Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods

Abstract

We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L-plectasin and D-plectasin were prepared by total chemical synthesis; interestingly, L-plectasin showed the expected antimicrobial activity, while D-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L-plectasin molecule and one D-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.

Introduction

The antimicrobial natural product plectasin, recently discovered by Christensen and coworkers, is a small protein that has broad activity against several species of gram-positive bacteria making it a promising drug candidate.1 Plectasin is the first defensin isolated from a fungus (Pseudoplectania nigrella). In mice, this small protein was as effective as vancomycin and penicillin against S. pneumoniae; an important advantage of plectasin is that it showed potent activity against various clinical strains of S. pneumoniae that are resistant to conventional antibiotics.1 An NMR structure has been reported,1 but as yet no high resolution X-ray crystal structure has been reported for plectasin.

We set out to determine the X-ray crystal structure of plectasin. Recently, we showed that crystallization of a protein molecule from a racemic mixture (i.e. a solution containing equal proportions of L-protein and D-protein enantiomers) can result in formation of centrosymmetric crystals.2, 3 It has been suggested that the availability of highly diffracting centrosymmetric protein crystals will facilitate ab initio structure solution by direct methods, because all reflections from centrosymmetric crystals have quantized phases (e.g. in P1, all phases are 0 or π).4 However, of the centrosymmetric racemic protein structures reported to date,2, 3, 5, 6 only the small scorpion toxin protein BmBKTx1 has been solved by direct methods,3 although three small peptides with not more than 12 amino acid residues have also been solved as racemates by direct methods.7–9

To obtain a racemic protein crystal it is necessary to prepare the D-protein, i.e. the enantiomer of the native L-protein; this can only be achieved by total chemical synthesis of the protein molecule.10–12 Synthesis of a mirror image D-protein is more feasible now than it was just a short time ago, using modern methods based on native chemical ligation.13, 14 In this article, we report efficient total chemical syntheses of L-plectasin and D-plectasin, their activities in antimicrobial assays, and the determination by racemic crystallography of the X-ray structure of the plectasin molecule at atomic resolution (1.0 Å) using direct methods.

Results

Chemical synthesis of L-plectasin and D-plectasin

Plectasin is a protein of 40 amino acid residues containing six cysteine residues that form three disulfides in the folded protein molecule.1 The small size and the presence of six Cys residues make plectasin an ideal target for total synthesis by native chemical ligation, which involves the thioester-mediated covalent condensation of two unprotected peptide segments at cysteine.13, 14 The target sequence and the synthetic strategy used to prepare plectasin are shown in Scheme 1.

Scheme 1. (a) Amino acid sequence of plectasin.1 (b) Synthetic strategy used for the total chemical synthesis of plectasin by native chemical ligation. R = -CH₂CH₂CO-Ala-COOH.

The peptide-thioester and the Cys-peptide building blocks were prepared by manual stepwise solid phase peptide synthesis using Boc chemistry “in situ neutralization” protocols.15 The ligation of the peptide-^αthioester and the Cys-peptide was carried out on a multiple-tens-of-milligrams scale, followed by deformylation of the single Trp residue and solid phase extraction to remove residual thiols and salts. The resulting crude lyophilized full-length peptide was used in a folding reaction carried out at pH 8.4 in 1M guanidine hydrochloride aqueous buffer containing a cysteine/cystine redox couple; folding and concomitant disulfide bond formation was essentially complete within 2–3 h, as evidenced by earlier elution in reverse phase HPLC and a mass decrease of 6 Daltons, corresponding to the formation of three disulfides, for the product compared with the linear peptide. Essentially identical results were obtained for both the native L-plectasin and the D-plectasin molecules. The folded protein molecules were purified by preparative HPLC and characterized by LC-MS (see Fig. 1). Amounts obtained were 51 mg (45%, based on the limiting peptide segment) of L-plectasin and 19 mg (42%) of D-plectasin. Data for the syntheses of native L-plectasin and D-plectasin are shown in Figures S1–S6 (Supporting Information). As expected, the circular dichroism (CD) spectra of the chemically synthesized protein enantiomers were found to be identical in shape and magnitude, but opposite in sign, as shown in Figure S7 (Supporting Information).

