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Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS. The A4V variant is responsible for the majority of familial ALS cases in North America, causing rapidly progressing paralysis once symptoms begin and the G93A SOD1 variant is overexpressed in often studied murine models of the disease. Here we report the three-dimensional structures of metal-free A4V and of metal-bound and metal-free G93A SOD1. In the metal-free structures, the metal-binding loop elements are observed to be severely disordered, suggesting that these variants may share mechanisms of aggregation proposed previously for other pathogenic SOD1 proteins.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons in the spinal cord and brain, eventually leading to paralysis and death . Approximately 90% of cases are termed “sporadic” (sALS), arising without family history or known cause. About 10% of cases have a genetic component and are termed “familial” (fALS), with approximately 20% of fALS cases being linked to dominant mutations in the gene encoding the cytosolic antioxidant enzyme copper-zinc superoxide dismutase (SOD1) [2, 3]. To date, over 100 different pathogenic SOD1 mutations have been reported (reviewed in [4–9]). A steadily increasing list of mutations can be monitored at http://alsod.iop.kcl.ac.uk/Als.
Studies in transgenic mice have established that pathogenic SOD1 proteins elicit motor neuron dysfunction through a gain of a toxic property and not a loss of enzymatic function. Mice expressing human ALS SOD1 polypeptides in addition to their endogenous SOD1 (thereby retaining normal SOD1 activity) become paralyzed [10–13], while SOD1 knockout mice do not . Although the exact nature of this toxicity is not well understood, the observation that insoluble proteinaceous inclusions enriched in pathogenic SOD1 appear concomitant with the onset of paralytic symptoms in these animals suggests that SOD1-linked ALS is a protein misfolding disease [4–9].
Human SOD1 is a 32-kDa homodimeric enzyme that catalyzes the detoxification of superoxide anion (O2 −) to molecular oxygen and hydrogen peroxide (2O2 − + 2H+ → O2 + H2O2) [15, 16]. Figure 1 shows that each subunit folds as an eight-stranded Greek key β-barrel, binds one copper and one zinc ion, and contains an intrasubunit disulfide bond . The protein-bound copper ion is essential for catalysis, while the protein-bound zinc ion is important to maintain the structural integrity of the enzyme and for the proper positioning of charged residues that assist in the electrostatic guidance of the superoxide substrate into the active site [18, 19]. SOD1 is abundant in brain and spinal cord, presumably because these highly respiring tissues produce copious levels of superoxide as a normal byproduct of respiration.
The binding of metal ions and the oxidation of the intrasubunit disulfide bond by nascent SOD1 have been demonstrated to favor dimerization [20–22] and are also known to impart enormous thermal stability to the enzyme. The importance of these posttranslational modifications to the stability of SOD1 in the context of pathogenic SOD1 misfolding and aggregation has focused attention on the copper chaperone for SOD1 (CCS), a helper protein that recognizes and binds to newly translated SOD1, inserts the catalytic copper ion, and catalyzes the oxidation of the intrasubunit disulfide bond in each SOD1 monomer [23, 24]. Hindering these posttranslational modifications results in immature, metal-deficient and/or disulfide-reduced forms of SOD1 that are increasingly observed as components of the higher order oligomers believed to be relevant in ALS toxicity [25–28]. However, whether the oligomers of pathogenic SOD1 mutants are the causative agents or are symptomatic of motor neuron dysfunction is not yet clear.
A rapidly progressing form of ALS arises from mutations at position A4 (A4V, A4T, and A4S), of which A4V is the most common in North American patients. ALS patients harboring the A4V mutation have a mean survival time of only about 1.5 years post diagnosis . Figure 1 shows that A4 is located in the first β-strand that forms part of the dimer interface. Mutations at the A4 position are predicted to affect SOD1 dimerization  and/or to destabilize the Greek key β- barrel because A4 packs into the hydrophobic core of the SOD1 monomer. There are six known pathogenic substitutions at G93 (G93A, G93C, G93D, G93R, G93S, and G93V). As shown in Figure 1, G93 is positioned in the short loop V connecting the anti-parallel β-strands 5 and 6, and which is near residues 37 – 42 of loop III. Substitution of G93 is also predicted to destabilize the β-barrel by interfering with the packing of residues that form the “apolar plug” found at one end of the β-barrel .
