulin to be separated by the modulation of HSA adsorption/desorption onto the copolymer brush grafted silica beads. These results suggested that the prepared copolymer brush grafted silica beads adsorbed negatively-charged proteins both through electrostatic and hydrophobic interactions by the modulation of column temperature and gave attractive adsorption materials for protein purification process.

The Methanothermobacter thermautotrophicus MCM Helicase Is Active as a Hexameric Ring

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukarya. The structure of the single MCM protein homologue from the archaeon Methanothermobacter thermautotrophicus is not yet clear, and hexameric, heptameric, octameric, and dodecameric structures, open rings, and filamentous structures have been reported. Using a combination of biochemical and structural analysis, it is shown here that the M. thermautotrophicus MCM helicase is active as a hexamer.

Modelación Fenomenológica de Cromatografía de Interacción Hidrofóbica

The aim of this work was to obtain a phenomenological model to describe the elution profile in hydrophobic interaction chromatography of a protein injected as a pulse. The plate model, which was solved numerically, was used, and good fit to experimental data was found. The plate model allowed simulating the chromatographic process under different operating conditions, such as flow rate, elution gradient and sample volume. In is concluded that it is possible to apply the plate theory to describe elution profiles of proteins in hydrophobic interaction chromatography. The proposed model could be used to optimize the separation of protein mixtures.

Preface to the special issue on regulation of gene function

Crystals of native Drosophila melanogaster translin diffracted to 7 Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size-exclusion chromatography analyses but the methylated protein displayed reduced DNA-binding activity. Crystals of the methylated protein diffracted to 4.2 Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P21 with eight protomers in the asymmetric unit. Only 2% of low-resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to Rwork (Rfree) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel-filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up-down dimer as the basic structural subunit of translin-like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel-filtration columns. This study demonstrates clearly that low-resolution X-ray structure can be useful in understanding complex biological oligomers.