Structural Insights into Iron Ions Accumulation in Dps Nanocage

Dps (DNA-binding protein from starved cells) is well known for the structural protection of bacterial DNA by the formation of highly ordered intracellular assemblies under stress conditions. Moreover, this ferritin-like protein can perform fast oxidation of ferrous ions and subsequently accumulate clusters of ferric ions in its nanocages, thus providing the bacterium with physical and chemical protection. Here, cryo-electron microscopy was used to study the accumulation of iron ions in the nanocage of a Dps protein from Escherichia coli. We demonstrate that Fe2+ concentration in the solution and incubation time have an insignificant effect on the volume and the morphology of iron minerals formed in Dps nanocages. However, an increase in the Fe2+ level leads to an increase in the proportion of larger clusters and the clusters themselves are composed of discrete ~1–1.5 nm subunits.


Introduction
The DNA-binding protein from starved cells (Dps) interacts with DNA condensing, and compacting it into a higher ordered structure and protecting it from the destructive effects of high temperature, electromagnetic and UV radiation, as well as oxidative stress [1][2][3]. Dps is a multifunctional protein belonging to the ferritin superfamily, which includes prokaryotic and eukaryotic ferritins and heme-containing bacterioferritin and is called miniferritin [3][4][5][6]. While ferritins use molecular oxygen to catalyze Fe 2+ oxidation, Dps utilizes hydrogen peroxide as a physiological iron oxidant, thus contributing to a decrease in the effectiveness of the Fenton reaction [7][8][9][10]. The formation of minerals in the ferritin cavity occurs in several stages: the penetration of Fe 2+ , the catalytic redox process with O 2 , the nucleation of Fe 3+ O and the growth of the mineral, and then the dissolution of minerals with the release of Fe 2+ . Moreover, the catalytic binding of 2Fe-/O 2 occurs in ms, and nucleation/mineralization takes from minutes to hours. In eukaryotic ferritins, biomineral order/crystallinity is influenced by nucleation channels between active sites and the mineral growth cavity [10]. Ferritin can accommodate about 4500 iron atoms [11], forming nanominerals with diameter~5 to~8 nm [12]. Dps consists of 12 identical subunits and forms a dodecamer with 23 (tetrahedral) point group symmetry, which also has a hollow core and pores at the three-folds resulting in one of the smallest

Results and Discussion
While it is well established that the cavity of Dps can store up to 500 iron atoms [3,5,[12][13][14], for detailed study of fast iron accumulation processes five concentrations of FeSO 4 (50,200,350, 500 and 2000 iron atoms per Dps dodecamer) were used. The prepared iron-containing samples are referred to in the following text as Dps-Fe50, Dps-Fe200, Dps-Fe350, Dps-Fe500 and Dps-Fe2000. Figure 1 demonstrates cryo-EM images and 2D class averages of Dps particles upon the addition of FeSO 4 to the mixture and 24h incubation. Dark spots on cryo-EM images indicate the formation of the iron clusters inside the Dps cavities ( Figure 1, 1st line). The obtained class averages suggest that the clusters (shown in white in Figure 1, 2nd line) are composed of discrete~1-1. 5

