Single-wavelength Anomalous Diffraction Research Articles

DSAS: a new macromolecular substructure solution program based on the modified phase-retrieval algorithm

Abstract Considering the pivotal role of single-wavelength anomalous diffraction (SAD) in macromolecular crystallography, our objective was to introduce DSAS, a novel program designed for efficient anomalous scattering substructure determination. DSAS stands out with its core components: a modified phase-retrieval algorithm and automated parameter tuning. The software boasts an intuitive graphical user interface (GUI), facilitating seamless input of essential data and real-time monitoring. Extensive testing on DSAS has involved diverse datasets, encompassing proteins, nucleic acids, and various anomalous scatters such as sulfur (S), selenium (Se), metals, and halogens. The results confirm DSAS's exceptional performance in accurately determining heavy atom positions, making it a highly effective tool in the field.

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.

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Se-MAG Is a Convenient Additive for Experimental Phasing and Structure Determination of Membrane Proteins Crystallised by the Lipid Cubic Phase (In Meso) Method

Both intensity and phase information are needed for structure determination by macromolecular X-ray crystallography. The diffraction experiment provides intensities. Phases must be accessed indirectly by molecular replacement, or by experimental phasing. A popular method for crystallising membrane proteins employs a lipid cubic mesophase (the in meso method). Monoolein is the most popular lipid for in meso crystallisation. Invariably, the lipid co-crystallises with the protein recapitulating the biomembrane from whence it came. We reasoned that such a lipid bearing a heavy atom could be used for experimental phasing. In this study, we replaced half the monoolein in the mesophase with a seleno-labelled analogue (Se-MAG), which has a selenium atom in the fatty acyl chain of the lipid. The lipid mixture formed the cubic mesophase and grew crystals by the in meso method of the alginate transporter, AlgE, and the lipoprotein N-acyltransferase, Lnt. Se-MAGs co-crystallised with both proteins and were used to obtain phases for high-resolution structure determination by the selenium single-wavelength anomalous diffraction method. The use of such a mixed lipid system may prove to be a general strategy for the experimental phasing part of crystallographic structure determination of membrane proteins that crystallise via the in meso method.

Facing the phase problem.

The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we - myself, my laboratory and the diffraction community - have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today's structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.

Biochemical characterization of type I-E anti-CRISPR proteins, AcrIE2 and AcrIE4

In bacteria and archaea, CRISPRs and Cas proteins constitute an adaptive immune system against invading foreign genetic materials, such as bacteriophages and plasmids. To counteract CRISPR-mediated immunity, bacteriophages encode anti-CRISPR (Acr) proteins that neutralize the host CRISPR–Cas systems. Several Acr proteins that act against type I-E CRISPR–Cas systems have been identified. Here, we describe the biochemical characterization of two type I-E Acr proteins, AcrIE2 and AcrIE4. We determined the crystal structure of AcrIE2 using single-wavelength anomalous diffraction and performed a structural comparison with the previously reported AcrIE2 structures solved by different techniques. Binding assays with type I-E Cas proteins were carried out for the target identification of AcrIE2. We also analyzed the interaction between AcrIE4 and its target Cas component using biochemical methods. Our findings corroborate and expand the knowledge on type I-E Acr proteins, illuminating diverse molecular mechanisms of inhibiting CRISPR-mediated prokaryotic anti-phage defense.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experiment.

To determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F-) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.

Crystal structure of LGR ligand α2/β5 from Caenorhabditis elegans with implications for the evolution of glycoprotein hormones

A family of leucine-rich-repeat-containing G-protein-coupled receptors (LGRs) mediate diverse physiological responses when complexed with their cognate ligands. LGRs are present in all metazoan animals. In humans, the LGR ligands include glycoprotein hormones (GPHs) chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (hFSH), and thyroid-stimulating hormone (hTSH). These hormones are αβ heterodimers of cystine-knot protein chains. LGRs and their ligand chains have coevolved. Ancestral hormone homologs, present in both bilaterian animals and chordates, are identified as α2β5. We have used single-wavelength anomalous diffraction and molecular replacement to determine structures of the α2β5 hormone from Caenorhabditis elegans (Ceα2β5). Ceα2β5 is unglycosylated, as are many other α2β5 hormones. Both Hsα2β5, the human homolog of Ceα2β5, and hTSH activate the same receptor (hTSHR). Despite having little sequence similarity to vertebrate GPHs, apart from the cysteine patterns from core disulfide bridges, Ceα2β5 is generally similar in structure to these counterparts; however, its α2 and β5 subunits are more symmetric as compared with α and β of hCG and hFSH. This quasisymmetry suggests a hypothetical homodimeric antecedent of the α2β5 and αβ heterodimers. Known structures together with AlphaFold models from the sequences for other LGR ligands provide representatives for the molecular evolution of LGR ligands from early metazoans through the present-day GPHs. The experimental Ceα2β5 structure validates its AlphaFold model, and thus also that for Hsα2β5; and interfacial characteristics in a model for the Hsα2β5:hTSHR complex are similar to those found in an experimental hTSH:hTSHR structure.