Figure 1. The LC-MS profiles of the purified synthetic plectasin enantiomers. (a) L-plectasin (obs. = 4401.2 ± 0.5 Da, calc. = 4400.8 Da (high point of isotope distribution) (b) D-plectasin (obs. = 4401.2 ± 0.5 Da, calc. = 4400.8 Da (high point of isotope distribution). The chromatographic separations were performed using a linear gradient (5%–65%) of buffer B in buffer A over 15 min (buffer A = 0.1% trifluoroacetic acid (TFA) in water; buffer B = 0.08% TFA in acetonitrile) on an in-house packed 3 μm C-4, 2.1 × 50 mm column at 40°C with detection at 214 nm and on-line ion trap electrospray MS.

Assays for antimicrobial activity

Although the mechanism by which plectasin exerts its antimicrobial activity is not well understood, plectasin has previously been shown to exhibit antibacterial activity against Gram-positive but not Gram-negative bacteria.1 Consistent with this, we found that synthetic L-plectasin was able to inhibit growth of three gram-positive bacteria (Staphylococcus aureus clinical strains newman and USA300 as well as Bacillus cereus); however, synthetic D-plectasin did not inhibit bacterial growth in the same assays at similar concentrations (see Supporting Information).

Determination of the X-ray crystal structure of plectasin

Racemic plectasin was crystallized from a solution containing equal amounts of the L- and D-protein enantiomers. Sparse-matrix crystallization screening was carried out at 19°C. Twelve of the 96 initial crystallization conditions produced crystals overnight at a protein concentration of 12 mg/mL (i.e. 6 mg of each enantiomer), and seven at 6 mg/mL (i.e. 3 mg of each enantiomer). Four sets of conditions were optimized to produce crystals suitable for X-ray diffraction. Diffraction data from four crystals were collected at the Advanced Photon Source. For structure determination we selected a racemic crystal that diffracted to atomic resolution (1.0 Å).

Diffraction intensity statistics revealed that the protein racemate had crystallized in the centrosymmetric space group P1. There was one enantiomer in the asymmetric unit and two molecules (one D-plectasin, one L-plectasin) in the unit cell. With atomic-resolution diffraction data in hand, the crystal structure was solved by direct methods16, 17 using the program SHELXS18 in a 5-h computational run on a standard dual core Xeon processor. The best structure solution had a figure of merit of 0.205 and, after preliminary refinement with SHELXL,18 produced interpretable electron density maps for the entire molecule, including both N- and C-termini. The L-polypeptide including all the side chains of the amino acid sequence was then manually built using the program TURBO-FRODO.19 After placement of solvent molecules and hydrogen atoms in riding positions, the final model was refined to crystallographic R-factor and R-free values of 0.202 and 0.224 respectively by REFMAC5.20 The B-factor distribution in the crystal structure and the comparison with the NMR structure are shown in Figure 2(a,b), respectively. The quality of the electron-density map of the final model is shown in Figure 2(c).

Figure 2. X-ray structure of L-plectasin determined by racemic crystallography. (a) Ribbon representation of L-plectasin; the ribbon is colored by B-factor values. (b) Superposition of the NMR structure (PDB ID: 1zfu) of plectasin (in red) and the present X-ray crystal structure (in blue). The Cα-traces are shown. The disulfide bridges are included as sticks with larger radius; the coloring scheme matches the Cα-traces. The selected NMR model (#4) displays the minimal r.m.s. deviation of 1.1 Å. (c) SigmaA-weighted 2Fo-Fc electron density map of plectasin crystallized in P1 space group, contoured at 2σ encompassing three disulfide bridges. (b, c) were made with Pymol21 and (a) was prepared with Ribbons.22

For comparison, we also crystallized L-plectasin alone and collected diffraction data to a resolution of 1.35 Å. Crystals of L-plectasin belonged to the chiral hexagonal space group P6₁; the structure was solved by molecular replacement using the L-enantiomer structure from the racemic crystal as a search model. The hexagonal L-plectasin structure was refined to crystallographic R-factor and R-free values of 0.157 and 0.182 respectively using REFMAC5. X-ray data collection and refinement statistics for both the racemate and the L-plectasin are summarized in Table S1.

