[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: We report experimental results on x-ray diffraction of quantum-state-selected
and strongly aligned ensembles of the prototypical asymmetric rotor molecule
2,5-diiodobenzonitrile using the Linac Coherent Light Source. The experiments
demonstrate pioneering steps toward a new bottom-up approach to diffractive
imaging of distinct structures of individual, isolated gas-phase molecules. We
confirm several key ingredients of single molecule diffraction experiments: the
abilities to detect and count individual scattered x-ray photons in single shot
diffraction data, to deliver state-selected, e.g., structural-isomer-selected,
ensembles of molecules to the x-ray interaction volume, and to strongly align
the scattering molecules. Our approach, using ultrashort x-ray pulses, is
suitable to study ultrafast dynamics of isolated molecules.
[show abstract][hide abstract] ABSTRACT: The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host
protein degradation, is a promising target to develop new treatments against sleeping sickness, a
fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB
has so far not provided sufficient information for the design of a safe and specific drug against T.
brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond
crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully
glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native
TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the
“diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands
of individual microcrystals.
[show abstract][hide abstract] ABSTRACT: X-ray free-electron lasers enable high-resolution imaging of biological
materials by using short enough pulses to outrun many of the effects of
radiation damage. Experiments conducted at the LCLS have obtained
diffraction data from single particles and protein nanocrystals at doses
to the sample over 3 GGy. The details of the interaction of the X-ray
FEL pulse with the sample determine the limits of this new paradigm for
imaging. Recent studies suggest that in the case of crystalline samples,
such as protein nanocrystals, the atomic displacements and loss of bound
electrons in the crystal (due to the high X- ray intensity) has the
effect of gating the diffraction signal, and hence making the experiment
less radiation sensitive. Only the incident photon intensity in the
first part of the pulse, before the Bragg diffraction has died out, is
relevant to acquiring signal and the rest of the pulse will mainly
contribute to a diffuse background. In this work we use a plasma based
non-local thermodynamic equilibrium code to explore the displacement and
the ionization of a protein nanocrystal at various X-ray wavelengths and
[show abstract][hide abstract] ABSTRACT: The solubility of organic molecules is a well established property, founded on decades of measurements, the results of which have been tabulated in handbooks. Under atmospheric conditions water droplets may form containing small amounts of other molecules. Such droplets typically have a very large area to volume ratio, which may shift the solvation equilibrium towards molecules residing on the droplet surface. The presence of organic molecules on droplet surfaces is extremely important for reactivity--it is well established that certain chemical reactions are more prevalent under atmospheric conditions than in bulk. Here we present a thermodynamic rationalization of the surface solvation properties of methanol, ethanol, propanoic acid, n-butylamine, diethyl ether, and neopentane based on potential of mean force (PMF) calculations--we have previously demonstrated that an energetic description is a very powerful means of disentangling the factors governing solvation (Caleman et al., Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 6838-6842). All organic molecules investigated here are preferentially solvated on the surface of the droplets rather than in the inside, yet the magnitude of surface preference may differ by orders of magnitude. In order to dissect the energetic contributions that govern surface preference, we decompose the PMF into enthalpic and entropic components, and, in a second step, into contributions from water-water and solute-water interactions. The analysis demonstrates that surface preference is primarily an enthalpic effect, but the magnitude of surface preference of solutes containing large apolar groups is enhanced due to entropy. We introduce an analysis of the droplet PMFs that allows one to extrapolate the results to larger droplets. From this we can estimate the solubility of the solutes in water droplets, demonstrating that the solubility in droplets can be orders of magnitude larger than in bulk water. Our findings have implications for understanding the process of electrospray ionization, an important technique in biological mass spectrometry, since our work strongly suggests that in equilibrium biomolecules would be adsorbed on the droplet surface as well.
Physical Chemistry Chemical Physics 06/2012; 14(27):9537-45. · 3.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
[show abstract][hide abstract] ABSTRACT: The molecular dynamics simulation package GROMACS is a widely used tool used in a broad range of different applications within physics, chemistry and biology. It is freely available, user friendly and extremely efficient. The GROMACS software is force field agnostic, and compatible with many molecular dynamics force fields; coarse-grained, unified atom, all atom as well as polarizable models based on the charge on a spring concept. To validate simulations, it is necessary to compare results from the simulations to experimental data. To ease the process of setting up topologies and structures for simulations, as well as providing pre-calculated physical properties along with experimental values for the same we provide a web-based database, containing 145 organic molecules at present.
Liquid properties of 145 organic molecules have been simulated using two different force fields, OPLS all atom and Generalized Amber Force Field. So far, eight properties have been calculated (the density, enthalpy of vaporization, surface tension, heat capacity at constant volume and pressure, isothermal compressibility, volumetric expansion coefficient and the static dielectric constant). The results, together with experimental values are available through the database, along with liquid structures and topologies for the 145 molecules, in the two force fields.
The database is freely available under http://virtualchemistry.org.
[show abstract][hide abstract] ABSTRACT: We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
[show abstract][hide abstract] ABSTRACT: The chemical composition of small organic molecules is often very similar to amino acid side chains or the bases in nucleic acids, and hence there is no a priori reason why a molecular mechanics force field could not describe both organic liquids and biomolecules with a single parameter set. Here, we devise a benchmark for force fields in order to test the ability of existing force fields to reproduce some key properties of organic liquids, namely, the density, enthalpy of vaporization, the surface tension, the heat capacity at constant volume and pressure, the isothermal compressibility, the volumetric expansion coefficient, and the static dielectric constant. Well over 1200 experimental measurements were used for comparison to the simulations of 146 organic liquids. Novel polynomial interpolations of the dielectric constant (32 molecules), heat capacity at constant pressure (three molecules), and the isothermal compressibility (53 molecules) as a function of the temperature have been made, based on experimental data, in order to be able to compare simulation results to them. To compute the heat capacities, we applied the two phase thermodynamics method (Lin et al. J. Chem. Phys.2003, 119, 11792), which allows one to compute thermodynamic properties on the basis of the density of states as derived from the velocity autocorrelation function. The method is implemented in a new utility within the GROMACS molecular simulation package, named g_dos, and a detailed exposé of the underlying equations is presented. The purpose of this work is to establish the state of the art of two popular force fields, OPLS/AA (all-atom optimized potential for liquid simulation) and GAFF (generalized Amber force field), to find common bottlenecks, i.e., particularly difficult molecules, and to serve as a reference point for future force field development. To make for a fair playing field, all molecules were evaluated with the same parameter settings, such as thermostats and barostats, treatment of electrostatic interactions, and system size (1000 molecules). The densities and enthalpy of vaporization from an independent data set based on simulations using the CHARMM General Force Field (CGenFF) presented by Vanommeslaeghe et al. (J. Comput. Chem.2010, 31, 671) are included for comparison. We find that, overall, the OPLS/AA force field performs somewhat better than GAFF, but there are significant issues with reproduction of the surface tension and dielectric constants for both force fields.
Journal of Chemical Theory and Computation 01/2012; 8(1):61-74. · 5.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis(1). For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information(1-4). Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology(5) should enable structural determination from submicrometre protein crystals with atomic resolution.
[show abstract][hide abstract] ABSTRACT: Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.
[show abstract][hide abstract] ABSTRACT: X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.
[show abstract][hide abstract] ABSTRACT: X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.
Physical Review B 12/2011; 84(21):214111. · 3.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Powerful free electron lasers (FELs) operating in the soft X-ray regime are offering new possibilities for creating and probing materials under extreme conditions. We describe here simulations to model the interaction of a focused FEL pulse with metallic solids (niobium, vanadium, and their deuterides) at 13.5 nm wavelength (92 eV) with peak intensities between 10(15) to 10(18) W/cm(2) and a fixed pulse length of 15 femtoseconds (full width at half maximum). The interaction of the pulse with the metallic solids was modeled with a non-local thermodynamic equilibrium code that included radiation transfer. The calculations also made use of a self-similar isothermal fluid model for plasma expansion into vacuum. We find that the time-evolution of the simulated critical charge density in the sample results in a critical depth that approaches the observed crater depths in an earlier experiment performed at the FLASH free electron laser in Hamburg. The results show saturation in the ablation process at intensities exceeding 10(16) W/cm(2). Furthermore, protons and deuterons with kinetic energies of several keV have been measured, and these concur with predictions from the plasma expansion model. The results indicate that the temperature of the plasma reached almost 5 million K after the pulse has passed.
[show abstract][hide abstract] ABSTRACT: X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.
[show abstract][hide abstract] ABSTRACT: X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
[show abstract][hide abstract] ABSTRACT: Structural studies of biological macromolecules are severely limited by radiation damage. Traditional crystallography curbs the effects of damage by spreading damage over many copies of the molecule of interest in the crystal. X-ray lasers offer an additional opportunity for limiting damage by out-running damage processes with ultrashort and very intense X-ray pulses Such pulses may allow the imaging of single molecules, clusters; Or nanoparticles: Coherent flash Imaging Will also open up new avenues for structural studies on nano- and microcrystalline substances. This paper addresses the theoretical potentials and limitations of nanocrystallography with extremely intense coherent X-ray pulses. We use urea nanocrystals as a model for generic biological substances and simulate the primary and secondary ionization dynamics in the crystalline sample. The results establish conditions for ultrafast single shot nanocrystallography diffraction experiments as a function of X-ray fluence, pulse duration, and the size of nanocrystals. Nanocrystallography using ultrafast X-ray pulses has the potential to open up a new route in protein crystallography to solve atomic structures of many systems that remain Inaccessible using conventional X-ray sources.
[show abstract][hide abstract] ABSTRACT: Evidence from mass-spectrometry experiments and molecular dynamics simulations suggests that it is possible to transfer proteins, or in general biomolecular aggregates, from solution to the gas-phase without grave impact on the structure. If correct, this allows interpretation of such experiments as a probe of physiological behavior. Here, we survey recent experimental results from mass spectrometry and ion-mobility spectroscopy and combine this with observations based on molecular dynamics simulation, in order to give a comprehensive overview of the state of the art in gas-phase studies. We introduce a new concept in protein structure analysis by determining the fraction of the theoretical possible numbers of hydrogen bonds that are formed in solution and in the gas-phase. In solution on average 43% of the hydrogen bonds is realized, while in vacuo this fraction increases to 56%. The hydrogen bonds stabilizing the secondary structure (α-helices, β-sheets) are maintained to a large degree, with additional hydrogen bonds occurring when side chains make new hydrogen bonds to rest of the protein rather than to solvent. This indicates that proteins that are transported to the gas phase in a native-like manner in many cases will be kinetically trapped in near-physiological structures. Simulation results for lipid- and detergent-aggregates and lipid-coated (membrane) proteins in the gas phase are discussed, which in general point to the conclusion that encapsulating proteins in "something" aids in the conservation of native-like structure. Isolated solvated micelles of cetyl-tetraammonium bromide quickly turn into reverse micelles whereas dodecyl phosphocholine micelles undergo much slower conversions, and do not quite reach a reverse micelle conformation within 100 ns.