Multi-crystal native-SAD phasing at 5 keV with a helium environment.

De novo structure determination from single-wavelength anomalous diffraction using native sulfur or phospho-rus in biomolecules (native-SAD) is an appealing method to mitigate the labor-intensive production of heavy-atom derivatives and seleno-methio-nyl substitutions. The native-SAD method is particularly attractive for membrane proteins, which are difficult to produce and often recalcitrant to grow into decent-sized crystals. Native-SAD uses lower-energy X-rays to enhance anomalous signals from sulfur or phospho-rus. However, at lower energies, the scattering and absorption of air contribute to the background noise, reduce the signals and are thus adverse to native-SAD phasing. We have previously demonstrated native-SAD phasing at an energy of 5 keV in air at the NSLS-II FMX beamline. Here, the use of a helium path developed to reduce both the noise from background scattering and the air absorption of the diffracted X-ray beam are described. The helium path was used for collection of anomalous diffraction data at 5 keV for two proteins: thaumatin and the membrane protein TehA. Although anomalous signals from each individual crystal are very weak, robust anomalous signals are obtained from data assembled from micrometre-sized crystals. The thaumatin structure was determined from 15 microcrystals and the TehA structure from 18 microcrystals. These results demonstrate the usefulness of a helium environment in support of native-SAD phasing at 5 keV.

Glycoside hydrolase subfamily GH5_57 features a highly redesigned catalytic interface to process complex hetero-β-mannans.

Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage β-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-β-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-β-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing β6-α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the -1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

Extending the novel |ρ|-based phasing algorithm to the solution of anomalous scattering substructures from SAD data of protein crystals.

Owing to the importance of the single-wavelength anomalous diffraction (SAD) technique, the recently developed |ρ|-based phasing algorithm (SM,|ρ|) incorporating the inner-pixel preservation (ipp) procedure [Rius & Torrelles (2021). Acta Cryst A77, 339-347] has been adapted to the determination of anomalous scattering substructures and its applicability tested on a series of 12 representative experimental data sets, mostly retrieved from the Protein Data Bank. To give an idea of the suitability of the data sets, the main indicators measuring their quality are also given. The dominant anomalous scatterers are either SeMet or S atoms, or metals/clusters incorporated by soaking. The resulting SAD-adapted algorithm solves the substructures of the test protein crystals quite efficiently.

Native SAD phasing at room temperature.

Single-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving macromolecular structures. This technique requires the accurate measurement of intensities to determine differences between Bijvoet pairs. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, a strategy is presented to obtain high-quality data from room-temperature, single-crystal experiments. To illustrate the strengths of this approach, native SAD phasing at 6.55 keV was used to solve four structures of three model systems at 295 K. The resulting data sets allow automatic phasing and model building, and reveal alternate conformations that reflect the structure of proteins at room temperature.

Crystallization, X-ray diffraction analysis and structure of ICMP from Pseudomonasaeruginosa

Insulin-cleaving membrane protease (ICMP), an outmember protein of Pseudomonas aeruginosa (P. aeruginosa), plays a critical role in the pathogenesis of the bacterium. ICMP has been reported to be involved in the process of iron uptake. In this study, we report the high-resolution structure of ICMP determined by single-wavelength anomalous diffraction (SAD), which shows an atypical HxxE motif that differs from the canonical zinc dependent M75 peptidases and a “V-shaped” cleft that is observed to coordinate the metal ion for the first time. Crystals from the selenomethionine-substituted ICMP(Se-Met ICMP) diffract to 1.9 Å resolution and belong to space group P21, with unit-cell parameters a = 87.93, b = 78.14, c = 9.92 Å, α = 90°, β = 113.5°, γ = 90°. ICMP consists of two up-and-down helix bundles, which are arranged into an inverted "V" shape. Unexpectedly, no electron densities of metal ions are observed around the ICMP HxxE motif, which is shown to be involved in metal coordination in zinc-dependent M75 peptidases. In contrast, we find a metal ion at the opening cleft of the V-shaped structure of ICMP, where the ICMP residues Asp211, Glu316, Cys319, Asp322, and Asp397 are observed to coordinate the metal via hydrogen-bond interactions. Such observations might imply new potential substrate-binding and catalytic sites. The current work therefore provides novel insights into the diversity of the HxxE-motif-containing peptidase and paves the way for future studies aiming to delineate the mechanism of ICMP catalysis.

