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Article

Effects of Radiation Damage on Metal-Binding Sites in Thermolysin

by
Ki Hyun Nam
College of General Education, Kookmin University, Seoul 02707, Republic of Korea
Crystals 2024, 14(10), 876; https://doi.org/10.3390/cryst14100876
Submission received: 22 July 2024 / Revised: 23 August 2024 / Accepted: 3 October 2024 / Published: 4 October 2024
(This article belongs to the Section Biomolecular Crystals)
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Abstract

:
Radiation damage is an inherent problem in macromolecular crystallography because it impairs the diffraction quality of crystals and produces inaccurate structural information. Understanding radiation damage in protein structures is crucial for accurate structural interpretation and effective data collection. This study undertook X-ray data collection and structure determination of thermolysin (TLN), which contains Zn and Ca ions, by using three different X-ray doses to improve our understanding of the radiation damage phenomena on metal ions in proteins. Data processing revealed typical global radiation damage in TLN, such as an increase in unit cell volume, Rmerge value, and Wilson B-factor. An analysis of the B-factor indicated that radiation damage at the Zn and Ca sites in TLN increased with higher X-ray doses. However, the distance between the metal ions and their interacting residues in TLN was not significantly affected, suggesting that radiation damage to the metal ions has a minimal effect on these interactions. Moreover, the increase in the B-factor of the metal ions according to the X-ray dose was similar to that in the B-factor of the residues interacting with the metal ions. These results expand our understanding of radiation damage phenomena in macromolecules and can be used to improve data collection strategies.

1. Introduction

Macromolecular crystallography provides valuable information for understanding the structure and function of biological macromolecules [1,2]. This structural information offers insights for developing new drugs and engineering proteins to improve industrial enzymes [3,4,5,6,7]. Providing accurate structural information at high resolution is crucial for understanding the exact molecular mechanisms and for designing successful applied research [8,9,10]. Collecting high-resolution data involves many factors, including crystal quality, X-ray properties, detector specifications, and data processing programs. Among these factors, providing intense X-rays with a high photon density to the crystal sample is important in increasing the diffraction intensity of the crystal.
The achievable beam size from low-emission third- and fourth-generation light sources has been reduced [11,12,13]. The development of optical devices such as Kirkpatrick–Baez mirrors enables the focusing of X-rays at sizes from a few microns to nanometers [14,15]. This microfocused beam not only increases the diffraction intensity of the crystal sample by providing a high density of photons, but also shortens the data collection time, thereby increasing the efficiency of the macromolecular crystallography process [16]. However, despite these advantages, intense X-rays cause significant global and specific radiation damage to crystal samples, resulting in degradation of data quality, structural errors, and even crystal destruction [17,18,19,20]. During the process of global radiation damage, free radicals break or modify amino acid bonds [21,22]. This process changes the conformation of amino acids and increases the nonisomorphism of the crystal [21,22]. This disruption of the crystal packing reduces the diffraction intensity of the crystal, leading to the collection of low-resolution data. Typically, global radiation damage is indicated by an increase in unit cell volume, Wilson B-factor, or Rmerge value [21,22]. Specific radiation damage causes incorrect structural information at specific atoms or amino acids, such as by breaking or elongating disulfide bonds, decarboxylating side chains of aspartic and glutamic acids, photoreducing metal centers, disorganizing sulfur atoms in methionine, and increasing the B-factor of selenium atoms in selenomethionine [21,22,23,24,25,26,27,28].
Therefore, crystal structures that sustain radiation damage may provide biologically irrelevant or inaccurate structural information [29,30,31]. This could potentially produce incorrect structural information for subsequent research, such as in drug design or protein engineering for industrial and medical applications. Various experimental and theoretical studies have been conducted to understand the effects and phenomena of radiation damage [18,26,32]. The radiation damage phenomenon that occurs in macromolecular crystallography varies depending not only on the crystal sample, but also on the X-rays used in the experiment, the experimental environment, and the data collection method. Accordingly, conducting radiation damage research based on various data collection scenarios contributes to establishing a data collection strategy that minimizes radiation damage.
Various proteins contain metal ions, which are commonly involved in enzymatic reactions, protein stability, and transport [33,34,35,36]. In general, high-Z atoms, such as metal ions, are known to be more sensitive to radiation damage than atoms (C, N, and O) that constitute amino acids [28,37]. Several studies have investigated radiation damage to metal ions in proteins, including the Mn4Ca complex in photosystem II [38], heme in peroxidase [39], and nickel-containing superoxide dismutase [40]. These studies offer valuable insights into the effects of radiation damage on metal coordination. However, the phenomenon of radiation damage to metal ions contained in proteins is still insufficiently explored in terms of structural analysis.
To extend our knowledge of radiation damage, we investigated radiation damage to the crystal structure of thermolysin (TLN), which contains Zn and Ca ions. X-ray diffraction experiments were performed with large TLN crystals and a microfocusing X-ray beam using a single-point data collection method. Three diffraction datasets of TLN crystals were collected based on three different X-ray doses, and crystal structures were determined at a 1.40 Å resolution. The global and specific radiation damage to TLN, depending on the X-ray dose, was analyzed. Furthermore, the specific radiation damage to metal ions and their interacting residues in TLN was examined. These results provide an improved understanding of the radiation damage phenomenon.