Discussion

High yield total chemical syntheses of plectasin were achieved using native chemical ligation of two unprotected peptide segments prepared by highly optimized stepwise solid phase synthesis. Our results demonstrate efficient and correct in-vitro folding of plectasin in the absence of the N-terminal prosequence.1 The L-enantiomer of plectasin, but not D-plectasin, showed antimicrobial activity toward gram-positive bacteria; this may provide an important insight into plectasin's mechanism of inhibition, which likely differs from the mechanism of action of other known defensins.23

It is noteworthy that crystals of racemic plectasin grown under completely different conditions all belonged to the P1 space group. Including the current work, to date six2, 5–9 of the seven previously reported racemic peptides or proteins have crystallized in space group P1, as predicted by Wukovitz and Yeates.24 With the high-resolution diffraction data from crystals of racemic plectasin, we were able to use direct methods to solve the structure of the plectasin molecule to atomic resolution. The higher R-factor than might have been expected for an atomic resolution structure is a phenomenon that has also been observed for other centrosymmetric protein structures.2, 3 A higher R-factor is an inherent feature of the refinement process applied to centrosymmetric space groups, because the range of structure amplitudes for a centrosymmetric structure is greater than that for a noncentrosymmetric one. Strictly, a racemic protein crystal need not be perfectly centrosymmetric: small differences in the structures of the enantiomeric protein populations might occur.6 However, in the present case, refinement in the space group P1 using (L-plectasin+D-plectasin) as the model did not significantly reduce the R-factor.

A cartoon representation of the X-ray structure of the plectasin molecule derived from the racemic P1 crystal form is shown in Figure 2(a). The structure of plectasin features an α-helix (Met¹³-Ser²¹) and a β-sheet contributed by two antiparallel β-strands. The overall topology of the molecule is stabilized by three disulfide bridges (Cys⁴-Cys³⁰, Cys¹⁵-Cys³⁷, and Cys¹⁹-Cys³⁹) with left-, right-, and left-hand chiral conformations [Fig. 2(c)].

A comparison of the crystal structure presented here with the previously reported NMR solution model (PDB ID 1zfu)1 of plectasin shows a general similarity of the two structures, but revealed some significant differences [Fig. 2(b)]. Superposition of the main chain atoms of the X-ray structure on to the corresponding atoms of the ten NMR best models shows root mean square (r.m.s) deviations ranging between 1.6 Å and 1.1 Å. The structural features that differ most are in the flexible N-terminus, particularly the first few residues, and in the orientation of the first β-strand. The handedness of the disulfide bridges is also different: the first and second disulfide bridges in the NMR model have different conformations as compared with the same disulfides in the crystal structures obtained for both the racemate and the L-plectasin [Fig. 2(b)].

Crystals of L-plectasin in hexagonal space group P6₁ diffracted to a somewhat poorer resolution (1.20–1.35 Å) than crystals of the protein racemate. Although the L-plectasin crystal diffracted to near-atomic resolution (∼1.2 Å), more than half of reflections in the resolution shells above 1.3 Å that are crucial for successful use of direct methods were weak with intensity-to-sigma ratios lower than three (these shells are marked with * in Table S2). This resulted in failure to obtain a direct method solution using a SHELXS automatic run. Therefore, we truncated the resolution for the L-plectasin data set to 1.35 Å and solved the structure by molecular replacement. In contrast, the quality of the racemic crystal was much higher and we were able to solve the structure in SHELXS using the same set of parameters as for the attempted solution of L-plectasin. The molecular structures of the plectasin crystallized as an L-enantiomer or as a racemic mixture are essentially the same, with average r.m.s. deviation of 0.6 Å for all main chain atoms.

