Multiwavelength Anomalous Diffraction Research Articles

Modes and model building in SHELXE.

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson-Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.

Fast, automated, continuous energy scans for experimental phasing at the BioMAX beamline.

In X-ray macromolecular crystallography (MX), single-wavelength anomalous dispersion (SAD) and multi-wavelength anomalous dispersion (MAD) techniques are commonly used for obtaining experimental phases. For an MX synchrotron beamline to support SAD and MAD techniques it is a prerequisite to have a reliable, fast and well automated energy scan routine. This work reports on a continuous energy scan procedure newly implemented at the BioMAX MX beamline at MAX IV Laboratory. The continuous energy scan is fully automated, capable of measuring accurate fluorescence counts over the absorption edge of interest while minimizing the sample exposure to X-rays, and is about a factor of five faster compared with a conventional step scan previously operational at BioMAX. The implementation of the continuous energy scan facilitates the prompt access to the anomalous scattering data, required for the SAD and MAD experiments.

Structurally Precise Two-Transition-Metal Water Oxidation Catalysts: Quantifying Adjacent 3d Metals by Synchrotron X-Radiation Anomalous Dispersion Scattering.

Mixed 3d metal oxides are some of the most promising water oxidation catalysts (WOCs), but it is very difficult to know the locations and percent occupancies of different 3d metals in these heterogeneous catalysts. Without such information, it is hard to quantify catalysis, stability, and other properties of the WOC as a function of the catalyst active site structure. This study combines the site selective synthesis of a homogeneous WOC with two adjacent 3d metals, [Co2Ni2(PW9O34)2]10- (Co2Ni2P2) as a tractable molecular model for CoNi oxide, with the use of multiwavelength synchrotron X-radiation anomalous dispersion scattering (synchrotron XRAS) that quantifies both the location and percent occupancy of Co (∼97% outer-central-belt positions only) and Ni (∼97% inner-central-belt positions only) in Co2Ni2P2. This mixed-3d-metal complex catalyzes water oxidation an order of magnitude faster than its isostructural analogue, [Co4(PW9O34)2]10- (Co4P2). Four independent and complementary lines of evidence confirm that Co2Ni2P2 and Co4P2 are the principal WOCs and that Co2+(aq) is not. Density functional theory (DFT) studies revealed that Co4P2 and Co2Ni2P2 have similar frontier orbitals, while stopped-flow kinetic studies and DFT calculations indicate that water oxidation by both complexes follows analogous multistep mechanisms, including likely Co-OOH formation, with the energetics of most steps being lower for Co2Ni2P2 than for Co4P2. Synchrotron XRAS should be generally applicable to active-site-structure-reactivity studies of multi-metal heterogeneous and homogeneous catalysts.

Multiwavelength anomalous X-ray diffraction for combined imaging of atomic displacement and strain.

The X-ray Bragg coherent diffractive imaging (CDI) technique assumes that the structure factor holds constant over the measured crystal. This approximation breaks down for materials exhibiting variations in the unit-cell configuration, such as piezo- and ferroelectrics. In that case, the strain field cannot be reliably determined from the reconstruction because the lattice deformation and the structure factor contribute concomitantly. Proposed here is a solution to this problem achieved by combining Bragg CDI and the multiwavelength anomalous diffraction approach that measures a Friedel pair of reflections at two different photon energies near an absorption edge. Comparing the obtained reconstructions with a parametric model that includes calculating the scattering amplitude as a function of wavelength and the unit-cell configuration, the contributions of the lattice deformation and the structure factor are separated. Simulations of the ferroelectric material BaTiO3 demonstrate the possibility of simultaneous probing of the strain and displacement of the Ti atoms. The proposed method opens up an opportunity to apply coherent X-ray diffraction for nanoscale-resolved 3D mapping of polarization domains in micro- and nanocrystals.

Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid.

The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

SHELIXIR: automation of experimental phasing procedures using SHELXC/D/E

The program SHELIXIR represents a simple and efficient tool for routine phase-problem solution using data for experimental phasing by the single-wavelength anomalous dispersion, multiwavelength anomalous dispersion, single isomorphous replacement with anomalous scattering and radiation-damage-induced phasing methods. As indicated in its name, all calculation procedures are performed with the SHELXC/D/E program package. SHELIXIR provides screening for alternative space groups, optimal solvent content, and high- and low-resolution limits. The procedures of SHELXE are parallelized to minimize the computational time. The automation and parallelization of such procedures are suitable for phasing at synchrotron beamlines directly or for finding the optimal parameters for further data processing. A simple graphical interface is designed to make use easier and to increase efficiency during beam time.

