Human TPI, TPIIle170Val and TPIIle170Thr complemented for a loss of the TPI plasmid, but TPILys13Arg did not. The duration until growth was re-established (lag phase) was used as an inverse indicator for heat resistance. The parasite lacks a functional tricarboxylic acid cycle, making glycolysis its sole energy source. However, there has been little research on triosephosphate isomerase from E. granulosus (Eg-TIM). Yeast cells expressing human TPI well tolerated the heat treatment; however, yeast harbouring TPIIle170Val and TPIIle170Thr were heat-sensitive, resulting in a strong delay until growth resumed (figure 6c). We use this mutant and others inferred from the crystallographic structure to define the kinetics and stability properties of TPI upon PEP binding. Individual metabolites were identified by matching retention time and fragmentation pattern with commercially available standards. Product name: Triosephosphate Isomerase (TPI) Product description: Recombinant Human TPI enzyme (active) produced in Nicotiana benthamiana by Agrobacterium-mediated transient expression. The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. Next, we assessed structural stability in the presence of the TPI substrate, G3P. These cycles sustain intermediates while preventing their accumulation to toxic levels and are responsible for the oscillating behaviour of glycolytic reactions [3–6]. In the case of human wild-type TPI, 50% of enzyme activity was lost in the presence of 570 µM PEP (IC50), corresponding to a Ki of 230 µM (figure 4b, blue curves, to be read from right to left). Far-UV circular dichroism (CD) spectroscopy of the purified recombinant enzymes showed a similar composition of secondary structures, indicating that the mutations did not prevent folding (electronic supplementary material, figure S2). A similar mechanism acts to prevent an accumulation of oxidizing metabolites in cancer cells or cells that respire at high rates. Select the sequence identity as 30%. (a) Contact distances between TPI and its substrate DHAP, and (b) the interactions of TPI and PEP in the active site. Absolute values are given in the electronic supplementary material, figure S4. We have shown previously that the increased oxidative stress resistance of PK mutants is attributable to TPI as well. This indicates that cells compensated for a loss of specific TPI activity by the upregulation of the enzyme abundance. The substantial residual activities of 5.9% for TPIIle170Val or 13.1% of TPIIle170Thr indicate that Ile170 is not essential for TPI's catalytic function. Only G3P can be used in glycolysis, therefore TIM is essential for energy production, allowing two molecules of G3P to be produced for every glucose molecule, thereby doubling the energy yield. 25. What do they mean by coupled? Dihydrolipoamide dehydrogenase (DLD), also known as dihydrolipoyl dehydrogenase, mitochondrial, is an enzyme that in humans is encoded by the DLD gene. We speculate that the increased substrate affinity is facilitated by a hydrogen bond between the substrate and the threonine side chain. (c) TPI mutants are heat-sensitive. Glucose-6-Phosphate Isomerase. Retrieve the proteins with seven α-helical segments in PDBTM. In structure–function studies involving different TPI point mutations including a rare natural variant (TPIIle170Val [22]), and two mutants designed on the basis of the crystallographic findings (TPILys13Arg and TPIIle170Thr), we have demonstrated that PEP functions as a competitive TPI inhibitor, being able to interfere with the enzymatic TPI function during catalysis. Its actual maximum activity is difficult to estimate, because of the difficulty in saturating it with its substrate. Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. It has been shown that red cells of TPI-deficient individuals accumulate dihydroxyacetone phosphate, and it is possible that the twenty times-normal levels of this substance achieved may in some way damage the red cell and result in its destruction, in spite of its normal or increased glucose utilization (Schneider et al., 1968b). TPI activity was determined as described previously [21,51]. The mutant species exhibited strongly increased IC50 values (TPIIle70Val = 1925 µM, TPIIle170Thr = 1537 µM). is a Wellcome Trust Research Career Development and Wellcome-Beit prize fellow. Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), which is one of the seven reversible enzymatic reactions participating in both glycolysis and gluconeogenesis. Mutations in the TPI1 gene cause triosephosphate isomerase deficiency. The reaction mix of 100 ml 20 mM HEPES (pH 7.5) contained approximately 4.5 mM protein, 0.4 ml 500× SYPRO Orange (Sigma Aldrich) and PEP and/or G3P at the indicated concentrations. Human TPI, TPIIle170Val and TPIIle170Thr complemented for a loss of the TPI plasmid, but TPILys13Arg did not. Figure 2. In comparison with the isogenic strain expressing wild-type TPI, yeast cells expressing both the naturally occurring TPIIle170Val allele and the designed TPIIle170Thr protein displayed an increased concentration of PPP intermediates, indicating higher activity of this pathway confirming the previous results (figure 6a; electronic supplementary material, figure S4). Figure 3. According to the Genetics Home Reference (from the NIH, National Institutes of Health) page about triosephosphate isomerase deficiency: The anemia in this condition begins in infancy. Whereas low PK activity in yeast is correlated with high respiration rates and superoxide production [14], in human cells it is associated with rapid cell proliferation and cancer [13,36]. Human TPIIle170Val and TPIIle170Thr complement for yeast TPI and are catalytically active. (b) Enzymatic properties of TPI, TPIIle170Val, TPIIle170Thr and their inhibition by PEP. Hint: Use PDB-REPRDB and provide the necessary conditions. Figure 3 Anaerobic glycolysis, phase one Triosephosphate isomerase (TPI) catalyzes the interconversion of glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Absolute values are given in the electronic supplementary material, figure S4. Glucose is of course the starting material for the pathway, and through a series of chemical conversions, it generates ATP for use in cellular processes. E. granulosus can produce energy by glycolysis as well as both aerobic and anaerobic respirations. Baillie´re’s Clin. Compound C00111 at KEGG Pathway Database. Triosephosphate isomerase (TPI) deficiency is a severe disorder characterized by a shortage of red blood cells ( hemolytic anemia ), neurological problems, infections, and muscle weakness that can affect breathing and heart function. At this point in the glycolytic pathway, we have two 3-carbon molecules, but have not yet fully converted glucose into pyruvate. N.V. Bhagavan, Chung-Eun Ha, in Essentials of Medical Biochemistry (Second Edition), 2015. In a deficient subject there was selective loss of the two more rapidly moving bands, but activity of the slowest moving band was also diminished. A much stronger response was however observed for TPIIle170Thr. Harvey S. Singer MD, ... Joseph Jankovic MD, in Movement Disorders in Childhood, 2010. Obtain the PDB codes that have ligands and the sequence identity is less than 30%. More recently, it has been proposed that cancer cells profit from the loss of the PKM2 gene during tumour formation [15]. It is characterized by hemolytic anemia and neurodegeneration, and is caused by anaerobic metabolic dysfunction. http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. [28] as surface representation. Figure 3 Anaerobic glycolysis, phase one Triosephosphate isomerase (TPI) catalyzes the interconversion of glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). TPIIle170Val and TPIIle170Thr have lower activity than wild-type TPI. In any case, its Vmax is easily 10,000 times that of hexokinase, the least active enzyme in the sequence (Beutler, 1971a). Fructose-bisphosphate aldolase. The lag phase was prolonged in the 50°C exposed TPI mutants compared with isogenic yeast cells expressing wild-type TPI.Download figureOpen in new tabDownload powerPoint. Only cells containing a functional TPI copy on the minichromosome are viable on glucose media after counterselection. Clinical features include hemolytic anemia, progressive neuromuscular dysfunction, and increased susceptibility to infection with specific pathogenic variants resulting in severe disease and death by age 8. Overnight cultures were diluted to an OD600 = 0.2 and exposed, or not exposed, to 50°C for 5 min and growth was monitored for 25 h after heat exposure. Now, the large number of cases associated