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Title: Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

Abstract

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring inmore » the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.« less

Authors:
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Publication Date:
Research Org.:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1260399
DOE Contract Number:  
GM103485; DMR-1332208; STC-1231306; GM110580; OD009180; SLAC-LDRD-0014-13-2; MCB-1330685; GM094586; Postdoctoral Research Fellowship; GM103393; GM102520; BioXFEL Postdoctoral Fellowship; AC02-76SF00515; Collaborative Innovation Award (HCIA); Packard Fellow; Pew Scholar; Searle Scholar; GM095887; Graduate Research Fellowship
Resource Type:
Journal Article
Journal Name:
eLife
Additional Journal Information:
Journal Volume: 4; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Country of Publication:
United States
Language:
English

Citation Formats

Keedy, Daniel A., Kenner, Lillian R., Warkentin, Matthew, Woldeyes, Rahel A., Hopkins, Jesse B., Thompson, Michael C., Brewster, Aaron S., Van Benschoten, Andrew H., Baxter, Elizabeth L., Uervirojnangkoorn, Monarin, McPhillips, Scott E., Song, Jinhu, Alonso-Mori, Roberto, Holton, James M., Weis, William I., Brunger, Axel T., Soltis, S. Michael, Lemke, Henrik, Gonzalez, Ana, Sauter, Nicholas K., Cohen, Aina E., van den Bedem, Henry, Thorne, Robert E., and Fraser, James S. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. United States: N. p., 2015. Web. doi:10.7554/eLife.07574.
Keedy, Daniel A., Kenner, Lillian R., Warkentin, Matthew, Woldeyes, Rahel A., Hopkins, Jesse B., Thompson, Michael C., Brewster, Aaron S., Van Benschoten, Andrew H., Baxter, Elizabeth L., Uervirojnangkoorn, Monarin, McPhillips, Scott E., Song, Jinhu, Alonso-Mori, Roberto, Holton, James M., Weis, William I., Brunger, Axel T., Soltis, S. Michael, Lemke, Henrik, Gonzalez, Ana, Sauter, Nicholas K., Cohen, Aina E., van den Bedem, Henry, Thorne, Robert E., & Fraser, James S. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography. United States. https://doi.org/10.7554/eLife.07574
Keedy, Daniel A., Kenner, Lillian R., Warkentin, Matthew, Woldeyes, Rahel A., Hopkins, Jesse B., Thompson, Michael C., Brewster, Aaron S., Van Benschoten, Andrew H., Baxter, Elizabeth L., Uervirojnangkoorn, Monarin, McPhillips, Scott E., Song, Jinhu, Alonso-Mori, Roberto, Holton, James M., Weis, William I., Brunger, Axel T., Soltis, S. Michael, Lemke, Henrik, Gonzalez, Ana, Sauter, Nicholas K., Cohen, Aina E., van den Bedem, Henry, Thorne, Robert E., and Fraser, James S. 2015. "Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography". United States. https://doi.org/10.7554/eLife.07574.
@article{osti_1260399,
title = {Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography},
author = {Keedy, Daniel A. and Kenner, Lillian R. and Warkentin, Matthew and Woldeyes, Rahel A. and Hopkins, Jesse B. and Thompson, Michael C. and Brewster, Aaron S. and Van Benschoten, Andrew H. and Baxter, Elizabeth L. and Uervirojnangkoorn, Monarin and McPhillips, Scott E. and Song, Jinhu and Alonso-Mori, Roberto and Holton, James M. and Weis, William I. and Brunger, Axel T. and Soltis, S. Michael and Lemke, Henrik and Gonzalez, Ana and Sauter, Nicholas K. and Cohen, Aina E. and van den Bedem, Henry and Thorne, Robert E. and Fraser, James S.},
abstractNote = {Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.},
doi = {10.7554/eLife.07574},
url = {https://www.osti.gov/biblio/1260399}, journal = {eLife},
issn = {2050-084X},
number = ,
volume = 4,
place = {United States},
year = {Wed Sep 30 00:00:00 EDT 2015},
month = {Wed Sep 30 00:00:00 EDT 2015}
}

Works referenced in this record:

Progress in rational methods of cryoprotection in macromolecular crystallography
journal, March 2010


NMR spectroscopy brings invisible protein states into focus
journal, October 2009


Modelling dynamics in protein crystal structures by ensemble refinement
journal, December 2012


Protein imperfections: separating intrinsic from extrinsic variation of torsion angles
journal, December 2004


Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals
journal, March 2007


An automated system to mount cryo-cooled protein crystals on a synchrotron beamline, using compact sample cassettes and a small-scale robot
journal, November 2002


Goniometer-based femtosecond crystallography with X-ray free electron lasers
journal, October 2014


The Backrub Motion: How Protein Backbone Shrugs When a Sidechain Dances
journal, February 2006


Multiscale Conformational Heterogeneity in Staphylococcal Protein A: Possible Determinant of Functional Plasticity
journal, October 2014


Dynamical transition of myoglobin revealed by inelastic neutron scattering
journal, February 1989


The protein-solvent glass transition
journal, January 2010


Biomolecular Simulation: A Computational Microscope for Molecular Biology
journal, June 2012