Revealing redox isomerism in trichromium imides by anomalous diffraction.

In polynuclear biological active sites, multiple electrons are needed for turnover, and the distribution of these electrons among the metal sites is affected by the structure of the active site. However, the study of the interplay between structure and redox distribution is difficult not only in biological systems but also in synthetic polynuclear clusters since most redox changes produce only one thermodynamically stable product. Here, the unusual chemistry of a sterically hindered trichromium complex allowed us to probe the relationship between structural and redox isomerism. Two structurally isomeric trichromium imides were isolated: asymmetric terminal imide (tbsL)Cr3(NDipp) and symmetric, μ3-bridging imide (tbsL)Cr3(μ3–NBn) ((tbsL)6− = (1,3,5-C6H9(NC6H4-o-NSitBuMe2)3)6−). Along with the homovalent isocyanide adduct (tbsL)Cr3(CNBn) and the bisimide (tbsL)Cr3(μ3–NPh)(NPh), both imide isomers were examined by multiple-wavelength anomalous diffraction (MAD) to determine the redox load distribution by the free refinement of atomic scattering factors. Despite their compositional similarities, the bridging imide shows uniform oxidation of all three Cr sites while the terminal imide shows oxidation at only two Cr sites. Further oxidation from the bridging imide to the bisimide is only borne at the Cr site bound to the second, terminal imido fragment. Thus, depending on the structural motifs present in each [Cr3] complex, MAD revealed complete localization of oxidation, partial localization, and complete delocalization, all supported by the same hexadentate ligand scaffold.

Nanoscale x-ray investigation of composition fluctuations in AlGaN nanowires

In the present study we combined, in the same synchrotron x-ray experiment, reciprocal space mapping, multiwavelength anomalous diffraction and diffraction anomalous fine structure, to determine the strain, crystallographic polarity, alloy composition and ordering at the atomic scale in [0001]-oriented AlGaN nanowires grown by molecular beam epitaxy on GaN nanowire bases. The information that we obtained was averaged over a macroscopic ensemble of NWs. We found from the diffraction anomalous fine structure that there were an isotropic increased number of Ga-Ga pairs in the Ga next nearest coordination shell (cation sublattice) with respect to what is expected for the AlGaN alloy composition determined by anomalous diffraction. This significant deviation from random alloy atomic distribution is present whatever the AlN molar fraction and growth conditions. Our results are consistent with nanoscale composition fluctuations expected from both alloy disorder or kinematically driven spontaneous ordering, both effects being suspected to account for the physical properties of AlGaN ternary alloys.

Heterotrimetallic Precursor with 2:2:1 Metal Ratio Requiring at Least a Pentanuclear Molecular Assembly.

This work represents an important step in the quest to make heteromultimetallic molecules featuring specific metal types and complicated metal ratios. The rational design, synthesis, and characterization of a complex heterotrimetallic single-source molecular precursor for the next generation sodium-ion battery cathode material, Na2Mn2FeO6, is described. A unique pentametallic platform [MnII(ptac)3-Na-MnIII(acac)3-Na-MnII(ptac)3] (1) was derived from the known polymeric structure of [NaMnII(acac)3]∞, through a series of elaborate design procedures, such as mixed-ligand, unsymmetric ligand, and mixed-valent approaches. Importantly, the application of those techniques results in a molecule with distinctively different transition metal positions in terms of ligand environment and oxidation states. An isovalent substitution of FeIII for the central MnIII ion forms the target heterotrimetallic precursor [MnII(ptac)3-Na-FeIII(acac)3-Na-MnII(ptac)3] (3) with an appropriate metal ratio of Na:Mn:Fe = 2:2:1. The arrangement of metal ions and ligands in this pentametallic assembly was confirmed by single crystal X-ray investigation. The unambiguous assignment of the positions and oxidation states of the Periodic Table neighbors Fe and Mn in 3 has been achieved by a combination of investigative techniques that include synchrotron resonant diffraction, X-ray multiwavelength anomalous diffraction, X-ray fluorescence spectroscopy, Mössbauer spectroscopy, and gas-phase DART mass spectrometry. The heterotrimetallic single-source precursor 3 was shown to exhibit a clean decomposition pattern yielding the phase-pure P2-Na2Mn2FeO6 quaternary oxide with high uniformity of metal ion distribution as confirmed by electron microscopy.

