Development of an X-ray fluorescence holographic measurement system for protein crystals

Ayana Sato-Tomita, Naoya Shibayama, Naohisa Happo, Koji Kimura, Takahiro Okabe, Tomohiro Matsushita, Sam Yong Park, Yuji C. Sasaki, Kouichi Hayashi

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)


Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α2β2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Original languageEnglish
Article number063707
JournalReview of Scientific Instruments
Issue number6
Publication statusPublished - 2016 Jun 1

ASJC Scopus subject areas

  • Instrumentation


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