2. Materials and Methods

2.1. Sample Preparation

TLN from Bacillus thermoproteolyticus (Cat No. HR7-098, Hampton Research, Aliso Viejo, CA, USA) was dissolved in 50 mM NaOH. The TLN solution was diluted in 10 mM Tris-HCl (pH 8.0) and 200 mM NaCl and then concentrated to 20 mg/mL using a Centricon (MWCO, 10 kDa, Millipore, Burlington, MA, USA) for crystallization. Crystallization was performed using the hanging-drop vapor diffusion method at 22 °C. The TLN solution (2 µL) was mixed with a crystallization solution (2 µL) containing 0.1 M Tris-HCl (pH 8.5), 10% (v/v) glycerol, and 1.5 M ammonium sulfate, and equilibrated with 0.5 mL of a reservoir solution. Suitable crystals for X-ray diffraction were obtained within a week. The dimensions of the rod-shaped TLN crystals were approximately 150 × 150 × 400 μm3.

2.2. X-ray Diffraction and Data Processing

X-ray diffraction data were collected at Beamline 11C at Pohang Light Source II (PLS-II, Pohang, Republic of Korea) [16]. The X-ray wavelength and photon flux were 0.9794 Å and ~5 × 1011 photons/sec, respectively. The X-ray beam size was approximately 3.5 × 8.5 μm (vertical × horizontal, full width at half maximum) at the sample position. TLN crystals were cryoprotected using a crystallization solution supplemented with 20% (v/v) glycerol for 5 s and then mounted on a goniometer under a 100 K liquid nitrogen stream. X-ray diffraction data were collected using a Pilatus 6M detector (Dectris, Baden-Daettwil, Switzerland). Diffraction data were collected by exposing X-rays to three fresh regions of a single rod-shaped crystal for each of the three exposure times. The X-ray exposure times for the TLN crystal were 100 ms, 500 ms, and 1 s per 1° oscillation, and the total collected oscillation was 360° per dataset. The absorbed X-ray doses were calculated with RADDOSE-3D [41]. The diffraction images were indexed, integrated, and processed using the HKL2000 program [42].

2.3. Structure Determination

The phase problem was solved by the molecular replacement method using MOLREP [43] from the CCP4 program suite [44]. The crystal structure of thermolysin (PDB: 6LZN) [45] was used as the search model. Manual model building was performed with COOT [46]. Structure refinement was performed with REFMAC5 [47]. Water molecules were automatically added to the model with default parameters during structure refinement. The final coordinates were validated by MolProbity [48]. The coordination of metal ions in TLN structures was validated using CheckMyMetal [49]. The structure figures were generated with PyMOL (http://www.pymol.org, accessed on 8 March 2024).