It is interesting to note that the racemic plectasin crystals had an exceptionally low solvent content (13%–15%), significantly lower than found for the L-plectasin crystals (40%–42%). The solvent content of protein crystals usually falls in the range 27%–65%.25 A schematic diagram illustrating the arrangement of L- and D-plectasin molecules in the centrosymmetric P1 crystal is shown in Figure 3(a). The L- and D-enantiomers form a dimer-like series of contacts involving two side chains of the β-sheet region (Tyr29 and Lys38), one side chain from the α-helix (Asp12), and two side chains of residues in the N-terminal region (Trp8 and Glu10), either through direct hydrogen bonds or mediated by water molecules [Fig. 3(b) and Table S3 (Supporting Information)]. There are six hydrogen bonds that link the enantiomers by direct contact and four more hydrogen bonds that mediate the intermolecular interaction through well-ordered water molecules, as summarized in Table S3 (Supporting information). The area buried at the interface between the L- and D-enantiomers is ∼500 Ų; this is 2.5 times larger than the interface area (∼200 Ų) between L-plectasin molecules found in the chiral hexagonal P6₁ crystal, where most crystal contacts are mediated by water molecules [Fig. 3(c,d) and Table S4 (Supporting Information)]. It is interesting to note that the same amino acid residues are involved in the crystal contacts in both P1 and P6₁ space groups. However, because of the inversion center in the centrosymmetric P1 crystal, the number of interactions is doubled; this obligate doubling may help to explain the preferential formation of centrosymmetric crystals and the low solvent content.

Figure 3. Crystal packing. (a) Centrosymmetric P1 unit cell. The L-plectasin molecule is shown in blue and the D-plectasin molecule is in gold. (b) Close-up view of the packing interface [boxed in panel (a)] between the L- and D-enantiomers forming the P1 unit cell. (c) Chiral P6₁ unit cell. Six neighboring L-plectasin molecules are shown in blue and cyan. (d) The packing interface (boxed in panel c) between the two closest L-plectasin molecules in P6₁ space group. In panels (b) and (d) water molecules are shown as small magenta spheres and hydrogen bonds are shown as dashed lines. The figures were prepared in Ribbons. The hydrogen bond distances are listed in Supporting Information Tables S3 and S4 for P1 and P6₁ space groups, respectively.

Here we have used total chemical synthesis to prepare the enantiomeric forms of the natural product plectasin; we have shown that the mirror image D-plectasin form is devoid of antimicrobial activity; and, we have used racemic protein crystallography to obtain the first X-ray crystal structure of plectasin. Atomic resolution X-ray diffraction data from the highly ordered racemic crystals enabled determination of the protein structure by ab initio methods. To the best of our knowledge, this is the largest racemic protein structure solved to date by direct methods. In our hands, racemic protein crystallography is proving to be a useful approach to the determination of novel protein structures. We have identified a series of important proteins of ever-increasing size, amenable to total chemical synthesis, for which no X-ray structure had been reported. Several of these could only be crystallized as racemic mixtures. Additionally, a general finding has been that racemic protein crystals are more highly ordered and diffract to higher resolution.

Materials and Methods

Reagents

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N^α-Boc protected amino acids (Peptide Institute, Osaka) were obtained from Peptides International. Side-chain protecting groups used were Arg(Tos), Asn(Xan), Asp(OcHex), Cys(4-CH₃Bzl), Glu(OcHex), His(Bom), Lys(2Cl-Z), Ser(Bzl), Trp(CHO), Tyr(2Br-Z). Boc-D-His(Bom) was purchased from Bachem Bioscience Inc. Aminomethyl-resin was prepared from Biobeads S-X1 (BioRad, California).26 Boc-L-Tyr(2BrZ)-OCH₂-phenylacetic acid was purchased from NeoMPS, Strasbourg France. Boc-D-Tyr(2BrZ)-OCH₂-phenylacetic acid was prepared following the literature procedure.27N,N-Diisopropylethylamine (DIEA) was obtained from Applied Biosystems Inc. (Foster City, California) N,N-Dimethylformamide (DMF), dichloromethane, diethyl ether, HPLC-grade acetonitrile, and guanidine hydrochloride were purchased from Fisher. Trifluoroacetic acid (TFA) was obtained from Halocarbon Products (New Jersey). HF was purchased from Matheson. All other reagents were purchased from Sigma-Aldrich.