Crystal structure of the flagellin protein FlaG from Helicobacter pylori

AbstractFlagella, comprising flagellin proteins, are essential virulence factors for Helicobacter pylori to colonize human stomach mucosa. The flagellin‐associated flaG operon of H. pylori consists of the flaG, fliD, and fliS genes under the control of a σ28‐dependent promoter. The flaG gene is involved in chemotaxis and motility. We cloned, expressed, and purified the H. pylori flaG gene encoding the flagellin protein HpFlaG. Sequence alignment revealed that HpFlaG exhibits low sequence identity with other FlaG proteins. Overall, N‐terminal sequences of FlaG proteins are mostly divergent, and C‐terminal regions might be important for dimer interactions between protein subunits. Here, we report the crystal structure of the N‐terminal truncated protein (NT‐HpFlaG), as determined by multiwavelength anomalous dispersion at a resolution of 2.7 å. The overall structure of NT‐HpFlaG consists of two helices and three strands, folded into a palm‐like conformation. Two monomers strongly interact as a dimer by hydrophobic coiled‐coil interactions. Based on our structure, we suggest that the functional state of HpFlaG is as a dimer.

Enhancement of MAD/MIR phasing at low resolution and a new procedure for automatic phase extension**Project supported by the National Basic Research Program of China (Grant No. 2011CB911100) of the Ministry of Science and Technology of China.

To achieve de novo protein structure determination of challenging cases, multi-wavelength anomalous diffraction (MAD) and multiple isomorphous replacement (MIR) phasing can be powerful tools to obtain low-resolution initial phases from heavy-atom derivative datasets, then phase extension is needed against high-resolution data to obtain accurate structures. In this context, we propose a direct-methods procedure here that could improve the initial low-resolution MAD/MIR phase quality. And accordingly, an automated process for extending initial phases to high resolution is also described. These two procedures are both implanted in the newly released IPCAS pipeline. Three cases are used to perform the test, including one set of 4.17 Å MAD data from a membrane protein and two sets of MAD/MIR data with derivatives truncated down to 6.80 Å and 6.90 Å, respectively. All the results have shown that the initial phases generated from the direct-methods procedure are better than that from the conventional MAD/MIR methods. The automated phase extensions for the latter two cases starting from 6.80 Å to 3.00 Å and 6.90 Å to 2.80 Å are proved to be successful, leading to complete models. This may provide convenient and reliable tools for phase improvement and phase extension in difficult low-resolution tasks.

PF505 FIRST‐IN‐HUMAN SINGLE‐ AND MULTIPLE‐ASCENDING DOSE STUDY OF THE ORAL FERROPORTIN INHIBITOR VIT‐2763 IN HEALTHY SUBJECTS