3. Results

3.1. Data Collection

To improve our understanding of radiation damage to metal ions in proteins, the X-ray diffraction data of TLN were collected using a single-point data collection method. The TLN crystal (150 × 150 × 400 μm³) used was larger than the X-ray beam size (3.5 × 8.5 μm, FWHM). Accordingly, the center of the crystal aligned with the X-ray beam was continuously exposed to X-rays during rotation for data collection. This caused accumulated radiation damage in the central region of the crystal. Since X-rays penetrate the crystal sample and the crystal volume was larger than the X-ray size, the unexposed crystal volume was continuously exposed during 180° rotation, and data collected after 180° received accumulated X-ray exposure. The TLN crystals were exposed to X-rays for 100 ms, 500 ms, and 1 s per 1° to investigate X-ray dose-dependent radiation damage, and diffraction data were collected for 360° oscillation. The calculated average diffraction-weighted dose/average doses (exposed region) for TLN100ms, TLN500ms, and TLN1s were approximately 13.01/7.39, 65.06/36.99, and 130.13/73.98 MGy, respectively. The criteria for the resolution cutoff of data for the TLN dataset were set at 100% completeness, >1.6 I/sigma (highest shell), and >0.7 CC1/2 (highest shell). TLN100ms, TLN500ms, and TLN1s were processed to 1.30, 1.35, and 1.30 Å resolutions, respectively (Table 1).
All TLN crystals belonged to the hexagonal space group P6122. The lengths of the a and b axes of TLN100ms, TLN500ms, and TLN1s determined via unit cell analysis were 92.91, 93.08, and 93.14 Å, respectively, while the lengths of the c axis were 128.378, 128.694, and 128.777 Å. This indicated that the unit cell volume increased with X-ray exposure time, showing typical evidence of global radiation damage. Data processing showed that the overall I/sigma(I) values of TLN100ms, TLN500ms, and TLN1s were 28.25, 31.94, and 32.89, respectively. This indicated an increase in the overall I/sigma value with longer X-ray exposure, especially at a low resolution with X-ray exposure (Figure 1A). In theory, global radiation damage should reduce the diffraction intensity of Bragg peaks with increasing exposure time of microfocused X-rays on the crystal sample; however, the I/sigma increases with extended X-ray exposure time because the crystal is larger than the X-ray beam. The quality of the statistical values of Rmerge, Rmeas, and CC1/2 significantly decreased with the increased amount of X-ray exposure (Figure 1B–D). The overall Rmerge/Rmeas values of TLN100ms, TLN500ms, and TLN1s were 0.144/0.146, 0.169/0.172, and 0.317/0.322, respectively, indicating that the Rmerge and Rmeas values increased in proportion to the X-ray exposure time. No significant difference occurred in Rmerge/Rmeas between data up to 50–3 Å, whereas at < 3 Å, the R-values significantly increased because of X-ray exposure (Figure 1B,C). No significant difference was present in CC1/2 values for the TLN data within the 50–3 Å range based on X-ray dose. However, at resolutions < 2 Å, a decrease in CC1/2 values was observed with increasing X-ray dose (Figure 1D).

