SPring-8, the large synchrotron radiation facility

Inquiry number

SOL-0000001092

Beamline

BL41XU (Macromolecular Crystallography I)

Scientific keywords

A. Sample category	biology, medicine
B. Sample category (detail)	biomolecule, crystal, protein, pharmaceuticals
C. Technique	X-ray diffraction
D. Technique (detail)	single crystal, MAD
E. Particular condition	low-T (~ liquid N2)
F. Photon energy	X-ray (4-40 keV)
G. Target information	chemical state, chemical bonding, structure analysis, function and structure

Industrial keywords

level 1---Application area	Pharmaceuticals
level 2---Target	drug design, process analytical technology (PAT)
level 3---Target (detail)	protein, drug
level 4---Obtainable information	crystal structure, local structure
level 5---Technique	diffraction, X-ray diffraction

Classification

M10.10 single crystal diffraction

Body text

Multiwavelength anomalous diffraction (MAD) method is the powerful method to obtain the phase information from one sample crystal composed of molecule with heavy atoms.

Most protein is composed by light atoms, such as carbon, oxygen, nitrogen and so on. In multiwavelength anomalous diffraction (MAD) experiment, therefore, it is necessary to bind some heavy atom to protein artificially. When the purpose protein is expressed as a selenomethionyl protein, it has some selen as heavy atoms intrinsically. It is not necessary to make heavy atom derivatives by using this selenomethionyl protein, and expected highly improvement of throughput.

In this case, structure of selenomethionyl heme oxygenase-1 from rat was determined by MAD experiment using Se.

This protein has eight methionines. The protein replaced all methionine to selenmethionine did not crystallized in reproducible. Four methionines were replaced to other amino acid by genomic engineering technique, and reminder were replaced to selenomethionine.

In this result, it could be discussed the differences of activities to substrates between this enzyme from rat and human (Fig. 1).

Figure 1. Stereo-view of heme oxygenase-I
(green: from rat, blue: crossed form from human, red: open form from human)

[ M. Sugishima, Y. Omata, Y. Kakuta, H. Sakamoto, M. Noguchi and K. Fukuyama, FEBS Letters 471, 61-66 (2000), Fig. 4,
©2000 Federation of European Biochemical Societies ]

Source of the figure

Original paper/Journal article

Journal title

M. Sugishima, et al., FEBS Lett. 471(1), 61-66, Apr. 7 (2000)

Figure No.

4

Technique

Multiwavelength anomalous dispersion (MAD) method is that the X-ray diffraction data are collected at near several energies of the absorption edge of heavy atom binding to molecule consisting crystal. By comparing and analyzing those diffraction data, the phase information is decided, and three-dimensional structure is able to determined.

Most protein is composed by light atoms, such as carbon, oxygen, nitrogen and so on. In multiwavelength anomalous dispersion (MAD)  experiment, therefore, it is necessary to bind some heavy atom to protein artificially. It costs large time and labor to search species and conditions of heavy atoms bound to the protein.

Methionine is one of the amino acid composing the protein, and selenomethionine is synthesized amino acid that replaced sulfur atom of methionine to selen. When the methione auxotrophic E. Coli is grown in the medium containing selenomethionine instead of methione, all protein expressed by that E. coli are synthesized using selenomethione. This "selenomethionyl" protein has the selen as heavy atoms intrinsically. By using this "selenomethionyl" protein as a sample for MAD experiment, it is not necessary to make heavy atom derivatives, and expected highly improvement of throughput.

Source of the figure

No figure

Required time for experimental setup

0 hour(s)

Instruments

Instrument	Purpose	Performance
Protein Crystal Diffractometer	To record diffraction data
Peltier-cooled Si-PIN photodiode	To measure X-ray absorption spectrum

References

Document name
M. Sugishima, et al., FEBS Lett. 471(1), 61-66, Apr. 7 (2000)

Related experimental techniques

Questionnaire

This solution is an application of a main instrument of the beamline.
Similar experiments account for more than 30% of the beamline's subject.

Ease of measurement

Middle

Ease of analysis

Middle

How many shifts were needed for taking whole data in the figure?

Two-three shifts