Water Window Ptychographic Imaging with Characterized Coherent X-rays

We report on a ptychographical coherent diffractive imaging experiment in the water window with focused soft X-rays at $500~\mathrm{eV}$. An X-ray beam with high degree of coherence was selected for ptychography at the P04 beamline of the PETRA III synchrotron radiation source. We measured the beam coherence with the newly developed non-redundant array method. A pinhole $2.6~\mathrm{\mu m}$ in size selected the coherent part of the beam and was used for ptychographic measurements of a lithographically manufactured test sample and fossil diatom. The achieved resolution was $53~\mathrm{nm}$ for the test sample and only limited by the size of the detector. The diatom was imaged at a resolution better than $90~\mathrm{nm}$.Comment: 22 pages. 7 figure

Quantum Imaging with Incoherently Scattered Light from a Free-Electron Laser

The advent of accelerator-driven free-electron lasers (FEL) has opened new avenues for high-resolution structure determination via diffraction methods that go far beyond conventional x-ray crystallography methods. These techniques rely on coherent scattering processes that require the maintenance of first-order coherence of the radiation field throughout the imaging procedure. Here we show that higher-order degrees of coherence, displayed in the intensity correlations of incoherently scattered x-rays from an FEL, can be used to image two-dimensional objects with a spatial resolution close to or even below the Abbe limit. This constitutes a new approach towards structure determination based on incoherent processes, including Compton scattering, fluorescence emission or wavefront distortions, generally considered detrimental for imaging applications. Our method is an extension of the landmark intensity correlation measurements of Hanbury Brown and Twiss to higher than second-order paving the way towards determination of structure and dynamics of matter in regimes where coherent imaging methods have intrinsic limitations

Ultrafast structural changes direct the first molecular events of vision

視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation

Time-Resolved Crystallography: Developing the Method and Solving the Mechanism of an Optogenetic Pump at Swissfel

During my PhD studies at the ETH Zurich and the Paul Scherrer Institute I worked in the field of time-resolved serial femtosecond X-ray crystallography (TR-SFX), an emerging structural biology method that is performed at X-ray free-electron lasers (XFELs). The first advantage of XFELs is their provision of ultrafast 10-100 fs pulses, which allow radiation damage effects to be avoided, and obtaining structures from data collected at room temperature. The second advantage of XFELs comes upon installing a pump laser that induces protein conformational changes and allows obtaining crystallographic datasets at different times after protein activation. The protein that was subject to the study, Krokinobacter eikastus rhodopsin 2 (KR2), is the first discovered light-driven sodium pump. It was intriguing to elucidate the protein’s new sodium-pumping mechanism. It had been long debated if such a non-H+ cation pump could exist, because Na+ was supposed to be electrostatically repulsed from a positively charged retinal chromophore Schiff base. Due to the fact that outward pumping of Na+ silences neuronal signalling, KR2 is considered a next-generation tool for optogenetics

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

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

Genome expansion by a CRISPR trimmer-integrase

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences

Ptychographic Imaging of Fossil Diatom Structures with Soft X-Rays

Imaging of biological samples in the water window offer high chemical contrast between Oxygen and Carbon. To avoid resolution limitations due to lens based microscopy techniques the coherent diffractive imaging (CDI) method is favourable for high resolution imaging. In order to measure extended samples the ptychographic coherent diffractive imaging technique (PCDI) is applied. Images of a test pattern and a fossil diatom are reconstructed with the extended ptychographic iterative engine (ePIE) at 55 nm and 286 nm resolution respectively

Synchrotron coherence measurements utilizing Non-Redundant Arrays of slits

We present a method to characterize the spatial coherence of soft X-ray radiation from a single diffraction pattern. The technique is based on scattering from non-redundant arrays (NRA) of slits and records the degree of spatial coherence at several relative separations [1]. Using NRAs we measured the spatial coherence of the X-ray beam at the XUV X-ray beamline P04 of the PETRA III storage ring [2] and at the U49/2 PGM1 beamline of the BESSY II synchrotron [3]. In both cases, the spatial coherence was measured for different beam parameters, including the photon energy and the monochromator exit slits separation (see Fig.1). We demonstrated that a rapid high-throughput analysis of the beam spatial coherence prior to any experiment utilizing coherence is possible. We also foresee that NRA method could become a viable tool for single pulse coherence characterization measurements at X-ray free-electron lasers [4, 5].References:[1] Y. Mejía et al., Opt. Commun. 273, 428 (2007).[2] P. Skopintsev et al., J. Synchrotron Rad. (2014) (accepted).[3] P. Skopintsev et al. (2014) (in preparation)[4] I.A. Vartanyants et al., Phys. Rev. Lett. 107, 144801 (2011).[5] A. Singer et al., Opt. Express 20(16), 17480 (2012)