3.2. Structure Analysis

The crystal structures of TLN were determined to understand the radiation damage to the overall structure and metal-binding sites depending on the X-ray dose. The refinement statistics and quality of electron density maps, as well as temperature factor values, varied depending on the data resolution. Accordingly, the refinement resolution range of all TLN datasets was unified to 35–1.40 Å (Table 2). The electron density map of all TLN samples was clear enough to build the entire sequence from Ile1 to Lys316. All water molecules were automatically added to the TLN structures during structure refinement with default parameters to avoid bias. TLN100ms, TLN500ms, and TLN1s contained 404, 371, and 344 defined water molecules, respectively. The Rwork/Rfree values of the TLN100ms, TLN500ms, and TLN1s structures were 14.82/16.73, 14.94/16.84, and 15.18/14.22, respectively. This indicated that the R-values tended to increase as the X-ray dose increased. The B-factors of TLN100ms, TLN500ms, and TLN1s were 11.64, 12.00, and 13.06 Ų, respectively, while those of the water molecules for TLN100ms, TLN500ms, and TLN1s were 26.29, 26.68, and 27.51 Å2, respectively. This indicates that the quality of the electron density maps corresponding to amino acid and water molecules deteriorated as the X-ray dose increased.
TLN comprises two approximately globular N- and C-terminal domains with a gap spanning the center of the molecule (Figure 2A). A Zn ion, which is involved in protease activity, is located in the central α-helix between the two domains, and four Ca ions involved in protein stability and enzyme activity are located in the outer loop regions (Figure 2A). Superimposition of TLN100ms, TLN500ms, and TLN1s showed structural similarity, with root mean square deviation of 0.02–0.05 Å, indicating no significant radiation damage to the main chain of the TLN structure. The B-factor is used to evaluate protein radiation damage. The B-factor putty representation showed that the B-factors of the loop regions of TLN slightly increased with the increasing X-ray dose (Figure 2B–D). B-factor plot analysis revealed that TLN100ms and TLN500ms were similar, whereas TLN1s had a relatively high B-factor value (Figure 2E). The normalized B-factor analysis showed no significant change in the B-factor for any specific region (Figure 2E). These results indicate that the TLN B-factor value increases with increasing X-ray exposure but without a particular tendency for that of specific amino acid regions to increase further.