Sequence analysis and crystal structure of a glycosylated protease from Euphorbia resinifera latex for its proteolytic activity aspect.

The investigation of a plant glycosylated serine protease (EuRP-61) isolated from Euphorbia resinifera latex for potential antiplatelet and anticoagulation activities has been previously reported. In the present study, the protein sequence and native crystal structure of EuRP-61 were characterized. The structure was identified using single-wavelength anomalous diffraction with a refinement resolution of 1.7Å (PDB ID: 7EOX). The main structural components of EuRP-61 were composed of three domains: catalytic, protease-associated (PA), and fibronectin type III (Fn3)-like domains. The crystal structure revealed that some loops in the PA and catalytic domains of EuRP-61 were different from the other subtilisin-like proteases (cucumisin and SBT3). These different loops might be involved in the general monomer formation of EuRP-61, substrate specificity, and maintenance of the catalytic domain. The Fn3-like domain may provide flexibility to the enzyme to bind with various substrates and cell receptors. Additionally, the active site of EuRP-61 consisted of the catalytic triad of Ser434, His106, and Asp32, similar to other serine proteases. The present study provides additional information and insight into the protease and antithrombotic activities of EuRP-61, which could contribute to further development of this enzyme for biomedical treatment.

A novel class of Candida glabrata cell wall proteins with β-helix fold mediates adhesion in clinical isolates.

Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-regions from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed β-helix domain that is linked to a C-terminal β-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with β-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.

Likelihood-based estimation of substructure content from single-wavelength anomalous diffraction (SAD) intensity data.

SAD phasing can be challenging when the signal-to-noise ratio is low. In such cases, having an accurate estimate of the substructure content can determine whether or not the substructure of anomalous scatterer positions can successfully be determined. Here, a likelihood-based target function is proposed to accurately estimate the strength of the anomalous scattering contribution directly from the measured intensities, determining a complex correlation parameter relating the Bijvoet mates as a function of resolution. This gives a novel measure of the intrinsic anomalous signal. The SAD likelihood target function also accounts for correlated errors in the measurement of intensities from Bijvoet mates, which can arise from the effects of radiation damage. When the anomalous signal is assumed to come primarily from a substructure comprising one anomalous scatterer with a known value of f'' and when the protein composition of the crystal is estimated correctly, the refined complex correlation parameters can be interpreted in terms of the atomic content of the primary anomalous scatterer before the substructure is known. The maximum-likelihood estimation of substructure content was tested on a curated database of 357 SAD cases with useful anomalous signal. The prior estimates of substructure content are highly correlated to the content determined by phasing calculations, with a correlation coefficient (on a log-log basis) of 0.72.

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing.

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Structural characterization of Myxococcus xanthus MglC, a component of the polarity control system, and its interactions with its paralog MglB

The δ-proteobacteria Myxococcus xanthus displays social (S) and adventurous (A) motilities, which require pole-to-pole reversal of the motility regulator proteins. Mutual gliding motility protein C (MglC), a paralog of GTPase-activating protein Mutual gliding motility protein B (MglB), is a member of the polarity module involved in regulating motility. However, little is known about the structure and function of MglC. Here, we determined ∼1.85 Å resolution crystal structure of MglC using Selenomethionine Single-wavelength anomalous diffraction. The crystal structure revealed that, despite sharing <9% sequence identity, both MglB and MglC adopt a Regulatory Light Chain 7 family fold. However, MglC has a distinct ∼30° to 40° shift in the orientation of the functionally important α2 helix compared with other structural homologs. Using isothermal titration calorimetry and size-exclusion chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using small-angle X-ray scattering and molecular docking studies, we show that the MglBC complex consists of a MglC homodimer sandwiched between two homodimers of MglB. A combination of size-exclusion chromatography and site-directed mutagenesis studies confirmed the MglBC interacting interface obtained by molecular docking studies. Finally, we show that the C-terminal region of MglB, crucial for binding its established partner MglA, is not required for binding MglC. These studies suggest that the MglB uses distinct interfaces to bind MglA and MglC. Based on these data, we propose a model suggesting a new role for MglC in polarity reversal in M. xanthus.

Crystal structure of the N-terminal domain of TagH reveals a potential drug targeting site

Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting.