実験報告書様式（一般利用課題・成果公開利用）

MLF Experimental Report

提出日 Date of Report
24 October, 2014

課題番号 Project No.

装置責任者 Name of responsible person

2012B0106

Kusaka, Katsuhiro

実験課題名 Title of experiment

装置名 Name of Instrument/（BL No.）

Neutron diffraction analysis of trigonal ferro cytochrome c of horse

IBARAKI biological crystal diffracto-heart

meter/(BL-03)

実験責任者名 Name of principal investigator

実施日 Date of Experiment

Yoko Sugawara

27 February – 16 March, 2013

所属 Affiliation
Kitasato University
試料、実験方法、利用の結果得られた主なデータ、考察、結論等を、記述して下さい。（適宜、図表添付のこと）
Please report your samples, experimental method and results, discussion and conclusions. Please add figures andtables for better explanation.
1. 試料 Name of sample(s) and chemical formula, or compositions including physical form.
Oxidized form of cytochrome c from horse heart(104 amino acid residues with a heme)

2. 実験方法及び結果 （実験がうまくいかなかった場合、その理由を記述してください。）
Experimental method and results. If you failed to conduct experiment as planned, please describe reasons.
Cytochrome c, a class of heme-containing proteins, mediates the transfer of electrons in the redox systems inmitochondria. It was proposed that hydration water molecules would participate in the inter-molecular electrontransfer process. In order to make the hydration and hydrogen bonding schemes around the heme moiety clear,
we are carrying out X-ray and neutron crystallographic analysis of oxidized form of cytochrome c from horse
heart.
We had prepared crystals of trigonal (space group P3121, a = 80.8, c = 90.6 Å) and tetragonal (space group
P43, a = 58.4, c = 42.1 Å) cytochrome c with approximate volumes of 1 mm3 using ammonium sulfate-sodium
nitrate and ammonium sulfate-sodium chloride as precipitants, respectively. The test neutron data collection
revealed that the tetragonal crystal was found to be better in crystallinity (Project Number: 2012A0097).
Therefore full neutron intensity data collection using a tetragonal crystal with approximate volume of 2.4 mm3(2.0 x 1.2 x 1.0 mm) was carried out.
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2. 実験方法及び結果（つづき） Experimental method and results (continued)
The single crystal sealed in a quartz capillary tube was irradiated by the pulsed white neutron, and Braggreflections were collected with TOF technique at room temperature using 30 detectors. The accelerator power
was 300 kW. Wavelength of the incident neutron was 3.0 – 6.0 Å for the 1st frame and 5.5 – 8.0 Å for the 2ndframe. Twenty data sets were collected for each frames, and the exposure time of 1st frame was 18 hours per one
crystal orientation and that for 2nd frame was 1.8 hours. Total beam time was 16 days.
Data reduction was carried out using STARGazer. Intensities of 8647 symmetrically independent reflections
up to 2.0 Å resolution were collected. Structure refinement was carried out using Phenix starting from the X-ray
structure of tetragonal cytochrome c (PDB-ID: 1HRC), and R and Rfree values at the present stage are 0.23, 0.24,
respectively. The nuclear density distribution was calculated by maximum entropy method (MEM) using 2765
reflections (|Fo|>10σ(Fo)) up to 2.0 Å resolution. The MEM map around the hem moiety is shown in Fig.1.
Several hydrogen atoms are recognized in the nuclear density map as negative densities.
The quality of the data was insufficient to discuss the precise hydration scheme around the heme moiety. We
are continuing trials to improve quality and size of crystals. In 2015, the accelerator power will increase to 600
kW and we are going to carry out remeasurements.

Fig. 1 The nuclear density distribution around the hem moiety calculated
by maximum entropy method (MEM). (Yellow cage, positiveregion (0.08 cm-14/Å3): light blue cage, negative region (−0.04cm-14/Å3).)

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