Here, we describe crystal structures of metal-free A4V and metal-bound and metal-free G93A pathogenic SOD1 variants. The structures reveal the destabilizing effects of metal ion deficiency on the conformations of two major loop elements of the enzyme and illuminate intriguing posttranslational modifications of C111 residues in A4V. These data, taken together with data coming from transgenic mouse and cell culture experiments, suggest the hypothesis that SOD1-linked ALS may arise from a general failure of a subset of newly translated pathogenic SOD1 mutants to mature.
DNA fragments encoding the human A4V and G93A mutations were amplified by the polymerase chain reaction (PCR) and ligated into the YEP351 plasmid, where expression of the human SOD1 is directed under the control of its own promoter. A4V and G93A SOD1 proteins were expressed, purified, and when appropriate, stripped of their metals as previously described . The metal contents of all SOD1 proteins used in this study were determined using inductively coupled plasma mass spectrometry (ICP-MS) at the mass spectrometry facility in the Department of Chemistry and Biochemistry at the University of California, Los Angeles as described previously .
Two distinct metal-containing SOD1 samples were used in crystallization experiments. The “metal-bound” G93A SOD1 sample contained the complement of metal ions present after purification, while the “metal-reconstituted” G93A SOD1 sample contained the complement of metal ions present after the purified G93A SOD1 protein is incubated with 1 mM copper and zinc sulfate overnight, followed by dialysis against 2.25 mM potassium phosphate pH 7.5, 150 mM sodium chloride. All crystals were grown using the hanging-drop vapor diffusion method  at room temperature. Metal-free A4V SOD1 crystals in space group P212121 were grown from a drop containing equal volumes of protein solution (protein concentration of 17 mg/ml in 10 mM sodium acetate pH 5.5, 125 mM sodium chloride) and reservoir solution (1.6 M ammonium sulfate, 0.1 M MES at pH 6, and 5 mM tris(2-carboxyethyl)phosphine (TCEP)). Block-like prisms appeared within two weeks. Metal-free G93A crystals in space group C2 were grown by mixing a 1:1 ratio of protein solution (protein concentration of 5 mg/ml in 10 mM sodium acetate, pH 5.5, 125 mM sodium chloride) and reservoir solution (2.4 M ammonium sulfate, 0.1 M HEPES, pH 7). Slender rod-like clusters appeared in approximately one month. The metal-bound G93A crystals in space group C2221 were obtained using the same conditions as metal-free G93A, except the reservoir solution contained 2.4 M sodium malonate, pH 6. Large trapezoids appeared after about three days. Crystals of metal-reconstituted G93A in space group P21 were grown from a drop containing 2 µL of protein solution (protein concentration 9 mg/mL in 2.25 mM potassium phosphate pH 7.5, 150 mM sodium chloride) and 2 µL of reservoir solution (20% PEG3350, 0.2 M sodium iodide). Rectangular prisms appeared within two weeks.
Crystals of metal-free A4V, metal-free G93A and metal-bound G93A in space group C2221 were quickly swiped through well solution made 20% (v/v) in glycerol as a cryoprotectant. The metal-reconstituted monoclinic G93A crystals were soaked for 10 s in reservoir solution made 25% (w/v) in sorbitol as a cryoprotectant. All cryoprotected crystals were flash-cooled by plunging into liquid nitrogen prior to X-ray data collection. Diffraction data from metal-free G93A crystals in space group C2 were collected at the Advanced Photon Source beam line 19-BM (Argonne, IL) on a custom charge-coupled device (CCD) detector. Diffraction data from metal-free A4V crystals in space group P212121 were collected at the Advanced Light Source beam line 8.2.2 (Berkeley, CA) on an Area Detector Systems Corporation Quantum 315 CCD detector. Diffraction data from metal-bound G93A crystals in space group C2221 were collected at the X-ray Crystallography Core Laboratory at the University of Texas Health Science Center at San Antonio using a Rigaku FR-D high flux X-ray generator equipped with Osmic High-flux mirrors on a Rigaku R-AXIS HTC imaging plate detector. Diffraction data from metal-reconstituted G93A crystals in space group P21 were collected at the Synchrotron Radiation Source beam line 9.6 (Daresbury, U.K.) on an Area Detector Systems Corporation CCD detector. All data sets were indexed, scaled, and merged using HKL2000 . Data collection statistics are presented in Table 1.