3.3. Metal Ion–Binding Sites

High-Z atoms, such as metal ions, are sensitive to radiation damage [28,37]. TLN contains a Zn ion, which is involved in hydrolase activity, and four Ca ions, which contribute to thermal stability [50]. To verify metal ion-binding to the TLN molecule, the initial structure refinement of TLN datasets was performed without metal ions. The results showed strong positive Fo–Fc electron density maps (>10 sigma) corresponding to one Zn and four Ca ions at previously described metal-binding sites in TLN (Supplementary Figure S1). Further structure refinement of TLNs with Zn and Ca ions was performed. The validation of metal ion coordination in TLNs, including geometry and gRMSD, is presented in Table S1. The refinement revealed partial negative Fo–Fc electron density maps for Zn and Ca1, indicating low occupancy. The volume of the negative Fo–Fc electron density map for Zn and Ca sites did not show a particular tendency to increase with increasing X-ray exposure time (Figure 3). The B-factors of the Zn ion in TLN100ms, TLN500ms, and TLN1s were 13.24, 13.47, and 15.23 Å2, respectively, while those of Ca1/Ca2/Ca3/Ca4 in TLN100ms, TLN500ms, and TLN1s were 8.61/12.91/8.46/18.16, 9.20/13.37/9.01/19.64, and 10.86/14.97/10.66/21.91 Å2, respectively. These results indicate that the B-factor increased with increasing X-ray exposure time. Meanwhile, positive Fo–Fc electron density maps were observed near the Zn-binding site (Supplementary Figure S2), which have also been observed in previous serial crystallography studies [51] but were not identified. Since it was unclear what this density corresponded to, no molecule was added to the final model, and the description is excluded here.
The distances between the metal ions and their interacting residues were measured to understand whether the increase in the B-factors of the metal-binding sites affected the interaction between the metal ions and TLN (Table 3).
The Zn-binding site was coordinated by His142 (distance range between TLN100ms, TLN500ms, and TLN1s: 2.09–2.10 Å), His146 (2.03–2.05 Å), and Glu166 (2.08–2.11 Å) (Figure 4A). The Ca1-binding site was coordinated by Asp138 (2.38–2.40 Å), Glu177 (2.53–2.54 Å/2.58–2.60 Å for OE1/OE2), Asp185 (2.42–2.45 Å), Glu187 (2.36–2.38 Å), and Glu190 (2.56–2.58/2.45–2.47 Å for OE1/OE2) (Figure 4B). The Ca2-binding site was coordinated by Tyr193 (2.33 Å), Thr194 (2.43–2.44 Å), Thr194 (2.40–2.42 Å), Ile197 (2.24–2.27 Å), and Asp200 (2.34–2.37 Å) (Figure 4C). The Ca3-binding site was coordinated by Asp57 (2.57 Å), Asp57 (2.38 Å), Asp59 (2.38–2.39 Å), and Gln61 (2.28–2.30 Å) (Figure 4D). The Ca4-binding site was coordinated by Glu177 (2.75–2.79 Å), Asn183 (2.27–2.29 Å), Asp185 (2.39–2.40 Å), and Glu190 (2.34–2.37 Å) (Figure 4E). The superimposition of the Zn and Ca ion-binding sites of TLN100ms, TLN500ms, and TLN1s showed almost identical positioning and conformation of metal-interacting residues (Figure 4). Consequently, B-factors at the metal ion site increased with the X-ray dose, but no significant trends or changes were observed in the distances between the amino acids interacting with these metal ions.
Next, the B-factors of the metal-binding amino acids were investigated according to the X-ray dose. All metal-binding residues showed a tendency for B-factors to increase as the X-ray dose increased. Compared with the overall B-factor and normalized B-factor of TLN100ms, those of TLN500ms and TLN1s increased by approximately < 10.8% and 15%–30%, respectively. Next, when the X-ray exposure time was increased to TLN100ms and TLN1s, the changes in the B-factors of the metal ions and metal-interacting residues were compared. At the Zn site, the B-factor of the Zn ion was larger than that of the interacting residues (Figure 5A). The B-factors (normalized B-factor values in TLN1s/TLN100ms) of the Zn ion and the interacting residues were 2.0 Å2 (1.15) and 1.4–2.0 Å2 (1.15–1.22), respectively. At the Ca1 site, the B-factor of the Asp185, Glu187, and Glu190 residues was larger than that of the Zn ion (Figure 5B) at 2.3 Å2 (1.27) and 1.9–4.1 Å2 (1.21–1.28), respectively. At the Ca2 site, the B-factor of the Ca2 ion was smaller than that of the interacting residues (Figure 5C) at 2.1 Å2 (1.16) and 2.2–3.5 Å2 (1.16–1.18), respectively. At the Ca3 site, the B-factor of the Ca3 ion was smaller than that of the interacting residues (Figure 5D) at 2.2 Å2 (1.26) and 1.8–2.3 Å2 (1.16–1.21), respectively. At the Ca4 site, the B-factor of the Ca4 ion was larger than that of the interacting residues (Figure 5E) at 7 Å2 (1.20) and 1.9–3.4 Å2 (1.15–1.31), respectively.
Upon increasing X-ray exposure, the increase in the absolute B-factor of the metal ion at the Ca4 site was greater than that of the interacting amino acids, whereas the absolute increase in the B-factors of metal ions at other Zn, Ca1, Ca2, and Ca3 sites was not always larger than that of their interacting amino acids. The normalized B-factor of the metal ion at the Ca3 site was larger than that of the interacting amino acids upon increasing X-ray exposure, whereas the normalized B-factor of metal ions at other Zn, Ca1, Ca2, and Ca4 sites was not always larger than that of their interacting amino acids. Consequently, the B-factors of metal ions and their interacting amino acids increased with increasing X-ray dose, but the metal ions did not exhibit a significantly larger change in B-factor compared with their interacting amino acids.

