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

Research output: Contribution to journalArticlepeer-review

10 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 - 2019 Jan 18

Bibliographical note

Funding Information:
This work was supported by National Research Foundation of Korea (NRF) Grants NRF-2017R1A2B2008483 (to W. L.), NRF-2017M3A9F6029755 (to H. C.), and the Basic Science Research Program NRF-2016R1A6A3A04010213 (to J.-H. Y.) through the Ministry of Education. This work was also supported by Brain Korea Plus(BK) program (to J.-H. P.) and by National Natural Science Foundation of China Grants 11575021, U1530401, and U1430237 (to H. L.). The authors declare that they have no conflicts of interest with the contents of this article. We are grateful to the staff scientists at the Coherent X-ray Imaging (CXI) station of the Linac Coherent Light Source of the SLAC National Accelerator Laboratory, BL17A beamline of the Photon Factory. We also thank the staff scientists at the Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) for their technical support on the initial screening. This research was supported by a Tianhe-2JK computing time award at the Beijing Computational Research Center (CSRC). Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. This LCLS beam time was part of the Protein Crystal Screening (PCS) program. LCLS is an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. Parts of the sample delivery system used at LCLS for this research were funded by the National Institutes of Health Grant P41GM103393, formerly P41RR001209.

Publisher Copyright:
© 2019 American Society for Biochemistry and Molecular Biology Inc. All Rights Reserved.

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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