The structure of metal-free A4V was determined by molecular replacement using the program MOLREP  with the S134N SOD1 variant (PDB code 1ozu) as the search model . Initial refinement was performed using the maximum likelihood method as implemented in REFMAC5  as part of the CCP4 program suite. Minor adjustments to the model were made using σA-weighted electron density maps  in the program O . Prior to refinement, 5% of the reflections were excluded for use in cross-validation for the calculation of Rfree . Solvent molecules were added with the aid of the program ARP/WARP . Individual anisotropic B-factor refinement was performed using the program PHENIX [41, 42]. In the later stages of structure refinement the graphics program COOT  was used for model adjustment.
The structure of metal-free G93A was determined by molecular replacement using the program MOLREP and wild type SOD1 subunits A and H (PDB code 1hl5 ) as the search model. The structure obtained from molecular replacement was subsequently refined using REFMAC5 and PHENIX with non-crystallographic-symmetry restraints. Fine tuning of the model proceeded using the program COOT.
The structure of metal-bound G93A SOD1 in space group C2221 was determined by molecular replacement using the program MOLREP and wild type SOD1 subunits A and F (PDB code 1sos ) as the search model The molecular replacement solution was subjected to several rounds of refinement using PHENIX. All ten subunits (five dimers) in the asymmetric unit were refined independently and no stereochemical restraints were applied to metal-ligand distances or bond angles. Water molecules were included in the later stages of refinement by automatic water picking routines within PHENIX and COOT, and some of them were positioned manually after visual inspection of σA-weighted electron density maps.
The structure of metal-reconstituted G93A SOD1 in space group P21 was determined by molecular replacement using one dimer from wild type SOD1 subunits A and F (PDB code 2c9v ) as the search model The molecular replacement solution was subjected to several rounds of refinement using REFMAC5. The two monomers were refined independently and no stereochemical restraints were applied to metal-ligand distances or bond angles. Water molecules were included in the later stages of refinement by automatic water picking routines in ARP/WARP and COOT. Three iodine anions from the crystallization medium were added manually to the model after visual inspection of 2(Fo-Fc) and (Fo-Fc) electron density maps.
Sedimentation velocity experiments were performed using a Beckman Optima XL-I centrifuge at the Center for Analytical Ultracentrifugation of Macromolecular Assemblies, University of Texas Health Science Center at San Antonio. A4V and G93A SOD1 variants at a concentration of 2.4 mg/ml in sodium acetate buffer pH 5.5 with and without the reducing agent TCEP were used in the experiments, which were conducted at 20 °C and at 50,000 rpm. Sedimentation velocity data were analyzed using the method of van Holde and Weischet  as implemented in the ULTRASCAN 6.0 software . This analysis method effectively removes the contributions of diffusion to boundary spreading to yield the integral distribution of s20,w of all species in the sample, G(s). Consequently, a G(s) plot of boundary fraction versus s20,w will be vertical if the sample is homogeneous, and will have a positive slope if the sample is heterogeneous [48, 49].
X-ray diffraction data collection and protein structure refinement statistics are given in Table 1. Metal-free A4V SOD1 crystallized in space group P212121 with one dimer (subunits A/B) per asymmetric unit. Metal-free G93A crystallized in space group C2 with two dimers (subunits A/B and C/D) per asymmetric unit. Figure 2A shows a ribbon representation of the dimeric human wild type holoenzyme (Protein Data Bank code 2c9v ). Each SOD1 subunit of the homodimer folds as an eight-stranded Greek key β-barrel, binds one copper and one zinc ion, and possesses an intrasubunit disulfide bond . Two rather lengthy loop elements protrude from the β-barrel to form the walls of the active site and to participate in metal binding. The first, termed the “zinc loop” (loop IV, residues 50–83) holds most of the zinc-binding ligands. The second, termed the “electrostatic loop” (loop VII, residues 121–142), contains charged amino acid residues that help guide the negatively charged superoxide substrate to the active site entrance while simultaneously excluding larger nonsubstrate anions [19, 51]. A third, smaller element, the “disulfide loop” (residues 50–62, a substructure of loop IV), is covalently linked to the β-barrel through a disulfide bond between C57 and C146. The disulfide loop and the eighth strand of the β-barrel from each subunit interact reciprocally and comprise most of the SOD1 dimer interface [17, 21].