4. Discussion

In this study, the effects of X-ray doses on radiation damage in TLN crystals were investigated to enhance our understanding of radiation damage to metal ions within proteins. As observed during data collection, the quality of statistical values such as I/sigma(I), Rmerge, Rmeas, and CC1/2 in TLN crystals deteriorated with increasing X-ray dose. Moreover, the structural analysis revealed an increase in B-factor values for metal ions with higher X-ray doses. However, unlike X-ray dose-dependent radiation damage, no significant changes were produced in the interaction distances between metal ions and interacting residues. This result indicates that despite radiation damage affecting the data collection and refinement statistics, as well as the B-factors, the interaction distances between metal ions and their interacting residues may remain relatively unaffected. Thus, although high-Z atoms are known to be sensitive to radiation damage, metal ions coordinated within the TLN protein may be less sensitive to radiation damage. Meanwhile, the B-factors for both proteins and metal ions increased as the X-ray dose increased. This suggests that the B-factor may become inaccurate because of radiation damage as the X-ray dose increases. Therefore, for data collection strategies, the X-ray dose should be reduced to minimize the increase in the B-factor caused by radiation damage.
In a similar previous study using larger thaumatin crystals exposed to microfocusing X-rays for 100 ms and 1 s per degree of rotation over 180° during data collection, specific radiation damage such as disulfide bond breaking was observed [52]. However, in this study, despite exposing the TLN crystals to a larger X-ray dose over 360° of rotation during data collection, no significant specific radiation damage was observed apart from an increase in B-factors. The difference in radiation damage may be due to the specific sensitivity of metal ions compared to that of disulfide bonds [53,54]. For example, the reduction of the metal center by X-ray irradiation can depend on the redox potential of the metal ions [39]. Redox-sensitive iron, copper, and disulfide bonds in proteins may be easily damaged by X-ray irradiation [55,56,57,58]. Meanwhile, Zn and Ca are known to be redox-insensitive [59,60], making it difficult to observe significant radiation damage caused by X-ray irradiation on electron density maps. Moreover, the observed specific radiation damage may vary depending on the properties of the crystal sample, such as crystal size and solvent content.
Despite observing radiation damage in this study, the absence of severe consequences, such as diffraction resolution reduction and extremely high B-factors, could be attributed to the protein and crystal properties. Additionally, the larger size of the crystal used in this experiment, compared to the microfocusing beam, may make it relatively less sensitive to radiation damage. For example, if the crystal sample is smaller than or equal to the X-ray beam, the entire crystal volume is continuously exposed to the X-ray, producing radiation damage throughout the sample. Conversely, if the crystal sample is larger than the X-ray beam, only the center aligned with the X-ray is continuously exposed, leading to localized radiation damage accumulation. For example, exposing cuboid-shaped insulin crystals of 10, 20, 50, 100, 200, and 300 µm in size to an X-ray beam (3.5 × 8.5 µm) with a photon flux of 1 × 1012 photons/s showed that the volumes of X-rays exposed in the crystals were 100%, 100%, 48%, 26%, 13.0%, and 8.7%, respectively. The average dose in these exposed regions for 10, 20, 50, 100, 200, and 300 µm crystals was approximately 164.14, 51.40, 17.31, 7.92, 3.89, and 2.54 MGy, respectively. Consequently, when using microfocused beams with single-point data collection, larger crystal sizes result in lower average radiation damage. This indicates that larger crystal sizes can reduce average radiation damage and can be a strategic consideration for data collection using microfocusing beamlines. To systematically analyze radiation damage to metal ions in TLN in more detail, future studies could increase the X-ray dose or use smaller crystal samples for an improved understanding of radiation damage to metal ions in these proteins.
This study investigated radiation damage to metal ions in TLN crystals larger than the microfocusing beam. An increase in the B-factors at metal ion sites was observed with increasing X-ray dose, while the interaction distances between metal ions and coordinating residues remained unaffected. These findings not only enhance our understanding of adiation damage to metal ions, but also provide valuable insights for data collection strategies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cryst14100876/s1, Figure S1: 2Fo-Fc (blue mesh, 1.5σ) and Fo-Fc (green, 10σ) electron density maps of metal binding sites in TLN100ms, TLN500ms, and TLN1s generated by refinement without metal ions; Figure S2: Undefined Fo-Fc (green, counted at 3σ) electron density map are observed around the Zn binding site of TLN; Table S1: Validation of metal coordination for TLN100ms, TLN500ms, and TLN1s.

Funding

This work was funded by the National Research Foundation of Korea (NRF) (NRF-2021R1I1A1A01050838).

