Publications by authors named "Petr Skopintsev"

13 Publications

  • Page 1 of 1

Advances in long-wavelength native phasing at X-ray free-electron lasers.

IUCrJ 2020 Nov 9;7(Pt 6):965-975. Epub 2020 Sep 9.

LeadXpro AG, Park InnovAARE, Villigen, 5234, Switzerland.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.
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http://dx.doi.org/10.1107/S2052252520011379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642782PMC
November 2020

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Nature 2020 07 20;583(7815):314-318. Epub 2020 May 20.

Laboratory of Biomolecular Research, Biology and Chemistry Division, Paul Scherrer Institut, Villigen, Switzerland.

Light-driven sodium pumps actively transport small cations across cellular membranes. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.
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http://dx.doi.org/10.1038/s41586-020-2307-8DOI Listing
July 2020

Soft X-ray diffraction patterns measured by a LiF detector with sub-micrometre resolution and an ultimate dynamic range.

J Synchrotron Radiat 2020 May 16;27(Pt 3):625-632. Epub 2020 Mar 16.

Moscow Engineering Physics Institute (MEPhI), Kashirskoe shosse 31, Moscow 115409, Russian Federation.

The unique diagnostic possibilities of X-ray diffraction, small X-ray scattering and phase-contrast imaging techniques applied with high-intensity coherent X-ray synchrotron and X-ray free-electron laser radiation can only be fully realized if a sufficient dynamic range and/or spatial resolution of the detector is available. In this work, it is demonstrated that the use of lithium fluoride (LiF) as a photoluminescence (PL) imaging detector allows measuring of an X-ray diffraction image with a dynamic range of ∼10 within the sub-micrometre spatial resolution. At the PETRA III facility, the diffraction pattern created behind a circular aperture with a diameter of 5 µm irradiated by a beam with a photon energy of 500 eV was recorded on a LiF crystal. In the diffraction pattern, the accumulated dose was varied from 1.7 × 10 J cm in the central maximum to 2 × 10 J cm in the 16th maximum of diffraction fringes. The period of the last fringe was measured with 0.8 µm width. The PL response of the LiF crystal being used as a detector on the irradiation dose of 500 eV photons was evaluated. For the particular model of laser-scanning confocal microscope Carl Zeiss LSM700, used for the readout of the PL signal, the calibration dependencies on the intensity of photopumping (excitation) radiation (λ = 488 nm) and the gain have been obtained.
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http://dx.doi.org/10.1107/S1600577520002192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285683PMC
May 2020

Publisher Correction: Seeded X-ray free-electron laser generating radiation with laser statistical properties.

Nat Commun 2019 Nov 13;10(1):5217. Epub 2019 Nov 13.

Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, D-22607, Hamburg, Germany.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-13249-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853951PMC
November 2019

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography.

Science 2019 07;365(6448):61-65

Division of Biology and Chemistry-Laboratory for Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland.

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.
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http://dx.doi.org/10.1126/science.aaw8634DOI Listing
July 2019

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers.

J Vis Exp 2019 02 28(144). Epub 2019 Feb 28.

Division of Biology and Chemistry - Laboratory for Biomolecular Research, Paul Scherrer Institut;

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.
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http://dx.doi.org/10.3791/59087DOI Listing
February 2019

Seeded X-ray free-electron laser generating radiation with laser statistical properties.

Nat Commun 2018 10 29;9(1):4498. Epub 2018 Oct 29.

Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, D-22607, Hamburg, Germany.

The invention of optical lasers led to a revolution in the field of optics and to the creation of such fields of research as quantum optics. The reason was their unique statistical and coherence properties. The emerging, short-wavelength free-electron lasers (FELs) are sources of very bright coherent extreme-ultraviolet and X-ray radiation with pulse durations on the order of femtoseconds, and are presently considered to be laser sources at these energies. FELs are highly spatially coherent to the first-order but in spite of their name, behave statistically as chaotic sources. Here, we demonstrate experimentally, by combining Hanbury Brown and Twiss interferometry with spectral measurements that the seeded XUV FERMI FEL-2 source does indeed behave statistically as a laser. The results may be useful for quantum optics experiments and for the design and operation of next generation FEL sources.
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http://dx.doi.org/10.1038/s41467-018-06743-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206026PMC
October 2018

Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution.

Structure 2018 11 13;26(11):1522-1533.e5. Epub 2018 Sep 13.