Background:Ferroportin (FPN), the only known iron transporter in mammals, is mainly expressed in tissues of iron absorption (duodenum), recycling and storage (liver and mononuclear phagocyte system). Through its action on FPN, hepcidin controls plasma iron levels. VIT‐2763 is the first oral FPN inhibitor that has been shown to lower plasma iron levels in preclinical pharmacology studies. In addition, VIT‐2763 ameliorated ineffective erythropoiesis in a thalassemia disease model.Aims:A Phase I, first‐in‐human, double–blind, randomized, placebo‐controlled study was performed to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of single‐ and multiple‐ascending doses (SAD and MAD) of VIT‐2763 in healthy male or female subjects. EudraCT no. 2017–003395‐31.Methods:In Part A (SAD), subjects received a single oral dose of VIT‐2763 in five cohorts: 5/15/60/120/240 mg. Part B (MAD) involved four VIT‐2763 dose cohorts: oral VIT‐2763 60/120 mg once daily (QD) or 60/120 mg twice daily (BID) for 7 days. In both parts, subjects were randomized 3:1 into each cohort to receive VIT‐2763 or placebo. Safety and tolerability (primary endpoint) were assessed throughout. Serial blood samples were collected for analysis of PK and PD (including but not limited to serum iron and hepcidin levels). PK parameters were determined by non‐compartmental analysis.Results:In Parts A and B, 38 (29 active, 9 placebo) and 34 subjects (26 active, 8 placebo) were enrolled respectively.Treatment with single and multiple oral doses was well tolerated. In Part A, 32 treatment‐emergent adverse events (TEAEs), all of which were mild, occurred in 53% of subjects; in Part B, 96 TEAEs (all mild/moderate) occurred in 85% of subjects. All TEAEs were transient and self‐limiting. There were no findings of clinical relevance with respect to vital signs, 12‐lead ECG, telemetry or physical examination. There were no serious or severe TEAEs nor discontinuations due to TEAEs following single or multiple dosing.The initial oral absorption of VIT‐2763 was relatively fast; most subjects had detectable levels 15–30 min post dose and median Tmax was 0.5–3 hours. A second peak in the concentration–time profile occurred 3–4 hours post dose. The exposure over the dose range of 5–240 mg was slightly more than dose proportional for Cmax, AUC0last and AUC0‐inf. Following multiple oral dosing up to 120 mg BID for 7 days there was no apparent change in absorption and accumulation was minimal. The geometric mean elimination t 1/2 was 1.8–5.3 hours following single dosing, and 2.1‐3.8 hours on Day 1 and 2.6–5.3 hours on Day 7, following repeated dosing.Single and multiple doses of VIT‐2763 ≥60 mg lowered serum iron, which remained below baseline values for up to 24 hours post‐dose, following BID dosing in the MAD phase (see Figure). There was also a temporary increase in mean serum hepcidin levels. This iron‐lowering pharmacodynamic effect is consistent with observations in preclinical models. No dose‐related effects were observed with respect to serum ferritin, transferrin, erythropoietin or soluble transferrin receptor levels on Day 1 or 7.Summary/Conclusion:VIT‐2763 administered at single oral doses up to 240 mg, or multiple oral doses up to 120 mg BID was safe and well tolerated and achieved rapid and prominent decreases in circulating iron levels in healthy subjects. This iron‐lowering pharmacodynamic effect is consistent with observations in preclinical models. Results of this study support the initiation of a proof‐of concept study of VIT‐2763 in patients with β‐thalassemia and documented iron overload.image

MAD Phase I Study to Investigate Contraloid Acetate

MAD Phase I Study to Investigate Contraloid Acetate

A practical method for efficient and optimal production of Seleno-methionine-labeled recombinant protein complexes in the insect cells.

The use of Seleno-methionine (SeMet) incorporated protein crystals for single or multi-wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared with native protein expression.

Structural basis of the cystein protease inhibitor Clonorchis sinensis Stefin-1

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel β-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

Multi-wavelength anomalous diffraction de novo phasing using a two-colour X-ray free-electron laser with wide tunability

Serial femtosecond crystallography at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems prone to radiation damage. However, de novo structure determination, i.e., without prior structural knowledge, is complicated by the inherent inaccuracy of serial femtosecond crystallography data. By its very nature, serial femtosecond crystallography data collection entails shot-to-shot fluctuations in X-ray wavelength and intensity as well as variations in crystal size and quality that must be averaged out. Hence, to obtain accurate diffraction intensities for de novo phasing, large numbers of diffraction patterns are required, and, concomitantly large volumes of sample and long X-ray free-electron laser beamtimes. Here we show that serial femtosecond crystallography data collected using simultaneous two-colour X-ray free-electron laser pulses can be used for multiple wavelength anomalous dispersion phasing. The phase angle determination is significantly more accurate than for single-colour phasing. We anticipate that two-colour multiple wavelength anomalous dispersion phasing will enhance structure determination of difficult-to-phase proteins at X-ray free-electron lasers.

Crystal structure of human WBSCR16, an RCC1-like protein in mitochondria.

