Non-cryogenic structure of a chloride pump provides crucial clues to temperature-dependent channel transport efficiency

Ji-Hye Yun, Xuanxuan Li, Jae-Hyun Park, Yang Wang, Mio Ohki, Zeyu Jin, Wonbin Lee, Sam-yong Park, Hao Hu, Chufeng Li, Nadia Zatsepin, Mark S. Hunter, Raymond G. Sierra, Jake Koralek, Chun Hong Yoon, Hyun-Soo Cho, Uwe Weierstall, Leihan Tang, Haiguang Liu*, Weontae Lee*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

11 Citations (Scopus)


Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.
Original languageEnglish
Pages (from-to)794-804
Number of pages11
JournalJournal of Biological Chemistry
Issue number3
Publication statusPublished - Jan 2019


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