Centre for Systems Structural Biology (CSSB), DESY and European Molecular Biology Laboratory, Notkestrasse 85, Hamburg D-22607, Germany; Department of Medical Biochemistry and Biophysics, Karolinska Insititutet, Scheels väg 2, Stockholm SE-17177, Sweden. Electronic address:

The scaffolding protein PDZK1 has been associated with the regulation of membrane transporters. It contains four conserved PDZ domains, which typically recognize a 3-5-residue long motif at the C terminus of the binding partner. The atomic structures of the individual domains are available but their spatial arrangement in the full-length context influencing the binding properties remained elusive. Here we report a systematic study of full-length PDZK1 and deletion constructs using small-angle X-ray scattering, complemented with biochemical and functional studies on PDZK1 binding to known membrane protein partners. A hybrid modeling approach utilizing multiple scattering datasets yielded a well-defined, extended, asymmetric L-shaped domain organization of PDZK1 in contrast to a flexible "beads-on-string" model predicted by bioinformatics analysis. The linker regions of PDZK1 appear to play a central role in the arrangement of the four domains underlying the importance of studying scaffolding proteins in their full-length context.
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http://dx.doi.org/10.1016/j.str.2018.07.016DOI Listing
November 2018

Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser.

Science 2018 07 14;361(6398). Epub 2018 Jun 14.

Division of Biology and Chemistry-Laboratory for Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland.

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.
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http://dx.doi.org/10.1126/science.aat0094DOI Listing
July 2018

Quantitative ptychographic bio-imaging in the water window.

Opt Express 2018 Jan;26(2):1237-1254

Coherent X-ray ptychography is a tool for highly dose efficient lensless nano-imaging of biological samples. We have used partially coherent soft X-ray synchrotron radiation to obtain a quantitative image of a laterally extended, dried, and unstained fibroblast cell by ptychography. We used data with and without a beam stop that allowed us to measure coherent diffraction with a high-dynamic range of 1.7·10. As a quantitative result, we obtained the refractive index values for two regions of the cell with respect to a reference area. Due to the photon energy in the water window we obtained an extremely high contrast of 53% at 71 nm half-period resolution. The dose applied in our experiment was 9.5·10 Gy and is well below the radiation damage threshold. The concept for dynamic range improvement for low dynamic range detectors with a beam stop opens the path for high resolution nano-imaging of a variety of samples including cryo-preserved, hydrated and unstained biological cells.
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http://dx.doi.org/10.1364/OE.26.001237DOI Listing
January 2018

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.

Nat Commun 2017 09 14;8(1):542. Epub 2017 Sep 14.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232, Villigen PSI, Switzerland.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.
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http://dx.doi.org/10.1038/s41467-017-00630-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599499PMC
September 2017

Quantifying Angular Correlations between the Atomic Lattice and the Superlattice of Nanocrystals Assembled with Directional Linking.

Nano Lett 2017 06 17;17(6):3511-3517. Epub 2017 May 17.

Eberhard Karls Universität Tübingen , Geschwister-Scholl-Platz, D-72074 Tübingen, Germany.

We show that the combination of X-ray scattering with a nanofocused beam and X-ray cross correlation analysis is an efficient way for the full structural characterization of mesocrystalline nanoparticle assemblies with a single experiment. We analyze several hundred diffraction patterns at individual sample locations, that is, individual grains, to obtain a meaningful statistical distribution of the superlattice and atomic lattice ordering. Simultaneous small- and wide-angle X-ray scattering of the same sample location allows us to determine the structure and orientation of the superlattice as well as the angular correlation of the first two Bragg peaks of the atomic lattices, their orientation with respect to the superlattice, and the average orientational misfit due to local structural disorder. This experiment is particularly advantageous for synthetic mesocrystals made by the simultaneous self-assembly of nanocrystals and surface-functionalization with conductive ligands. While the structural characterization of such materials has been challenging so far, the present method now allows correlating the mesocrystalline structure with optoelectronic properties.
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http://dx.doi.org/10.1021/acs.nanolett.7b00584DOI Listing
June 2017

Water window ptychographic imaging with characterized coherent X-rays.

J Synchrotron Radiat 2015 May 23;22(3):819-27. Epub 2015 Apr 23.

Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany.

A ptychographical coherent diffractive imaging experiment in the water window with focused soft X-rays at 500 eV is reported. An X-ray beam with high degree of coherence was selected for ptychography at the P04 beamline of PETRA III synchrotron radiation source. The beam coherence was measured with the newly developed non-redundant array method, and a coherence length of 4.1 µm and global degree of coherence of 35% at 100 µm exit slit opening in the vertical direction were determined. A pinhole, 2.6 µm in size, selected the coherent part of the beam that was used to obtain ptychographic reconstruction results of a lithographically manufactured test sample and a fossil diatom. The achieved resolution was 53 nm for the test sample and was only limited by the size of the detector. The diatom was imaged at a resolution better than 90 nm.
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http://dx.doi.org/10.1107/S1600577515005524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416689PMC
May 2015
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