WBSCR16 (Williams-Beuren Syndrome Chromosomal Region 16) gene is located in a large deletion region of Williams-Beuren syndrome (WBS), which is a neurodevelopmental disorder. Although the relationship between WBSCR16 and WBS remains unclear, it has been reported that WBSCR16 is a member of a functional module that regulates mitochondrial 16S rRNA abundance and intra-mitochondrial translation. WBSCR16 has RCC1 (Regulator of Chromosome Condensation 1)-like amino acid sequence repeats but the function of WBSCR16 appears to be different from that of other RCC1 superfamily members. Here, we demonstrate that WBSCR16 localizes to mitochondria in HeLa cells, and report the crystal structure of WBSCR16 determined to 2.0 Å resolution using multi-wavelength anomalous diffraction. WBSCR16 adopts the seven-bladed β-propeller fold characteristic of RCC1-like proteins. A comparison of the WBSCR16 structure with that of RCC1 and other RCC1-like proteins reveals that, although many of the residues buried in the core of the β-propeller are highly conserved, the surface residues are poorly conserved and conformationally divergent.

Structural basis for the regulatory interactions of proapoptotic Par-4

Par-4 is a unique proapoptotic protein with the ability to induce apoptosis selectively in cancer cells. The X-ray crystal structure of the C-terminal domain of Par-4 (Par-4CC), which regulates its apoptotic function, was obtained by MAD phasing. Par-4 homodimerizes by forming a parallel coiled-coil structure. The N-terminal half of Par-4CC contains the homodimerization subdomain. This structure includes a nuclear export signal (Par-4NES) sequence, which is masked upon dimerization indicating a potential mechanism for nuclear localization. The heteromeric-interaction models specifically showed that charge interaction is an important factor in the stability of heteromers of the C-terminal leucine zipper subdomain of Par-4 (Par-4LZ). These heteromer models also displayed NES masking capacity and therefore the ability to influence intracellular localization.

The Bial 10-2474 Phase 1 Study-A Drug Development Perspective and Recommendations for Future First-in-Human Trials.

BIA 10-2474 (a fatty acid amide hydrolase inhibitor) was evaluated in a first-in-human phase 1 study in normal volunteers to assess safety/tolerability, pharmacokinetics, pharmacodynamics, and food effect. The dose-escalation process consisted of a single-ascending-dose phase (SAD) and multiple-ascending-dose phase (MAD). Prospective determination of the starting dose and maximal escalated dose was consistent with the usual clinical pharmacology principles for extrapolation of preclinical toxicology data to human equivalent doses. After only 5-6 days of multiple-dose administration of 50mg daily in the MAD phase, several subjects became quite ill with central nervous system symptoms. One subject progressed to brain death within several days of symptom onset. Magnetic resonance imaging scans demonstrated signal abnormalities consistent with microbleeds affecting the hippocampus and pons, suggestive of possible cytotoxic or vasogenic edema compatible with a toxic/metabolic process. There were no findings at lower MAD doses or during the SAD phase. The toxicology program carried out in 4 preclinical species (mouse, rat, dog, and monkey) did not demonstrate significant neurotoxicity. The probable mechanism of neurologic toxicity demonstrated in humans at the 50-mg daily dose was inhibition of off-target cerebral receptors or through another mechanism. Additional recommendations have been proposed for future first-in-human studies to maximize subject safety. However, one must also accept the basic premise that, in general, first-in-human phase 1 studies are remarkably safe, and these rare events are not 100% avoidable during the drug development process.

Bio‐incorporation of Amino Acid Analogs into Target Proteins

The 3D structure of a protein is vital for understanding how it functions in both normal conditions as well as in disease. X‐ray crystallography has been the traditional method of determining protein structure, however, the small atoms that compose amino acids are problematic in this approach. This phase problem can be solved through multi‐wavelength anomalous diffraction (MAD) if a heavy atom exists in the protein. Incorporating telluromethionine (TeMet), which includes a tellurium atom in place of the lighter sulfur atom, into proteins will assist in MAD measurements. This is done by providing only TeMet to an auxotrophic Escherichia coli strain that is unable to produce methionine, causing the synthesized proteins to include TeMet. However, it has been shown that TeMet is toxic to cells, and if the cells die, an insufficient amount of the target protein will be synthesized for crystallization. To reduce the toxic effects of TeMet, the E. coli cells have been cultured in chemically defined media with components necessary for cell growth that seem to be lost with incorporation of TeMet. By measuring the absorbance of the cultures over time, the growth curve of the cells can be constructed. Once the optimum growing conditions have been determined, the target protein, dihydrofolate reductase, will be isolated and characterized. Successful incorporation of TeMet will make solving the 3D structure of proteins more accessible, allowing for enhanced disease treatment and drug design.