Data Availability Statement

The coordinates and structure-factor amplitudes for both structures have been deposited in the PDB under the accession codes 8ZM4 (TLN100ms), 8ZM5 (TLN500ms), and 8ZM6 (TLN1s).

Acknowledgments

I would like to thank the beamline staff at the 11C beamline at the Pohang Accelerator Laboratory for their assistance with data collection.

Conflicts of Interest

The author declares no conflicts of interest.

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Crystals 14 00876 g001
Figure 1. Analysis of data collection statistics of TLN dataset depending on X-ray exposure time. (A) I/sigma, (B) Rmerge, (C) Rmeas, and (D) CC1/2 values of TLN100ms, TLN500ms, and TLN1s.
Figure 1. Analysis of data collection statistics of TLN dataset depending on X-ray exposure time. (A) I/sigma, (B) Rmerge, (C) Rmeas, and (D) CC1/2 values of TLN100ms, TLN500ms, and TLN1s.
Crystals 14 00876 g001
Crystals 14 00876 g002
Figure 2. Analysis of temperature factor of TLN dependent on X-ray dose. (A) Crystal structure of TLN. B-factor putty representation of (B) TLN100ms, (C) TLN500ms, and (D) TLN1s. (E) Profile of B-factors and normalized B-factor of TLN100ms, TLN500ms, and TLN1s.
Figure 2. Analysis of temperature factor of TLN dependent on X-ray dose. (A) Crystal structure of TLN. B-factor putty representation of (B) TLN100ms, (C) TLN500ms, and (D) TLN1s. (E) Profile of B-factors and normalized B-factor of TLN100ms, TLN500ms, and TLN1s.
Crystals 14 00876 g002
Crystals 14 00876 g003
Figure 3. Analysis of electron density maps for metal-binding sites of TLNs. (A) 2Fo–Fc (blue mesh, 5σ) and Fo–Fc (green mesh, 3σ) omit maps for Zn- and Ca-binding sites of TLN100ms, TLN500ms, and TLN1s. (B) 2Fo–Fc (blue mesh, 5σ) and Fo–Fc (green mesh, 3σ; red mesh, −3σ) electron density maps for Zn- and Ca-binding sites of TLN100ms, TLN500ms, and TLN1s after refinement with metal ions.
Figure 3. Analysis of electron density maps for metal-binding sites of TLNs. (A) 2Fo–Fc (blue mesh, 5σ) and Fo–Fc (green mesh, 3σ) omit maps for Zn- and Ca-binding sites of TLN100ms, TLN500ms, and TLN1s. (B) 2Fo–Fc (blue mesh, 5σ) and Fo–Fc (green mesh, 3σ; red mesh, −3σ) electron density maps for Zn- and Ca-binding sites of TLN100ms, TLN500ms, and TLN1s after refinement with metal ions.
Crystals 14 00876 g003
Crystals 14 00876 g004
Figure 4. Superimposition of metal-binding site of TLNs at (A) Zn, (B) Ca1, (C) Ca2, (D) Ca3, and (E) Ca4 sites from TLN100ms (green), TLN500ms (cyan), and TLN1s (pink).
Figure 4. Superimposition of metal-binding site of TLNs at (A) Zn, (B) Ca1, (C) Ca2, (D) Ca3, and (E) Ca4 sites from TLN100ms (green), TLN500ms (cyan), and TLN1s (pink).
Crystals 14 00876 g004
Crystals 14 00876 g005
Figure 5. B-factor analysis of metal ions and their interacting residues of TLNs at (A) Zn, (B) Ca1, (C) Ca2, (D) Ca3, and (E) Ca4 sites from TLN100ms (green), TLN500ms (cyan), and TLN1s (pink).
Figure 5. B-factor analysis of metal ions and their interacting residues of TLNs at (A) Zn, (B) Ca1, (C) Ca2, (D) Ca3, and (E) Ca4 sites from TLN100ms (green), TLN500ms (cyan), and TLN1s (pink).