nm subunits.
While it is well established that the cavity of Dps can store up to 500 iron atoms [3,5,[12][13][14], for detailed study of fast iron accumulation processes five concentrations of FeSO4 (50,200,350, 500 and 2000 iron atoms per Dps dodecamer) were used. The prepared iron-containing samples are referred to in the following text as Dps-Fe50, Dps-Fe200, Dps-Fe350, Dps-Fe500 and Dps-Fe2000. Figure 1 demonstrates cryo-EM images and 2D class averages of Dps particles upon the addition of FeSO4 to the mixture and 24h incubation. Dark spots on cryo-EM images indicate the formation of the iron clusters inside the Dps cavities ( Figure 1, 1st line). The obtained class averages suggest that the clusters (shown in white in Figure 1, 2nd line) are composed of discrete ~1-1.5 nm subunits. All samples showed uneven filling of the Dps nanocages; even the mixture with the highest Fe concentration (Dps-Fe2000) demonstrated the presence of empty Dps particles. However, an increase in iron concentration leads to a distinguishable increase in the number of classes with a large (up to 30 nm 3 ) core size. Such cores possess high contrast, which strongly affects the image alignment procedure, resulting in the lack of the features corresponding to the elements of the protein secondary structure on class averages. It is worth mentioning that the differences between 2D classes obtained from the samples incubated for 30 s, 10 min, 3 h or 24 h (see Figures 2 and A1) are insignificant, which indicates that the cluster formation takes place almost immediately upon the addition of FeSO4 to the solution. Despite the high symmetry of the Dps dodecamer, the formation of the symmetrical mineral cores was observed for only 9.5% of the particles in the Dps-Fe2000 sample after 10 min incubation (Figures 2 and A2). The morphology of such cores corresponded to a uniform hollow cube with thin walls ( Figure A2, EMD-14952), differing significantly from all other samples but similar to the structure of hepatic ferritin [43]. All samples showed uneven filling of the Dps nanocages; even the mixture with the highest Fe concentration (Dps-Fe2000) demonstrated the presence of empty Dps particles. However, an increase in iron concentration leads to a distinguishable increase in the number of classes with a large (up to 30 nm 3 ) core size. Such cores possess high contrast, which strongly affects the image alignment procedure, resulting in the lack of the features corresponding to the elements of the protein secondary structure on class averages. It is worth mentioning that the differences between 2D classes obtained from the samples incubated for 30 s, 10 min, 3 h or 24 h (see Figures 2 and A1) are insignificant, which indicates that the cluster formation takes place almost immediately upon the addition of FeSO 4 to the solution. Despite the high symmetry of the Dps dodecamer, the formation of the symmetrical mineral cores was observed for only 9.5% of the particles in the Dps-Fe2000 sample after 10 min incubation (Figures 2 and A2). The morphology of such cores corresponded to a uniform hollow cube with thin walls ( Figure A2, EMD-14952), differing significantly from all other samples but similar to the structure of hepatic ferritin [43]. Further analysis was performed for the samples incubated for 24 h. To determine the morphology of iron minerals, ab initio models were generated using the stochastic gradient descent (SGD) method followed by 3D classification of single particles. The smallest mineral observed was approximately 1 nm in diameter and had a spherical shape, as described previously [34].  Further analysis was performed for the samples incubated for 24 h. To determine the morphology of iron minerals, ab initio models were generated using the stochastic gradient descent (SGD) method followed by 3D classification of single particles. The smallest mineral observed was approximately 1 nm in diameter and had a spherical shape, as described previously [34]. Figure 3 demonstrates diverse morphology of the iron minerals at different levels of Dps cavity filling (EMD-14948, EMD-14949, EMD-14950, EMD-14951). The accumulation of iron atoms occurs heterogeneously in the cavity of the Dps dodecamer, which leads to a displacement of the mass center from the center of the nanocage. Further analysis was performed for the samples incubated for 24 h. To determine the morphology of iron minerals, ab initio models were generated using the stochastic gradient descent (SGD) method followed by 3D classification of single particles. The smallest mineral observed was approximately 1 nm in diameter and had a spherical shape, as described previously [34]. Figure 3 demonstrates diverse morphology of the iron minerals at different levels of Dps cavity filling (EMD-14948, EMD-14949, EMD-14950, EMD-14951). The accumulation of iron atoms occurs heterogeneously in the cavity of the Dps dodecamer, which leads to a displacement of the mass center from the center of the nanocage.  Figure 4 shows a wide variety of mineral core projections, which can be described using the terminology introduced in [45] for classification of the ferritin iron core morphology based on 2D HAADF STEM (High Angle Annular Dark Field Scanning Transmission Electron Microscopy) images as 'small circle', 'dumbbell', 'crescent', 'doughnut' and 'full raft'. The majority of the clusters consist of subunits, except for those with a 'full  Figure 4 shows a wide variety of mineral core projections, which can be described using the terminology introduced in [45] for classification of the ferritin iron core morphology based on 2D HAADF STEM (High Angle Annular Dark Field Scanning Transmission Electron Microscopy) images as 'small circle', 'dumbbell', 'crescent', 'doughnut' and 'full raft'. The majority of the clusters consist of subunits, except for those with a 'full raft' morphology, and the morphology changes with iron loading, which was previously shown for ferritin using HAADF STEM [43] and GLC-TEM [44].
