Cryo-cooling has been nearly universally adopted to mitigate X-ray damage and facilitate crystal handling in protein X-ray crystallography. However, cryo X-ray crystallographic data provide an incomplete window into the ensemble of conformations that is at the heart of protein function and energetics. Room-temperature (RT) X-ray crystallography provides accurate ensemble information, and recent developments allow conformational heterogeneity (the experimental manifestation of ensembles) to be extracted from single-crystal data. Nevertheless, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical issue, increasingly X-ray-damaged high-resolution data sets (1.02–1.52 Å resolution) were obtained from single proteinase K, thaumatin and lysozyme crystals at RT (277 K). In each case a modest increase in conformational heterogeneity with X-ray damage was observed. Merging data with different extents of damage (as is typically carried out) had negligible effects on conformational heterogeneity until the overall diffraction intensity decayed to ∼70% of its initial value. These effects were compared with X-ray damage effects in cryo-cooled crystals by carrying out an analogous analysis of increasingly damaged proteinase K cryo data sets (0.9–1.16 Å resolution). X-ray damage-associated heterogeneity changes were found that were not observed at RT. This property renders it difficult to distinguish real from artefactual conformations and to determine the conformational response to changes in temperature. The ability to acquire reliable heterogeneity information from single crystals at RT, together with recent advances in RT data collection at accessible synchrotron beamlines, provides a strong motivation for the widespread adoption of RT X-ray crystallography to obtain conformational ensemble information.
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Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography
Proteins are the workhorses of the cell. The shape that a protein molecule adopts enables it to carry out its role. However, a protein’s shape, or 'conformation', is not static. Instead, a protein can shift between different conformations. This is particularly true for enzymes – the proteins that catalyze chemical reactions. The region of an enzyme where the chemical reaction happens, known as the active site, often has to change its conformation to allow catalysis to proceed. Changes in temperature can also make a protein shift between alternative conformations. Understanding how a protein shifts between conformations gives insight into how it works. A common method for studying protein conformation is X-ray crystallography. This technique uses a beam of X-rays to figure out where the atoms of the protein are inside a crystal made of millions of copies of that protein. At room temperature or biological temperature, X-rays can rapidly damage the protein. Because of this, most crystal structures are determined at very low temperatures to minimize damage. But cooling to low temperatures changes the conformations that the protein adopts, and usually causes fewer conformations to be present. Keedy, Kenner, Warkentin, Woldeyes et al. have used X-ray crystallography from a very low temperature (-173°C or 100 K) to above room temperature (up to 27°C or 300 K) to explore the alternative conformations of an enzyme called cyclophilin A. These alternative conformations include those that have previously been linked to this enzyme’s activity. Starting at a low temperature, parts of the enzyme were seen to shift from having a single conformation to many conformations above a threshold temperature. Unexpectedly, different parts of the enzyme have different threshold temperatures, suggesting that there isn’t a single transition across the whole protein. Instead, it appears the way a protein’s conformation changes in response to temperature is more complex than was previously realized. This result suggests that conformations in different parts of a protein are coupled to each other in complex ways. Keedy, Kenner, Warkentin, Woldeyes et al. then performed X-ray crystallography at room temperature using an X-ray free-electron laser (XFEL). This technique can capture the protein’s structure before radiation damage occurs, and confirmed that the alternative conformations observed were not affected by radiation damage. The combination of X-ray crystallography at multiple temperatures, new analysis methods for identifying and measuring alternative conformations, and XFEL crystallography should help future studies to characterize conformational changes in other proteins.
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- Award ID(s):
- 1330685
- NSF-PAR ID:
- 10019225
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Date Published:
- Journal Name:
- eLife
- Volume:
- 4
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.more » « less
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null (Ed.)Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure–function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as ∼5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09–1.54 Å resolution data over a temperature range of 293–363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.more » « less
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X-ray crystallography has been invaluable in delivering structural information about proteins. Previously, an approach has been developed that allows high-quality X-ray diffraction data to be obtained from protein crystals at and above room temperature. Here, this previous work is built on and extended by showing that high-quality anomalous signal can be obtained from single protein crystals using diffraction data collected at 220 K up to physiological temperatures. The anomalous signal can be used to directly determine the structure of a protein,more » « less
i.e. to phase the data, as is routinely performed under cryoconditions. This ability is demonstrated by obtaining diffraction data from model lysozyme, thaumatin and proteinase K crystals, the anomalous signal from which allowed their structures to be solved experimentally at 7.1 keV X-ray energy and at room temperature with relatively low data redundancy. It is also demonstrated that the anomalous signal from diffraction data obtained at 310 K (37°C) can be used to solve the structure of proteinase K and to identify ordered ions. The method provides useful anomalous signal at temperatures down to 220 K, resulting in an extended crystal lifetime and increased data redundancy. Finally, we show that useful anomalous signal can be obtained at room temperature using X-rays of 12 keV energy as typically used for routine data collection, allowing this type of experiment to be carried out at widely accessible synchrotron beamline energies and enabling the simultaneous extraction of high-resolution data and anomalous signal. With the recent emphasis on obtaining conformational ensemble information for proteins, the high resolution of the data allows such ensembles to be built, while the anomalous signal allows the structure to be experimentally solved, ions to be identified, and water molecules and ions to be differentiated. Because bound metal-, phosphorus- and sulfur-containing ions all have anomalous signal, obtaining anomalous signal across temperatures and up to physiological temperatures will provide a more complete description of protein conformational ensembles, function and energetics. -
PNAS (Ed.)While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C’-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.07574