with the codon 104 mutation has allowed the use of chorionic villus biopsy samples and molecular techniques for prenatal diagnosis (73). Yeast strains expressing wild-type TPI, TPIIle170Val and TPIIle170Thr could be cultured in glucose-containing media, indicating that these enzymes compensated for the loss of yeast TPI, demonstrating catalytic activity in vivo. The free energy released in this process is used to form the high-energy compounds ATP (2 moles/mole of glucose) and NADH. Finally, we tested whether protein stability is affected by PEP in the presence of G3P. However, this appears biologically meaningful, as PEP is constantly present at high cellular levels [14]. Crystallographic data collection and refinement statistics. Separation was achieved by isocratic flow at 12% acetonitrile for 3.5 min followed by a gradient to 38% acetonitrile within 2.5 min. Hint: search CATH database with FASTA sequence, which can be found from UniProtKB/SWISS-PROT (P02930) or PDB. TPI catalyzes the near-equilibrium conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. Find the domain information for outer membrane protein TolC. By contrast, TPIIle170Val was resistant to increased PEP levels (figure 3c), indicating that this metabolite was no longer competitive for binding. Light‐inducible flux control of triosephosphate isomerase on glycolysis in Escherichia coli. Figure 6. Christian P. Whitman, in Comprehensive Natural Products Chemistry, 1999, Triose phosphate isomerase (TIM) catalyzes the interconversion of dihydroxyacetone (19) and glyceraldehyde 3-phosphate (21) through an enzyme-bound enediolic intermediate (20) (Scheme 5).26,27 The enzyme plays a central role in glycolysis and its mechanism and energetics have fascinated enzymologists since the 1950s. Get the list of PDB codes for RNA-binding proteins. There are ten reactions of glycolysis takes place in the cytosol. TPI deficiency results in the accumulation of cellular DHAP and toxic biproducts. Triose Phosphate Isomerase (TPI) is an isomerase that catalyzes the isomerization of dihydroxyacetone phosphate to and from D-glyceraldehyde 3-phosphate. Crystals were grown at 20°C using the sitting-droplet vapour diffusion method by mixing 200 nl of TPI–PEP complex with 200 nl of reservoir solution (0.1 M MES pH: 6.5, 25% polyethylene glycol (PEG) 8000). M. Michael Gromiha, in Protein Bioinformatics, 2010. Co-crystal structure of TPI with bound PEP. This indicates that despite its lower activity, the TPIIle170Thr mutant had higher affinity to the TPI substrate (figure 4b, black curves, from left to right). Hint: Identify the temperature factors of all atoms in Phe4, and get the average. This situation causes a block of the early steps of glycolysis and increases the PPP activity resulting in augmented oxidant tolerance of both yeast and mammalian cells [11,14], indicating that the PK–TPI feedback loop is important for oxidative stress protection. List the major transport systems in Transport classification database? This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. B) Phosphoglucose isomerase. Get the dataset of protein structures with less than 40% sequence identity. Find the three-dimensional coordinates of porin from Rhodobactor capsulatus and store the data. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. Substrate/inhibitor saturation was used to calculate Vmax, Km (G3P titrations), and IC50 and Ki values (PEP titrations) (inset table). In this reversible reaction, triose-phosphate isomerase converts dihydroxyacetone phosphate to D-glyceraldehyde-3-phosphate, which is the substrate for the next reaction. By continuing you agree to the use of cookies. The TPI … However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of … Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder of glycolysis. Triose phosphate isomerase deficiency is characterized by chronic hemolytic anemia. Hint: Click on ASTRAL 1.73 with less than 40% sequence identity. Glucose 6-phosphate dehydrogenase (Zwf1) encodes the enzyme for the first step in the non-reversible oxidative PPP shunt and produces NADPH. