skip to main content

Explore Research Products in the NSF-PAR

  • NSF Public Access
  • Search Results
  • Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals
Title: Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals
Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.  more » « less
Award ID(s):
1264434 1706322
NSF-PAR ID:
10057071
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more » ; ; ; « less
Date Published:
Journal Name:
IUCrJ
Volume:
5
Issue:
1
ISSN:
2052-2525
Page Range / eLocation ID:
103 to 117
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ( , Journal of Applied Crystallography)
    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
  2. ; ; ; ; ; ; ; ; ; ; et al ( , Journal of Synchrotron Radiation)
    Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations. 
    more » « less
  3. ; ; ( , Acta Crystallographica Section D Structural Biology)
    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,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.

     
    more » « less
  4. ; ; ; ; ; ; ( , Electronic Materials)
    null (Ed.)
    We report an optimized chemical vapor transport method to grow single crystals of (Mn1−xNix)2P2S6 where x = 0, 0.3, 0.5, 0.7, and 1. Single crystals up to 4 mm × 3 mm × 200 μm were obtained by this method. As-grown crystals are characterized by means of scanning electron microscopy and powder X-ray diffraction measurements. The structural characterization shows that all crystals crystallize in monoclinic symmetry with the space group C2/m (No. 12). We have further investigated the magnetic properties of this series of single crystals. The magnetic measurements of the all as-grown single crystals show long-range antiferromagnetic order along all principal crystallographic axes. Overall, the Néel temperature TN is non-monotonous; with increasing Ni2+ doping, the temperature of the antiferromagnetic phase transition first decreases from 80 K for pristine Mn2P2S6 (x = 0) up to x = 0.5 and then increases again to 155 K for pure Ni2P2S6 (x = 1). The magnetic anisotropy switches from out-of-plane to in-plane as a function of composition in (Mn1−xNix)2P2S6 series. Transport studies under hydrostatic pressure on the parent compound Mn2P2S6 evidence an insulator-metal transition at an applied critical pressure of ~22 GPa. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ( , Physical review. B, Condensed matter)
    We characterize the properties of Ce1−xYbxRhIn5 single crystals with 0  x  1 using measurements of powder x-ray diffraction, energy dispersive x-ray spectroscopy, electrical resistivity, magnetic susceptibility, specific heat, x-ray absorption near edge structure (XANES), and neutron diffraction. The Yb valence vYb, calculated from the magnetic susceptibility and measured using XANES, decreases from 3+ at x = 0 to ∼2.1+ at xact = 0.2, where xact is the measured Yb concentration. A transition from incommensurate to commensurate antiferromagnetism is observed in neutron diffraction measurements along Q = (0.5, 0.5, l) between 0.2  xact  0.27; this narrative is supported by specific-heat measurements in which a second robust feature appears at a temperature TI (TI < TN) for the same concentration range. Magnetic susceptibility measurements also reveal features which provide additional evidence of magnetic ordering. The results of this study suggest that the evolution of the Yb valence plays a critical role in tuning the magnetic ground state of Ce1−xYbxRhIn5. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email