Figures 2B and 2C show ribbon representations of metal-free A4V and G93A SOD1, respectively, in the same orientation as the wild type enzyme, revealing conformational disorder in the electrostatic and zinc loop elements (detailed in Table 2). Figure 3A and Figure 4A show the A4V substitution site and the copper- and zinc-binding sites in subunit A of the metal-free A4V SOD1 structure, respectively, superimposed on σA-weighted electron density contoured at 1.2 σ.
Metal-bound G93A SOD1 crystallized in space group C2221 with five dimers (subunits A/B, C/D, E/F, G/H, and I/J) per asymmetric unit, while metal-reconstituted G93A SOD1 crystallized in space group P21 with one dimer (subunits A/F) in the asymmetric unit. The overall structures of all subunits in the metal-bound and metal-reconstituted G93A SOD1 structures are quite similar to that of the wild type enzyme. Although zinc ions are bound in the zinc-binding sites with full occupancy in all subunits, the occupancy of copper in the copper-binding sites was variable, ranging from 0.17 to 1.0, depending on the crystal form. Figure 3B and Figure 4B show the G93A substitution site and the copper- and zinc-binding sites, respectively, in subunit A of the monoclinic, metal-reconstituted G93A structure superimposed on σA-weighted electron density contoured at 1.2 σ.
Figure 5 shows the results of analytical ultracentrifugation sedimentation velocity runs of the A4V and G93A SOD1 protein samples used for crystallization in this study. Metal-bound G93A SOD1 sedimented at ~2.8 S, which is in agreement with previously reported values for the dimeric wild type enzyme . Metal-free A4V and G93A SOD1 proteins sedimented between 2.7 and 2.8 S at high concentrations (high boundary fraction %), but at smaller S values at lower concentrations (low boundary fraction %). The shape of the distributions of S for the metal-free A4V and G93A SOD1 proteins is suggestive of a self-associating system (i.e. an equilibrium between monomeric and dimeric forms of the protein). In the presence of the reducing agent TCEP, the distributions for metal-free A4V and G93A SOD1 proteins shift almost entirely to S values approaching 1.2, which is the value previously reported for monomeric SOD1 .
As expected, crystal structures of metal-bound forms of A4V SOD1  and metal-bound and metal-reconstituted forms of G93A SOD1 (this study) are similar to that of the wild type enzyme in that they possess well-ordered zinc loop (loop IV, residues 50–83) and electrostatic loop (loop VII, residues 121–142) elements. The molecular basis for why the binding of metal ions (particularly zinc) exerts a stabilizing effect on the conformation of these loop elements is shown in Figure 1. First, in the Cu(II) form of the enzyme, H63 of the zinc loop bridges the copper and zinc ions. This “primary bridge”, also known as the “bridging imidazolate”, is a metal ion coordinating feature unique to SOD1 . Second, the binding of zinc to the zinc-binding site by amino acid residues H63, H71, H80, and D83, induces the zinc loop to adopt a structure in which the proline residues at positions 62, 66, and 74 adopt the trans conformation [17, 52]. Third, when copper and zinc occupy their respective binding sites, they orient the nonliganding imidazole nitrogen atoms of copper ligand H46 and zinc ligand H71 such that they form hydrogen bonds with the side chain oxygen atoms of D124 coming from the electrostatic loop. Because D124 also directly links the copper- and zinc-binding sites, it has been termed the “secondary bridge” . Fourth, when zinc occupies the zinc-binding site, it sits precisely on an axis running down the center of the short α-helix consisting of residues 131–136 of the electrostatic loop, thereby capping the helix dipole at the C-terminus of this helix. Figure 1 shows that this electrostatic helix-capping interaction, plus the two hydrogen bonds of the secondary bridge, together with a hydrogen bond between the amide nitrogen of H71 and the carbonyl oxygen of T135, are the only non-van der Waals interactions linking the zinc and electrostatic loop elements. Thus, the conformation of the electrostatic loop appears to be strictly dependent on the conformation of the zinc loop, which is in turn dependent on the binding of zinc.