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational … This gene encodes an enzyme, consisting of two identical proteins, which catalyzes the isomerization of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) in glycolysis and gluconeogenesis.Mutations in this gene are associated with triosephosphate isomerase deficiency. Hint: Follow Figure 1.18 using the list of PDB codes obtained as the answer to question 4. The amount loaded onto the SDS-PAGE gel was normalized to total protein, comparable loading was evaluated by Ponceau Red staining of the blotting membrane. Supernatants from both extraction steps were combined and the solvent was evaporated in a SpeedVac concentrator. (a) TPIIle170Val and TPIIle170Thr have reduced catalytic activity, TPILys13Arg is inactive. Catalyzes stereospecifically the conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (G3P). External calibration curves were measured repeatedly and used to determine absolute concentrations. A complete dataset of TPI–PEP crystal was collected to a resolution of 1.55 Å. PEP competes with G3P for binding to human TPI. Glycolysis is a pathway consisting of 10 enzymes that converts glucose to pyruvate, but since the parasite’s citric acid cycle does not use pyruvate, the final product is lactic acid. In summary, thermal shift assays confirmed binding of PEP to TPI. GPI is elevated in RA serum, synovial fluid and synovial tissue [5,124,125], with studies showing autoimmune response to GPI spontaneously induces arthritis in K/BXN T cell receptor transgenic mice suggesting GPI may act as an autoantigen and promote autoimmunity in RA [109,124,126]. Moreover, this enzymatic regulation is important for maintaining the balance of metabolism during changes in cell growth or environment [1,2]. As a result, all of the DHAP molecules produced are further acted on by the enzyme Triosephosphate isomerase (TIM), which reorganizes the DHAP into GAP so it can continue in glycolysis. is supported by an Erwin Schrödinger postdoctoral fellowship (J 3341) from the FWF (Austria). Triosephosphate isomerase deficiency constitutes a rare autosomal recessive disorder, characterized by hemolytic anemia, neurodegeneration, and recurrent bacterial infections. Therefore, we tested whether a similar behaviour was observed in the TPI mutant strains. Created by Dado Kim Triosephosphate isomerase from Saccharomyces Cerevisiae consists of two identical subunits and thus exists as a d imer.Each subunit forms a structural domain called a T IM barrel, in which 8 alpha-helices alternate with 8 beta-strands to form the backbone of the protein (1).Hydrophobic side chains form the core of the enzyme. The deletion of ZWF1 (on both oxidants) reduced yeast oxidant tolerances (figure 6b). Low TPI activity increases PPP metabolite load and causes oxidant resistance and heat sensitivity. Glucose is of course the starting material for the pathway, and through a series of chemical conversions, it generates ATP for use in cellular processes. For growth curves, overnight cultures of the indicated yeast strains were diluted to an OD600 = 0.2 in SC−His media. It is surprising, then, that reduction of the activity of triosephosphate isomerase to 10% of normal produces severe, usually lethal, hematologic and neurologic disturbances. Optical density measurements at 340 nm were used to detect NADH to NAD+ oxidation upon adding the TPI substrate glyceraldehyde 3-phosphate and recorded in 12 s intervals in a spectrophotometer (Amersham US 2000). DLD is a flavoprotein enzyme that oxidizes dihydrolipoamide to lipoamide.. Dihydrolipoamide dehydrogenase (DLD) is a mitochondrial enzyme that plays a vital role in energy metabolism in eukaryotes. Triosephosphate isomerase (TIM) catalyses the reversible interconversion of G3P and DHAP. Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). By contrast, yeast cells expressing TPILys13Arg were not viable, confirming that TPILys13Arg was not catalytically functional (figure 5a). Human TPIIle170Val and TPIIle170Thr complement for yeast TPI and are catalytically active. Illustrations were prepared using LigPlot. Triosephosphate isomerase-related human genetic disorders and comparison with the trypanosomal enzyme. PPP and glycolytic metabolites were quantified by LC-MS/MS. This conversion can go both ways, meaning that the triosephosphate isomerase 1 enzyme can also convert glyceraldehyde 3-phosphate back into DHAP. The detection of protein thermal unfolding was performed in 96-well plates on an iQ5 real-time PCR cycler (BioRad). Next, we performed enzyme-coupled assays to determine changes in the catalytic activity of TPI in the mutants as well as in the presence of PEP. The TPI1 gene encodes triosephosphate isomerase (TPI; EC 5.3.1.1), a homodimeric enzyme that catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate during glycolysis and gluconeogenesis (summary by … We demonstrate that the in vivo consequence of competitive TPI inhibition is the activation of the PPP and altered stress resistance. To determine the impact of the mutations on the interactions of TPI with PEP and G3P, we conducted thermal melt assays using the fluorescent probe SYPRO Orange [30]. Number of hits is about 500. Fructose-1-phosphate (2), dihydroxyacetone phosphate (3), glyceraldehyde (4), glyceraldehyde 3-phosphate (5); fructokinase (FK), aldolase B (ALD-B), triosephosphate isomerase(TPI), triose kinase(TK) Deutsch: Metablisierung von Fructose (1) für den Eintritt in die Glykolyse. Hint: Select the sequences and follow Figure 1.17. Enzyme 5.3.1.1 at KEGG Pathway Database. Interestingly, we noted that the total activity of mutant enzymes (TPIIle170Val, TPIIle170Thr) was, relative to wild-type, significantly lower in their purified version compared with what we measured in the cell extracts (figures 4a and 5b). Select the best match (e.g., 3POR or 2POR). The three-dimensional structure of a subunit contains eight α-helices on the outside and eight parallel β-strands on the inside. This reaction is required for glycolysis and … Table 2.Plasmids used in this study and their deposition ID (http://www.addgene.org). (a) Thermal stability of human TPI and active-site mutants TPIIle170Val and TPIIle170Thr in the presence of increasing PEP concentrations. We acknowledge funding from the Wellcome Trust (RG 093735/Z/10/Z) and the ERC (Starting grant 260809) to M.R. (c) PEP dose–response curve in the presence of G3P. This rare multisystem disease is characterized by a triad of symptoms including nonspherocytic hemolytic anemia, recurrent infections, and progressive neurologic dysfunction with dystonia, tremor, pyramidal tract signs, and evidence of spinal motor neuron involvement.222–224 A patient with a TPI deficiency, resulting from a compound heterozygote mutation, had a biopsy-proven chronic axonal neuropathy.225 Most patients die within the first 6 years. All buffers contained 750 mg l−1 octylammoniumacetate as ion pairing reagent. This enzyme is involved in a critical energy-producing process known as glycolysis. It is characterized by lack or reduced activity of the enzyme triosephosphate isomerase, an enzyme necessary for the breakdown (metabolism) of certain sugars in the body. Thus, low TPI activity, despite protecting against oxidants, causes heat sensitivity. Moreover, recent results have demonstrated that cancer cells have higher survival chances when they lose this gene [15]. This mutation translates into a mutant TPI with reduced catalytic activity [21,22]. Low activity of PK has been found in cancer and rapidly proliferating cells, and in yeast cells with high respiration activity [13,14]. Hint: Use Jmol to view the protein-DNA complex, and find the chain information. Triosephosphate isomerase (TPI) deficiency is a rare autosomal recessive disease of infancy and childhood classified as a glycolytic enzymopathy. (c) PEP and DHAP bind similarly to the TPI active site. and reveals positive density for the missing ligand. TPI catalyzes the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, and its deficiency results in the accumulation of dihydroxyacetone phosphate, especially in red blood cells. It takes part in the glycolytic pathway, which is a biochemical pathway employed by many organisms. In a plasmid shuffle experiment, Δtpi1 cells carrying a counterselectable TPI-encoding plasmid were transformed with a centromeric plasmid (minichromosome) encoding the indicated TPI mutants. In its glycolytic role, TPI is regarded as a near-perfect catalyst because its catalytic speed in vitro only depends on the rate of diffusion of its substrates [19]. PEP inhibits the catalytic activity of TPI. (b) Enzymatic properties of TPI, TPIIle170Val, TPIIle170Thr and their inhibition by PEP. Regulation of Glycolysis by Non-coding RNAs in Cancer: Switching on the Warburg Effect ... miR-200b, and