M. Messerschmidt

Structure of the toxic core of α-synuclein from invisible crystals

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

Structural basis for bifunctional peptide recognition at human δ-opioid receptor

Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.

Femtosecond x-ray pulse length characterization at the Linac Coherent Light Source free-electron laser

Two-color, single-shot time-of-flight electron spectroscopy of atomic neon was employed at the Linac Coherent Light Source (LCLS) to measure laser-assisted Auger decay in the x-ray regime. This x-ray-optical cross-correlation technique provides a straightforward, non-invasive and on-line means of determining the duration of femtosecond (>40?fs) x-ray pulses. In combination with a theoretical model of the process based on the soft-photon approximation, we were able to obtain the LCLS pulse duration and to extract a mean value of the temporal jitter between the optical pulses from a synchronized Ti-sapphire laser and x-ray pulses from the LCLS. We find that the experimentally determined values are systematically smaller than the length of the electron bunches. Nominal electron pulse durations of 175 and 75?fs, as provided by the LCLS control system, yield x-ray pulse shapes of 120?20?fs full-width at half-maximum (FWHM) and an upper limit of 40?20?fs FWHM, respectively. Simulations of the free-electron laser agree well with the experimental results.

Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography

Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

Transient lattice contraction in the solid-to-plasma transition

Ultrafast x-ray heating of clusters leads to bond contraction in the solid-to-plasma transition. In condensed matter systems, strong optical excitations can induce phonon-driven processes that alter their mechanical properties. We report on a new phenomenon where a massive electronic excitation induces a collective change in the bond character that leads to transient lattice contraction. Single large van der Waals clusters were isochorically heated to a nanoplasma state with an intense 10-fs x-ray (pump) pulse. The structural evolution of the nanoplasma was probed with a second intense x-ray (probe) pulse, showing systematic contraction stemming from electron delocalization during the solid-to-plasma transition. These findings are relevant for any material in extreme conditions ranging from the time evolution of warm or hot dense matter to ultrafast imaging with intense x-ray pulses or, more generally, any situation that involves a condensed matter-to-plasma transition.

Pink-beam serial crystallography.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.Serial X-ray crystallography (SX) is used for data collection at X-ray Free Electron Lasers. Here the authors show that a polychromatic “pink” synchrotron X-ray beam can be used for SX, which is useful when crystal supply is limited and will allow time-resolved measurements at synchrotron sources in the future.

High-Z radiation shields for x-ray free electron laser detectors

The Linac Coherent Light Source (LCLS) produces brilliant x-ray in femtosecond pulses of high intensity. Many of the experiments performed at the LCLS use expensive pixel area detectors - the majority of which incorporate custom integrated circuit chips (ASIC). Such circuit chips are susceptible to radiation damage. To protect against this, micro-patterned tungsten foils were designed to cover the section of the circuit chip that extends beyond the sensor near the wire-bond pads. A description of the problem along with the details of how the tungsten foils were fabricated and installed will be given.

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

The resolution limit of serial diffraction from two-dimensional crystals at a free-electron laser was extended to the detector edge (4 Å) by exploiting the large redundancy of the data set.

Time-resolved x-ray photoelectron spectroscopy techniques for real-time studies of interfacial charge transfer dynamics

X-ray based spectroscopy techniques are particularly well suited to gain access to local oxidation states and electronic dynamics in complex systems with atomic pinpoint accuracy. Traditionally, these techniques are applied in a quasi-static fashion that usually highlights the steady-state properties of a system rather than the fast dynamics that often define the system function on a molecular level. Novel x-ray spectroscopy techniques enabled by free electron lasers (FELs) and synchrotron based pump-probe schemes provide the opportunity to monitor intramolecular and interfacial charge transfer processes in real-time and with element and chemical specificity. Two complementary time-domain xray photoelectron spectroscopy techniques are presented that are applied at the Linac Coherent Light Source (LCLS) and the Advanced Light Source (ALS) to study charge transfer processes in N3 dye-sensitized ZnO semiconductor nanocrystals, which are at the heart of emerging light-harvesting technologies.

LCLS Ultrafast Science Instruments:Conceptual Design Report

The Stanford Linear Accelerator Center (SLAC), along with Argonne National Laboratory (ANL), Lawrence Livermore National Laboratory (LLNL), and the University of California at Los Angeles (UCLA), is constructing a Free-Electron Laser (FEL) facility, which will operate in the wavelength range 1.5 nm - 0.15 nm. This FEL, the Linac Coherent Light Source (LCLS), utilizes the SLAC linac and will produce sub-picosecond pulses of short wavelength X-rays with very high peak brightness and almost complete transverse coherence. The final one-third of the SLAC linac will be used as the source of electrons for the LCLS. The high energy electrons will be transported across the SLAC Research Yard, into a tunnel which will house a long undulator. In passing through the undulator, the electrons will be bunched by the force of their own synchrotron radiation and produce an intense, monochromatic, spatially coherent beam of X-rays. By varying the electron energy, the FEL X-ray wavelength will be tunable from 1.5 nm to 0.15 nm. The LCLS will include two experimental halls as well as X-ray optics and infrastructure necessary to create a facility that can be developed for research in a variety of disciplines such as atomic physics, materials science, plasma physics and biosciences. This Conceptual Design Report, the authors believe, confirms the feasibility of designing and constructing three X-ray instruments in order to exploit the unique scientific capability of this new LCLS facility. The technical objective of the LCLS Ultrafast Science Instruments (LUSI) project is to design, build, and install at the LCLS three hard X-ray instruments that will complement the initial instrument included in the LCLS construction. As the science programs advance and new technological challenges appear, instrumentation needs to be developed and ready to conquer these new opportunities. The LCLS instrument concepts have been developed in close consultation with the scientific community through a series of workshops team meetings and focused reviews. In particular, the LUSI project instruments have been identified as meeting the most urgent needs of the scientific community based on the advice of the LCLS Scientific Advisory Committee (SAC) in response to an open call for letters of intent (LOI) from the breadth of the scientific community.