However, in contrast to the HAADF STEM approach, cryo-EM in the defocus mode does not allow for unequivocal determination of the amount of Fe atoms in the cluster using intensity. To compare relative volumes of the iron minerals using cryo-EM SPA the intensity threshold equal to three protein intensity values was chosen, which ensured both the absence of densities from the Dps and the comparability of the iron cluster sizes to the 2D projections on class averages ( Figure 5).
The cluster with the 'small circle' morphology has a volume <1.5 nm 3 , the size of the cluster with 'dumbbell' morphology is in the range from 1.5 nm 3 to 4 nm 3 (see Figures 5 and A3). Minerals with 4-10 nm 3 volume consist of several contacting subunits lining the inner surface of the Dps cavity, which leads to a large variety of the observed morphologies (see Figures 5 and A4). Nuclei larger than 10 nm 3 are characterized by greater homogeneity (absence of subunits due to fusion) and their various projections can correspond to the 'crescent', 'doughnut' and 'full raft' morphologies ( Figures 5 and A5). raft' morphology, and the morphology changes with iron loading, which was previously shown for ferritin using HAADF STEM [43] and GLC-TEM [44]. However, in contrast to the HAADF STEM approach, cryo-EM in the defocus mode does not allow for unequivocal determination of the amount of Fe atoms in the cluster using intensity. To compare relative volumes of the iron minerals using cryo-EM SPA the intensity threshold equal to three protein intensity values was chosen, which ensured both the absence of densities from the Dps and the comparability of the iron cluster sizes to the 2D projections on class averages ( Figure 5).
The cluster with the 'small circle' morphology has a volume <1.5 nm 3 , the size of the cluster with 'dumbbell' morphology is in the range from 1.5 nm 3 to 4 nm 3 (see Figures 5 and A3). Minerals with 4-10 nm 3 volume consist of several contacting subunits lining the inner surface of the Dps cavity, which leads to a large variety of the observed morphologies (see Figures 5 and A4). Nuclei larger than 10 nm 3 are characterized by greater homogeneity (absence of subunits due to fusion) and their various projections can correspond to the 'crescent', 'doughnut' and 'full raft' morphologies ( Figures 5 and A5). It was found that an increase in the iron concentration leads to an increase in the percentage of clusters with a larger volume; the results of the 3D classification are presented in the histogram in Figure 6. Despite the high symmetry of the Dps dodecamer, the formation of the symmetrical minerals was not observed (one could expect tetrahedral nuclei with vertices near the pores). In this case, the process of mineral formation can proceed by several reactions. The primary catalytic reaction mediated by miniferritins occurs at the ferroxidase centers. By using either hydrogen peroxide or molecular oxygen as electron acceptors, the Fe 2+ ions that reach the ferroxidase centers at the dimer interface are oxidized to Fe 3+ before moving to a nucleation site to start the mineralization process.
In our experiments, the co-substrate was oxygen and, accordingly, the ferroxidation It was found that an increase in the iron concentration leads to an increase in the percentage of clusters with a larger volume; the results of the 3D classification are presented in the histogram in Figure 6. Despite the high symmetry of the Dps dodecamer, the formation of the symmetrical minerals was not observed (one could expect tetrahedral nuclei with vertices near the pores). In this case, the process of mineral formation can proceed by several reactions. The primary catalytic reaction mediated by miniferritins occurs at the ferroxidase centers. By using either hydrogen peroxide or molecular oxygen as electron acceptors, the Fe 2+ ions that reach the ferroxidase centers at the dimer interface are oxidized to Fe 3+ before moving to a nucleation site to start the mineralization process. Thus, in the present study, cryo-EM SPA was successfully applied to explore the morphological diversity of the iron clusters formed in Dps nanocages. It was established that iron clusters have an inhomogeneous structure consisting of discrete subunits ~1-1.5 nm in diameter. The formation of the mineral occurred in a matter of seconds close to the inner surface of Dps cavity and further incubation up to 24 h did not lead to any significant changes. Our study proves that the entry of Fe 2+ into the Dps cavity occurs spontaneously in solution, similar to that of ferritin [10]. Moreover, it was found that the increase in the incubation time and Fe 2+ concentration does not affect the morphological diversity and volume distribution of the clusters. While the obtained results confirm the literature data that Dps loading does not exceed 500 Fe atoms per dodecamer [3,5,[12][13][14], we have shown that even at concentration of 2000 atoms per dodecamer the cavities of most Dps particles are only partially filled with iron minerals. This is possibly due to the presence of EDTA in the buffer, which is capable of forming salts of ethylenediaminetetraacetates with metal cations. It was demonstrated that the filling of the nanocage occurs unevenly and an increase in the concentration of iron in the solution results in an increase of the average size of the iron cluster.