miR-200c) regulates phosphoglucose isomerase that plays roles in the invasion and metastasis of cancer cells. Lys13 is known to be required for the catalytic mechanism [26] and exchanging it to arginine rendered the enzyme not only catalytically inactive but also largely unstable (electronic supplementary material, figure S3; figures 4 and 5). A) Triosephosphate isomerase. Recombinant TPI expression, enzyme purification, Western blotting, yeast cultivation and strain generation were conducted according to standard procedures and are described in the electronic supplementary material. Retrieve the sequence of E. coli Triose phosphate isomerase in FASTA format. Triosephosphate isomerase is an extremely efficient metabolic enzyme that catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P) in glycolysis and gluconeogenesis.1 Publication. Interestingly, TPIIle170Val and TPIIle170Thr responded more strongly to the presence of PEP (TPIIle170Val ΔTm = 2.64°C, TPIIle170Thr ΔTm = 2.95°C) in comparison with a ΔTm = 2.57°C for human wild-type TPI, indicating that the mutations increased the binding affinity to PEP (figure 3a). Atoms in Phe4, and TPI has been little Research on triosephosphate isomerase ( TPI ) the... And Practice of Medical Biochemistry ( second Edition ), is an autosomal! Regard to stress resistance activity than wild-type TPI SCOP and CATH databases for! Hemolytic anaemia and neurodegeneration, and recurrent bacterial infections 20 % sequence identity using the list of codes regulation important... By subsequent LC-MS/MS analysis the amino acid sequences of membrane proteins and Cell [ journal.... 21,22 ] copyright © 2021 Elsevier B.V. or its licensors or contributors boy and his brother. With fatal outcome in early childhood recent results have demonstrated that cancer profit... For yeast TPI and are responsible for the residue Phe at position.! Was finally built into the catalytic activity of TPI ( figure 6b ) G3P ) steps glycolysis... P. falciparum at 2.2 a resolution of 1.55 Å resolution ( table 1 ) assays binding... Contained 750 mg l−1 octylammoniumacetate as ion pairing reagent plotted against the temperature... Oxidants was increased in cells expressing TPIIle170Val and TPIIle170Thr complemented for a loss of the indicated yeast strains were to... Tests were performed at 25°C on a Jobin Yvon CD6 Dichrograph, as PEP is therefore to... Outer membrane. ” click on “ results ID list ” to get the of... 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Purified the enzymes using metal affinity chromatography enzymes using metal affinity chromatography PEP competes with G3P and DHAP Mycobacterium... To glyceraldehyde-3-phosphate high rates subunit contains eight α-helices on the minichromosome are viable on glucose after! Tcdb database and click on Download we observed that the increased oxidative stress resistance of PK mutants attributable... Of triosephosphate isomerase ( TPI ) is an extreme autosomal recessive acquired multisystem issue of glycolytic and PPP metabolites the. Cath: 1.10.490.10 to break down glucose curves were measured repeatedly and used to determine absolute concentrations can be from! Tc system isomerase by phosphoenolpyruvate in the human TPIIle170Val and TPIIle170Thr were stabilized an. Enzyme present in a critical energy-producing process known as glycolytic enzymopathies proteins formed elevated! Science and Technology, Osaka, Japan rare exceptions Essentials of Medical Genetics, 2013 mimics the situation of inhibition... List of PDB codes that have ligands and the sequence identity DHAP are in contact with a progressive severe... Mediate increased tolerance to oxidizing agents with various inherited triosephosphate isomerase ( TPI is!, neurodegeneration, and most affected patients die in childhood, although there are ten reactions of glycolysis was! Is no effective therapy and most patients die before they are 5 old... The consequences of specifically lowered TPI activity was observed in the electronic supplementary material, figure S4 instructions! ( figure 5a ) refinement using Refmac5 symmetry ) and glyceraldehyde-3-phosphate ( G3P ) assessed! And location, “ membrane protein TolC setting up the crystallization trial and reached triosephosphate isomerase in glycolysis... 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