Recent studies reveal that the binding of metal ions to the copper-binding site alone is insufficient to induce order in the zinc and electrostatic loop elements. For example, structures of the pathogenic SOD1 variants D124V and H80R [SVS and PJH under review], of a human SOD1 variant engineered to be monomeric and to have an ablated zinc-binding site , and of tomato SOD1 [AG and PJH in preparation] all reveal zinc ions bound in the copper-binding sites, empty zinc-binding sites, and disordered zinc and electrostatic loop elements. In contrast, wild type and mutant forms of SOD1 with empty copper-binding sites, but containing zinc in the zinc-binding site, reveal well-ordered zinc and electrostatic loop elements [54, 55]. Taken together, the structural data indicate that the binding of metal ions to the zinc-binding site (but not the copper-binding site) is the critical factor dictating whether or not the zinc and electrostatic loop elements will be ordered or disordered. Consistent with this observation, Figure 2 shows that metal-free forms of A4V and G93A pathogenic SOD1 variants possess disordered zinc and electrostatic loop elements. Disordered zinc and electrostatic loop elements in metal-deficient pathogenic SOD1 dimers have been observed to permit non-native SOD1-SOD1 interactions at deprotected edge strands in the Greek key β-barrel, leading to the formation of linear filamentous arrays  that are similar in appearance to the fibrillar SOD1 aggregates found in various murine models of ALS .
Although Figure 3 shows that the regions local to the A4V and G93A substitutions impart only relatively small displacements of surrounding residues in the Greek key β-barrels of these proteins, these two pathogenic substitutions are clearly destabilizing to both dimeric and monomeric forms of SOD1. For example, in previous analytical ultracentrifugation sedimentation velocity and sedimentation equilibrium experiments, disulfide-oxidized, wild type SOD1 proteins were observed to sediment as homogeneous dimers, whether metal-bound or metal-free. The wild type enzyme did not dissociate to monomers until the metal ions were removed and the intrasubunit disulfide bond was reduced . In contrast, as shown in the sedimentation velocity experiments in Figure 5, metal-free A4V and G93A SOD1 variants are prone to monomerization even though they still retain the intrasubunit disulfide bond.
The notion that monomeric pathogenic SOD1 proteins might be prone to aggregation in SOD1-linked ALS is gaining acceptance. Monomeric SOD1 was found to be a common intermediate in oxidation models of SOD1 aggregation . In biochemical studies on A4V SOD1, the introduction of an intersubunit disulfide bond designed to prevent dimer dissociation completely abolished the aggregation observed in A4V SOD1 proteins without this engineered crosslink . Small molecules designed to bind in a pocket at the SOD1 dimeric interface, thereby stabilizing the SOD1 dimer, were observed to protect A4V and G93A SOD1 from aggregation even when these proteins were metal-free . In studies probing the oligomeric state of SOD1 in murine models of ALS in which transgenic mice overexpressed G37R, G85R, and G93A SOD1, the presence of monomeric pathogenic SOD1 variants in the insoluble aggregates was established using an antibody that specifically recognizes monomeric, misfolded forms of SOD1 . Although all of the above-mentioned studies focused on the dissociation of pathogenic SOD1 dimers to monomers, the observations that SOD1 monomers are prone to aggregation are also consistent with the hypothesis that newly translated, monomeric pathogenic SOD1 proteins fail to mature to their dimeric forms via posttranslational modification by CCS [8, 9, 28, 55, 61] (see below).