Peptide synthesis

The L-amino acid and D-amino acid containing Cys-peptide segments were synthesized on Boc-Tyr(2Brz)-OCH₂-Pam-resin of the appropriate chirality, using manual in situ neutralization Boc chemistry protocols for stepwise SPPS,15 at 0.2 and 0.1 mmol scale, respectively. The L-amino acid and D-amino acid containing peptide thioester segments were synthesized on trityl-SCH₂CH₂CO-Ala-OCH₂-Pam-resin at 0.2 and 0.1 mmol scale, respectively. After removal of the N^α-Boc group, peptides were cleaved from the resin and deprotected by treatment with anhydrous HF at 0°C for 1 h.

HPLC and LC-MS analysis

LC-MS data for the crude and purified synthetic peptides are shown in the Supporting Information (Figs. S1-S4). Masses and yields of the synthetic peptide segments were: L-(1-18)-^αCOSCH₂CH₂Ala-COOH 69 mg, 15% (based on 0.2 mmol starting aminoacyl-resin); mass obs. 2249.0 Da, calc. 2249.3 Da (av isotopes). L-(19-40)-COOH 157 mg, 33% (based on 0.2 mmol starting aminoacyl-resin); mass obs. 2363.6 Da, calc. 2363.8 Da (av isotopes). D-(1-18)-^αCOSCH₂CH₂Ala-COOH 29 mg, 13% (based on 0.1 mmol starting aminoacyl-resin); mass obs. 2249.0 Da, calc. 2249.3 Da (av isotopes). D-(19-40)-COOH 83 mg, 35% (based on 0.1 mmol starting aminoacyl-resin); mass obs. 2363.6 Da, calc. 2363.8 Da (av isotopes).

Chemical synthesis of L-plectasin

L-plectasin was prepared by native chemical ligation of two unprotected peptide segments, followed by deformylation and folding. L-(1-18)-^αCOSCH₂CH₂Ala-COOH (30 mg, 13.3 μmol) and L-(19-40)-COOH (34.6 mg, 14.6 μmol) were dissolved in ligation buffer (2.5 mL) containing 6M guanidine hydrochloride, 200 mM Na₂HPO₄, 10 mM TCEP hydrochloride, and 10 mM MPAA. The pH was adjusted to 6.9. The reaction mixture was purged with argon for 20 min and sealed. After completion of the reaction (3 h, LC-MS monitoring), 30% (v/v) β-mercaptoethanol (1.5 mL), and 20% (v/v) piperidine (1 mL) were added to remove the formyl group from the Trp(CHO) residue. After 2-h incubation at 0°C the reaction mixture was acidified to pH 2 by addition of HCl and the crude peptide was separated from the salts and low MW contaminants on a solid phase extraction cartridge (C18, Alltech associates) and lyophilized.

The crude lyophilized linear (1–40) peptide was then dissolved in 6M guanidine hydrochloride at 3 mg/mL concentration and rapidly diluted 6-fold with a degassed solution of 100 mM tris-hydroxymethylaminomethane containing 9.2 mML-cysteine and 1.2 mML-cystine hydrochloride at pH 8.4, to give a final concentration of 0.5 mg/mL (1–40) peptide and 1M guanidine hydrochloride in the folding buffer. The folding was essentially complete within 3 h at room temperature, as determined by analytical LC-MS. The reaction mixture was acidified to pH 2 with dil. HCl and purified by reverse phase HPLC. Another parallel synthesis using 29 mg (12.9 micromol) of the limiting peptide was carried out following similar procedure and the products were combined after purification and lyophilized to give 52 mg (11.8 micromol, 45% based on limiting peptide) of the L-plectasin molecule. Mass obs. 4401.2 ± 0.5 Da, calc. 4400.8 Da (high point of isotope distribution).

Chemical synthesis of D-plectasin

Chemical synthesis of D-plectasin was performed using D-(19-40)-COOH (26 mg, 10.9 μmol) and 23 mg (10 μmol) of the limiting D-peptide (D-(1-18)-^αCOSCH₂CH₂Ala-COOH) following the same procedure as described for the L-plectasin. After Trp(CHO) removal, folding and purification, the overall yield of the purified D-plectasin was 19 mg (4.3 micromol, 42% based on limiting peptide). Mass obs. 4401.2 ± 0.5 Da, calc. 4400.8 Da (high point of isotope distribution).