Enzyme Dynamics During Catalysis
journal, February 2002


Intrinsic dynamics of an enzyme underlies catalysis
journal, November 2005


Features and development of Coot
journal, March 2010


Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR
journal, January 2014


Hidden alternative structures of proline isomerase essential for catalysis
journal, December 2009


Accessing protein conformational ensembles using room-temperature X-ray crystallography
journal, September 2011


A unified model of protein dynamics
journal, February 2009


Thermal expansion of a protein
journal, January 1987


Temperature-dependent X-ray diffraction as a probe of protein structural dynamics
journal, August 1979


The energy landscapes and motions of proteins
journal, December 1991


High-temperature and high-resolution crystallography of thermostable copper nitrite reductase
journal, January 2015


Radiation damage in macromolecular crystallography: what is it and why should we care?
journal, March 2010


Protein Sectors: Evolutionary Units of Three-Dimensional Structure
journal, August 2009


Biomolecular cryocrystallography: Structural changes during flash-cooling
journal, March 2004


Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers
journal, March 2014


Dynamic personalities of proteins
journal, December 2007


Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL
journal, May 2014


A beginner's guide to radiation damage
journal, February 2009


Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit
journal, October 2015


Crystal Cryocooling Distorts Conformational Heterogeneity in a Model Michaelis Complex of DHFR
journal, June 2014


Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy
journal, July 2014


Protein structural ensembles are revealed by redefining X-ray electron density noise
journal, December 2013


Microscopic origins of entropy, heat capacity and the glass transition in proteins
journal, May 2001


Direct observation of hierarchical protein dynamics
journal, April 2015


The ensemble nature of allostery
journal, April 2014


Flexible Backbone Sampling Methods to Model and Design Protein Alternative Conformations
book, January 2013


Protein conformational substates from X-ray crystallography
journal, January 1996


The ‘glass transition’ in protein dynamics: what it is, why it occurs, and how to exploit it
journal, September 2003


Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins
journal, March 2015


Characterizing and controlling the inherent dynamics of cyclophilin-A
journal, January 2009


Photosynthetic complex in close-up
journal, November 2014


Population Shuffling of Protein Conformations
journal, November 2014


Structural heterogeneity in protein crystals
journal, September 1986


X-ray lasers for structural and dynamic biology
journal, September 2012


Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses
journal, November 2014


On the nature of a glassy state of matter in a hydrated protein: Relation to protein function
journal, September 2001


Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals
journal, March 2015


Automated identification of functional dynamic contact networks from X-ray crystallography
journal, August 2013


Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers
journal, September 2009


Integrative, dynamic structural biology at atomic resolution—it's about time
journal, March 2015


Spatial distribution of radiation damage to crystalline proteins at 25–300 K
journal, August 2012


Global radiation damage: temperature dependence, time dependence and how to outrun it
journal, November 2012


Slow cooling of protein crystals
journal, August 2009


E pluribus unum, no more: from one crystal, many conformations
journal, October 2014


Population Shuffling of Protein Conformations
journal, November 2014


PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome
journal, December 2019


Protein imperfections: separating intrinsic from extrinsic variation of torsion angles
text, January 2005


Works referencing / citing this record:

How accurately do force fields represent protein side chain ensembles?
journal, September 2018


Conformational footprints
journal, October 2016


Rescue of conformational dynamics in enzyme catalysis by directed evolution
journal, April 2018


Nylon mesh-based sample holder for fixed-target serial femtosecond crystallography
journal, May 2019


Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus
journal, June 2019


Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature
journal, May 2016


Dynamic design: manipulation of millisecond timescale motions on the energy landscape of cyclophilin A
journal, January 2020


Microfluidics: From crystallization to serial time-resolved crystallography
journal, May 2017


Entropy in molecular recognition by proteins
journal, June 2017


Quantitative structural assessment of graded receptor agonism
journal, October 2019


X-ray free-electron laser: opportunities for drug discovery
journal, July 2018


RIDL : a tool to investigate radiation-induced density loss
journal, May 2018


A temperature-controlled cold-gas humidifier and its application to protein crystals with the humid-air and glue-coating method
journal, June 2019


Shining light on cysteine modification: connecting protein conformational dynamics to catalysis and regulation
journal, June 2019


Intermolecular correlations are necessary to explain diffuse scattering from protein crystals
journal, February 2018


Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography
journal, July 2016


Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography
journal, January 2019


Solvent flows, conformation changes and lattice reordering in a cold protein crystal
journal, October 2019


Data systems for the Linac coherent light source
journal, January 2017


Protein ensembles link genotype to phenotype
journal, June 2019


X-ray free electron laser: opportunities for drug discovery
journal, November 2017


Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles
journal, December 2020


Radiation Damage in Macromolecular Crystallography
journal, November 2015


Ice formation and solvent nanoconfinement in protein crystals
text, January 2019


Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme
journal, September 2019


Resolution and dose dependence of radiation damage in biomolecular systems
journal, July 2019


Strategies for sample delivery for femtosecond crystallography
text, January 2019


Ice formation and solvent nanoconfinement in protein crystals
text, January 2019


Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins
text, January 2019


Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus
text, January 2019


Ice formation and solvent nanoconfinement in protein crystals
journal, July 2019