Crystals 14 00876 g005
Table 1. Data collection statistics.
Table 1. Data collection statistics.
Data CollectionTLN100msTLN500msTLN1s
BeamlineBeamline 11C, PLS-IIBeamline 11C, PLS-IIBeamline 11C, PLS-II
Temperature (K)100100100
Exposure time (ms/°)1005001000
Space groupP6122P6122P6122
Unit cell dimension
a, b, c (Å)92.919, 92.919, 128.37893.081, 93.081, 128.69493.141, 93.141,128.777
α, β, γ (°)90.00, 90.00, 120.0090.00, 90.00, 120.0090.00, 90.00, 120.00
Resolution range (Å)50.0–1.35 (1.37–1.35)50.0–1.30 (1.32–1.30)50.0–1.35 (1.37–1.35)
No. of unique reflections72,111 (3536)81,578 (3991)74,793 (3665)
Completeness (%)100.0 (100.0)100.0 (100.0)100.0 (100.0)
Redundancy38.4 (33.3)35.7 (22.5)32.6 (14.0)
I/sigma(I)28.25 (2.00)31.94 (1.76)32.89 (1.60)
Rmerge0.144 (0.422)0.169 (0.472)0.317 (3.771)
Rmeas0.146 (1.617)0.172 (2.406)0.322 (3.907)
CC1/20.998 (0.859)0.998 (0.762)0.996 (0.703)
Values for the outer shell are given in parentheses.
Table 2. Structure refinement statistics.
Table 2. Structure refinement statistics.
RefinementTLN100msTLN500msTLN1s
Resolution range (Å)34.09–1.4034.16–1.4034.18–1.40
Rworka0.148220.149480.15182
Rfreeb0.167300.168430.17244
R.m.s. deviations
Bonds (Å)0.0120.0120.012
Angles (°)1.7801.8011.758
Average B factors (Å2)
Protein11.64212.00113.603
Water26.29426.68827.514
Ramachandran plot
Most favored (%)96.296.296.5
Allowed (%)3.83.83.5
PDB code8ZM48ZM58ZM6
a Rwork = Σ||Fobs| Σ |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. b Rfree was calculated as Rwork using a randomly selected subset of unique reflections not used for structural refinement.
Table 3. Distance between metal ions and metal ion-interacting residues of TLN.
Table 3. Distance between metal ions and metal ion-interacting residues of TLN.
Metal IonResidues (atom)TLN100msTLN500msTLN1s
ZnHis142 (NE2)2.102.092.09
His146 (NE2)2.032.032.05
Glu166 (OE2)2.082.102.11
Ca1Asp138 (OD2)2.402.382.38
Glu177 (OE1/OE2)2.53/2.582.53/2.602.54/2.59
Asp185 (OD1)2.422.452.45
Glu187 (O)2.382.362.36
Glu190 (OE1/OE2)2.56/2.452.57/2.472.58/2.45
Ca2Tyr193 (O)2.332.332.33
Thr194 (O/OG1)2.40/2.442.42/2.432.42/2.44
Ile197 (O)2.242.242.27
Asp200 (OD1)2.342.372.35
Ca3Asp57 (OD1/OD2)2.57/2.382.57/2.382.57/2.38
Asp59 (OD1)2.392.382.39
Gln61 (O)2.302.282.28
Ca4Glu177 (OE2)2.752.772.79
Asn183 (O)2.282.292.27
Asp185 (OD2)2.392.392.40
Glu190 (OE2)2.342.352.37
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Nam, K.H. Effects of Radiation Damage on Metal-Binding Sites in Thermolysin. Crystals 2024, 14, 876. https://doi.org/10.3390/cryst14100876

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Nam, Ki Hyun. 2024. "Effects of Radiation Damage on Metal-Binding Sites in Thermolysin" Crystals 14, no. 10: 876. https://doi.org/10.3390/cryst14100876

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Nam, K. H. (2024). Effects of Radiation Damage on Metal-Binding Sites in Thermolysin. Crystals, 14(10), 876. https://doi.org/10.3390/cryst14100876

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