Preparation of Fe-Containing Dps Samples
Expression, purification and analysis of E. coli Dps was performed as detailed in [49,50]

Cryo-Electron Microscopy
An amount of 3 µL of the Dps-Fe solution mixture was applied to a Lacey EM grid (Ted Pella, Northport, NY, USA) glow discharged for 30 s at 0.26 mbar pressure using current of 25 mA with Pelco EasiGlow (Ted Pella, Northport, NY, USA). The grids were then blotted with filter paper for 2.5 s from both sides at T = 20 °C and vitrified using Vitrobot Mark IV (Thermo Fisher Scientific, Hillsboro, OR, USA). Cryo-EM study was In our experiments, the co-substrate was oxygen and, accordingly, the ferroxidation reaction [46]: where [Fe 2 -Dps] Z+2 represents a dinuclear iron center and Fe 2 (OH) 2 -Dps represent the oxidized iron species. After ferroxidation, ferric species leave the ferroxidase centers towards the carboxylaterich lining of the inner cavity, where the ferrihydrite mineral core is formed. Following the initial iron turnovers by the apo-form of the protein, di-ferric oxo-species participate in a nucleation reaction that occurs in putative specific sites. The subsequent chain of ferroxidation and mineralization reactions leads to the formation of the ferrihydrite mineral core that grows from the initial nucleation sites [14].
The chemical composition of the mineral core does not depend on the co-substrate used in the iron oxidation reaction; in our study, the chemical equation for the reaction of mineralization with molecular oxygen [46][47][48]: where FeOOH represents the iron(III)oxide-hydroxide that composes the ferrihydrite mineral.
We can also assume that there could be a lack of molecular oxygen in the protein solution, in which case some part of the core was formed by the reaction: Fe 2+ + H 2 O + Cl − → FeOHCl (s) + H + Thus, in the present study, cryo-EM SPA was successfully applied to explore the morphological diversity of the iron clusters formed in Dps nanocages. It was established that iron clusters have an inhomogeneous structure consisting of discrete subunits~1-1.5 nm in diameter. The formation of the mineral occurred in a matter of seconds close to the inner surface of Dps cavity and further incubation up to 24 h did not lead to any significant changes. Our study proves that the entry of Fe 2+ into the Dps cavity occurs spontaneously in solution, similar to that of ferritin [10]. Moreover, it was found that the increase in the incubation time and Fe 2+ concentration does not affect the morphological diversity and volume distribution of the clusters. While the obtained results confirm the literature data that Dps loading does not exceed 500 Fe atoms per dodecamer [3,5,[12][13][14], we have shown that even at concentration of 2000 atoms per dodecamer the cavities of most Dps particles are only partially filled with iron minerals. This is possibly due to the presence of EDTA in the buffer, which is capable of forming salts of ethylenediaminetetraacetates with metal cations. It was demonstrated that the filling of the nanocage occurs unevenly and an increase in the concentration of iron in the solution results in an increase of the average size of the iron cluster.

Preparation of Fe-Containing Dps Samples
Expression, purification and analysis of E. coli Dps was performed as detailed in [49,50].