In a previous solution NMR study on metal-bound G93A, loops III and V exhibited higher mobility than wild type SOD1 on a time scale faster than the rate of protein tumbling. This increased mobility was interpreted as an indication that the β-barrel may be more easily opened than the wild type, especially when the protein is metal-free, potentially leading to fibrillogenesis through non-native SOD1-SOD1 interactions . In spite of the relatively small displacements of surrounding residues shown in Figure 3, the A4V and G93A pathogenic substitutions have dramatic destabilizing effects on metal-deficient, monomeric forms of these variants as has been demonstrated in differential scanning calorimetry (DSC) experiments [63, 64]. Disulfide-oxidized, dimeric, holo-wild type SOD1 has a melting temperature (Tm) of ~93 °C. Upon metal ion removal, disulfide-oxidized, dimeric, wild type SOD1 has a melting temperature of ~52 °C and upon reduction and monomerization, metal-free wild type SOD1 melts at ~42 °C. In contrast, metal-free, disulfide-oxidized A4V melts at ~40.5 °C and upon reduction, exhibits no endotherm in the DSC experiment, suggesting that it is predominantly unfolded. Although G93A was not tested in these DSC experiments, the disulfide-oxidized, metal-free pathogenic G93R variant melted at 44.3 °C and also exhibited no endotherm upon reduction [63, 64]. The unfolded state of these metal-free, monomeric pathogenic SOD1 variants suggested by the DSC experiments might hinder the ability of the copper chaperone for SOD1 to act upon these molecules (see below).
The metal-free, disulfide-reduced A4V and G93A/G93R pathogenic SOD1 variants that appear unfolded in the DSC experiments described above presumably correspond to the state in which these molecules exist when newly translated. These observations, combined with previous observations that CCS is unable to posttranslationally modify pathogenic SOD1 variants H46R/H48Q, D124V, and H80R, suggest the hypothesis that hindered CCS-mediated posttranslational modification of pathogenic SOD1 variants leads to metal-deficient, disulfide-reduced, immature pathogenic SOD1 molecules that are aggregation-prone. In support of this hypothesis, recent studies in cell culture and in transgenic mice reveal that much of the pathogenic SOD1 protein in the soluble fraction is metal-deficient, disulfide-reduced, and/or monomeric [25, 27, 60, 65]. Interestingly, in a pair of recent studies, it was found that overexpression of CCS greatly accelerated disease in a G93A SOD1 mouse model in the absence of visible proteinaceous inclusions [66, 67]. However, CCS overexpression failed to enhance oxidation of the G93A SOD1 disulfide bond and in fact, elevated the population of disulfide-reduced G93A SOD1 in the soluble fraction of brain and spinal cord of these animals . These data suggest that CCS may be interacting with nascent G93A, keeping it in the soluble fraction, but in a non-productive way in terms of the posttranslational insertion of copper and oxidation of the disulfide bond. However, it remains unclear why CCS overexpression in these animals results in elevated rather than diminished levels of disulfide reduced G93A SOD1, but this observation suggests that, like H46R/H48Q SOD1, CCS binding alone is not sufficient to convert nascent G93A SOD1 into its mature holo form. A more detailed description of how the various pathogenic SOD1 substitutions might interfere with CCS-mediated SOD1 maturation can be found in [8, 9, 55, 66].
In summary, the results presented here on the structure and biophysical properties of metal-bound and metal-free A4V and G93A SOD1 variants, together with data on pathogenic SOD1 proteins coming from cell culture and in transgenic mice, suggest that incomplete posttranslational modification of nascent SOD1 polypeptides may be a characteristic shared by fALS SOD1 mutants, resulting in a population of destabilized, folding intermediates that are toxic to motor neurons.
This work was supported by grants NIH-NINDS R01-NS39112 (P.J.H.), the Motor Neuron Disease Association, U.K. (S.S.H., R.W.S), and in part by NIH T32AG021890-03 (J.P.S.), the American Foundation for Aging Research (L.J.W.), the William and Ella Owens Medical Research Foundation and the Judith and Jean Pape Adams Charitable Foundation (A.G.). The U.K. group is grateful to BBSRC, MRC, EPSRC, NWDA and STFC for financial support and to STFC Daresbury Laboratory for provision of facilities and also thanks Peter A. Doucette and Joan S. Valentine for the gift of G93A SOD1. Support for the X-ray Crystallography Core Laboratory and the Center for Analytical Ultracentrifugation of Macromolecular Assemblies by the UTHSCSA Executive Research Committee and the San Antonio Cancer Institute is also gratefully acknowledged.
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Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 3GZO, 2WKO (metal-bound G93A), 3GZP (metal-free G93A),and 3GZQ (metal-free A4V).