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A multiwavelength X-ray diffraction study was performed at 6 Å resolution on the native cytochrome c′ crystal from Rhodospirillum rubrum, which contains heme irons. The X-ray intensity data from the crystal were collected on a four-circle diffractometer with three wavelengths, λ1 = 1.077, λ2 = 1.730 and λ3 = 1.757 Å, using the synchrotron radiation produced by the storage ring in the Photon Factory, National Laboratory for High Energy Physics. λ2 and λ3 are above and below the energy of the K absorption edge of iron (λe = 1.743 Å) respectively, while λ1 is far from the edge. The positions of the two iron atoms in the crystal could be determined from the difference Patterson maps based on either the real or imaginary components of the anomalous scattering effect caused by iron. The best phase angles for the crystal were calculated from the X-ray intensity data measured with λ1 and λ2 by the method which is basically the same as the isomorphous replacement method. Although the resulting best phase angles differed 75° on the average from those obtained by the multiple isomorphous replacement method, the molecular boundary and α helices could be recognized on the electron density map.

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