Cryo-Electron Microscopy
An amount of 3 µL of the Dps-Fe solution mixture was applied to a Lacey EM grid (Ted Pella, Northport, NY, USA) glow discharged for 30 s at 0.26 mbar pressure using current of 25 mA with Pelco EasiGlow (Ted Pella, Northport, NY, USA). The grids were then blotted with filter paper for 2.5 s from both sides at T = 20 • C and vitrified using Vitrobot Mark IV (Thermo Fisher Scientific, Hillsboro, OR, USA). Cryo-EM study was conducted using Titan Krios (Thermo Fisher Scientific, Hillsboro, OR, USA) equipped with Falcon 2 direct electron detector (Thermo Fisher Scientific, Hillsboro, OR, USA) and Image Corrector (CEOS, Heidelberg, Germany) operated at 300 kV. Images were obtained using EPU software (ThermoFisher Scientific, Hillsboro, OR, USA).
For SPA, movies of the Dps-Fe samples were obtained at 75,000× with 0.86 Å pixel size. Each movie consisted of 40 frames and was collected for 2 s. Total dose per movie was~80 e/Å 2 ; defocus values were in the range of 0.8-2.0 µm. Drift correction, CTF and defocus estimation, particle picking and extraction were conducted with Warp [51]; further processing was performed in CryoSPARC [52].
After the 1st round of 2D classification 267,406 particles of Dps-Fe50 sample (from 742 movies), 75,418 particles of Dps-Fe200 (from 108 movies), 321,356 particles of Dps-Fe350 (from 271 movies), 110,412 particles of Dps-Fe500 (from 164 movies), and 47,625 particles of Dps-Fe2000 (from 134 movies) were selected. Particles without Fe-containing core were removed after 2D classification and particles with iron core from all the samples were pooled into one set for further analysis. For this set 10 ab initio models were obtained and hetero refinement procedure was performed. The resolution of the obtained reconstructions was in the range of 7-10 Å (FSC = 0.143 criteria). Since alpha helices were not distinguishable on most reconstructions, it was impossible to determine the exact orientation of the core relative to the Dps dodecamer.
The resulting models were sorted by the core volume, the shape of the core was examined visually. For these density maps a mask was applied to the protein and the average intensity from the protein was determined; to determine the volume of the nucleus, a threshold was chosen equal to three protein intensity values (due to high contrast of the nucleus), at which no protein was observed for all reconstructions, and the sizes of the nuclei were comparable to those in 2D classes. Overall data curation procedure is shown in Figure 7

Conclusions
The Dps proteins are expressed in bacterial cells under nutritional and oxidative stress conditions to protect DNA. Since Dps is a key component of the protective Dps-DNA, on which the efficiency of bacterial protection depends, we investigated the process of formation of the iron minerals inside the Dps nanocage. Herein, for the first time, it was determined that the formation of mineral core in Dps nanocage occurs within 30 s, with no significant changes being observed within the next 24 h. It was found that the size and morphology of iron clusters essentially do not depend on the concentration of iron ions in solution, and the majority of clusters consist of discrete 1-1.5 nm nanoparticles. The obtained results can be useful for nanoparticle synthesis and practical applications in biotechnology.  The obtained density maps were divided into three groups depending on the volume of the nucleus: V 1 < 4 nm 3 , 4 nm 3 ≤ V 2 ≤ 10 nm 3 , V 3 > 10 nm 3 . The group V 1 was divided into five 3D classes; groups V 2 and V 3 were divided into 10 classes each. The classification results are shown in Figures 5 and A3-A5. The obtained metadata containing information about the number of particles from a particular sample in a particular class was used to build a histogram ( Figure 6) of the iron core volume distribution.
For the control Dps sample, 515 movies and 414,851 particles were obtained. After 2D classification, 246,751 Dps particles with an estimated resolution above 4 Å were selected for further processing. These particles were used to obtain ab initio model (using P23 symmetry) and 3D reconstruction of Dps dodecamer with a resolution of 3.16 Å. The subsequent 3D classification allowed the selection of 131,127 particles for the final reconstruction by the non-uniform refinement method, resulting in a 2.59 Å density map (using P23 symmetry; see Figure A6). The obtained cryo-EM map was deposited to EMDB under the code EMD-14943.

Conclusions
The Dps proteins are expressed in bacterial cells under nutritional and oxidative stress conditions to protect DNA. Since Dps is a key component of the protective Dps-DNA, on which the efficiency of bacterial protection depends, we investigated the process of formation of the iron minerals inside the Dps nanocage. Herein, for the first time, it was determined that the formation of mineral core in Dps nanocage occurs within 30 s, with no significant changes being observed within the next 24 h. It was found that the size and morphology of iron clusters essentially do not depend on the concentration of iron ions in solution, and the majority of clusters consist of discrete 1-1.5 nm nanoparticles. The obtained results can be useful for nanoparticle synthesis and practical applications in biotechnology.