Circular dichroism

CD spectra were recorded on an AVIV-202 CD spectrometer. 131.7 μM of L-plectasin and 136.3 μM of D-plectasin in water were transferred to a CD cuvette of path length 0.1 cm. CD spectra were measured at room temperature over the range 190–250 nm using 0.5 nm step with averaging of 10 scans.

Antimicrobial assays

S. aureus clinical isolates Newman (MSSA)28 or USA300 (MRSA)29 were grown in TSB at 37°C. S. aureus strain USA300 (MRSA) was obtained through the Network on Antimicrobial Resistance in S. aureus (NARSA, NIAID). B. cereus str. 14579 was obtained from the ATCC. Minimum inhibitory concentrations (MICs) were determined by the microdilution broth method according to National Commmittee for Clinical Laboratory Standards/Clinical and Laboratory Standards Institute (NCCLS/CLSI) guidelines [The Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards). Guideline M7–A6: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. khttp://www.nccls.orgl]. Briefly, fresh overnight colonies were suspended to a turbidity of 0.5 Absorbance (600 nm) and further diluted with Tryptic Soy broth for S. aureus species and LB broth for Bacillus species. The diluted bacterial suspensions were added to wells containing serial two-fold dilutions of synthetic protein. The polypropylene trays (Nunc) were incubated at 37°C in ambient air for 16–20 h for Staphylococcus and 30°C for 16–20 h for Bacillus cereus followed by absorbance readings at 600 nm to measure bacterial growth.

Crystallization

Crystallization of racemic plectasin was performed by mixing equal amounts (by weight) of purified lyophilized L-plectasin and D-plectasin in water at the following concentrations: 12 mg/mL (6 mg of L and 6 mg D) and 6 mg/mL (3 mg of L and 3 mg D). Each solution was centrifuged to remove any particulate matter present and then used directly for crystallizations. Crystallization screening was conducted at 19°C using the commercially available Hampton index. Crystallization screens were performed by the hanging drop vapor diffusion method. X-ray diffraction data were collected from crystals grown at 12 mg/mL protein concentration from either 0.2M NaCl, 15% (w/v) PEG-3350, 0.1M Tris, pH 8.5 or 0.1M HEPES, 22.5% (w/v) Jeffamine ED-2001, pH 7. Diffraction data obtained from a racemic crystal grown in the latter condition was used for structure solution.

Crystallization of L-plectasin was performed under the following conditions: 0.1M HEPES, 0.2M ammonium acetate, 21% PEG 3350, pH 7.5 with 18 mg/mL protein concentration.

Data collection

For data collection, selected crystals were briefly transferred to the cryoprotectant (reservoir solution plus 20% (v/v) glycerol) and flash-frozen in liquid nitrogen. The X-ray diffraction data were collected at 100 K using 0.97934 Å wavelength at the Argonne National Laboratory (Advanced Photon Source, beamline 23ID equipped with a MARCCD 300 detector). Crystal diffraction images were integrated, scaled, and merged with HKL2000.29

X-ray structure determination

The structure of racemic plectasin in the P1 space group was solved by direct methods using SHELXS.18 The structure of hexagonal L-plectasin in the P6₁ space group was solved by molecular replacement using the MOLREP program and the model derived from racemic crystals. Electron density and model examinations were done using TURBO-FRODO.19 The restrained positional and anisotropic B-factor refinement was performed in REFMAC5.20 The hydrogen atoms were included in the riding positions. Molecular graphics were generated using Ribbons or Pymol. The main chain torsion angles for all residues are in the allowed regions and additional allowed regions of the Ramachandran plot. The data collection and refinement statistics are summarized in Table S1.

PDB accession codes

The PDB accession numbers for the crystal structures are: 3E7R for racemic plectasin and 3E7U for L-plectasin.

Acknowledgements

Use of the GMCA-CAT beamline 23-ID at the Advanced Photon Source is supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Center for Advanced Radiation Sources at the University of Chicago.