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Charge-density analysis of an iron–sulfur protein at an ultra-high resolution of 0.48 Å


The fine structures of proteins, such as the positions of hydrogen atoms, distributions of valence electrons and orientations of bound waters, are critical factors for determining the dynamic and chemical properties of proteins. Such information cannot be obtained by conventional protein X-ray analyses at 3.0–1.5 Å resolution, in which amino acids are fitted into atomically unresolved electron-density maps and refinement calculations are performed under strong restraints1,2. Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with pre-existing common knowledge obtained by chemistry in small molecules. However, even now, computational calculation of such information with quantum chemistry also tends to be difficult, especially for polynuclear metalloproteins3. Here we report a charge-density analysis of the high-potential iron–sulfur protein from the thermophilic purple bacterium Thermochromatium tepidum using X-ray data at an ultra-high resolution of 0.48 Å. Residual electron densities in the conventional refinement are assigned as valence electrons in the multipolar refinement. Iron 3d and sulfur 3p electron densities of the Fe4S4 cluster are visualized around the atoms. Such information provides the most detailed view of the valence electrons of the metal complex in the protein. The asymmetry of the iron–sulfur cluster and the protein environment suggests the structural basis of charge storing on electron transfer. Our charge-density analysis reveals many fine features around the metal complex for the first time, and will enable further theoretical and experimental studies of metalloproteins.

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Figure 1: Crystal structure of HiPIP at 0.48 Å resolution.
Figure 2: The Fe4S4 cluster.
Figure 3: Topological analysis of charge density in the Fe4S4 cluster.
Figure 4: Interaction network around the Fe4S4 cluster.

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Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under accession number 5D8V.


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We thank K. Kusumoto and H. Ohno for their contributions in the initial steps of the work, and T. Tsujinaka and S. Niwa for their contributions in the preparation of the manuscript. We also thank the BL41XU beamline staff of SPring-8 for their help in data collection. This work was supported by a Grant-in-Aid for Scientific Research (number 23657073 to K.T.) and the Photon and Quantum Basic Research Coordinated Development Program (to K.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author information

Authors and Affiliations



K.M. initiated and supervised the project. K.T. designed the experiments. Y.H. prepared crystals. Y.H. and K.T. performed data collection and the crystallographic analysis. Y.H., K.T. and K.M. discussed the results. Y.H. wrote the initial draft, and K.T. and K.M. revised the manuscript.

Corresponding author

Correspondence to Kunio Miki.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Quality of the diffraction data at 0.48 Å resolution.

a, The diffraction image. Right, zoom view of the boxed region at left. The resolution for each circle is indicated. b, Rsym (blue) and <I>/<σ(I)> (pink) values are plotted for 30 resolution bins. c, Changes of Rsym at the highest-resolution shell (0.50–0.48 Å) and relative B factor in the course of the data collection. The Rsym (blue) and relative B factor (red) are plotted as functions of frame number.

Extended Data Figure 2 Residual electron density for each refinement step.

The left panels show the residual density after the ISAM refinement; the right panels show the residual density after the MAM refinement. a, The plane of the peptide bond between Asn45 and Cys46. Maximum and minimum peaks are 0.33 and −0.22 electrons per cubic ångström for the ISAM analysis, and 0.18 and −0.20 electrons per cubic ångström for the MAM analysis. b, The plane of the aromatic ring of Trp74. Maximum and minimum peaks are 0.34 and −0.29 electrons per cubic ångström for the ISAM analysis, and 0.23 and −0.23 electrons per cubic ångström for the MAM analysis. c, The Fe4S4 cluster. The plane consists of FE1, S3 and Cys43-Sγ atoms. Maximum and minimum peaks are 0.60 and −0.35 electrons per cubic ångström for the ISAM analysis, and 0.35 and −0.29 electrons per cubic ångström for the MAM analysis. The contour interval is 0.05 electrons per cubic ångström for all figures. Blue solid, red dashed and yellow dashed lines denote positive, negative and zero contours, respectively.

Extended Data Figure 3 Interaction network around the Fe4S4 cluster.

a, Deformation electron density around the Cys43-Sγ atom. The main-chain oxygen atom of Asn70, the main-chain carboxyl carbon atom of Gly73 and the H atom of Ile69-Cδ1 are located close to Cys43-Sγ. The static deformation maps are shown as grey and cyan surfaces contoured at the levels of +0.1 and +0.3 electrons per cubic ångström, respectively. The omit map of hydrogen atoms is shown as a pink mesh contoured at the 3.0σ level. The dashed lines indicate interactions between valence densities of sulfur atoms and hydrogen atoms. b, Deformation electron density around Cys61-Sγ. The main-chain amide of Leu63 and the H atom of Phe64-Cδ2 are located close to Cys61-Sγ. c, Deformation electron density around Cys75-Sγ. The main-chain amide of Ser77 is located close to Cys75-Sγ. d, Deformation electron density around S1 of the Fe4S4 cluster. The H atom of Phe48-Cδ2 and the Cδ1 atom of Leu63 are located close to S1. e, Deformation electron density around S2 of the Fe4S4 cluster. The H atoms of Tyr19-Cδ1, Phe64-Cε2 and Ile69-Cγ2 are located close to S2. f, Deformation electron density around S4 of the Fe4S4 cluster. The H atom of Cys43-Cβ, the H atom of Cys46-Cβ and the amide nitrogen atom of Met49 are located close to S4.

Extended Data Figure 4 The local axes for Fe atoms of Fe4S4(Cys-Sγ)4.

a, Whole view of the local axes of the four Fe atoms. b, Close-up views of the local axes of each Fe atom (FE1−FE4). The static deformation maps of Fe4S4(Cys-Sγ)4 are represented as grey isosurfaces contoured at the level of +0.2 electrons per cubic ångström.

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Dihedral and improper angles
Extended Data Table 3 Geometrical parameters in Fe4S4(Cys-Sγ)4
Extended Data Table 4 Atomic properties of Fe4S4(Cys-Sγ)4
Extended Data Table 5 Topological parameters at BCPs of Fe–S bonds
Extended Data Table 6 The d-orbital populations of iron atoms

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Hirano, Y., Takeda, K. & Miki, K. Charge-density analysis of an iron–sulfur protein at an ultra-high resolution of 0.48 Å. Nature 534, 281–284 (2016).

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