Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers and Synchrotron Radiation.

Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

Current advances in synchrotron radiation instrumentation for MX experiments.

Following pioneering work 40 years ago, synchrotron beamlines dedicated to macromolecular crystallography (MX) have improved in almost every aspect as instrumentation has evolved. Beam sizes and crystal dimensions are now on the single micron scale while data can be collected from proteins with molecular weights over 10 MDa and from crystals with unit cell dimensions over 1000 Å. Furthermore it is possible to collect a complete data set in seconds, and obtain the resulting structure in minutes. The impact of MX synchrotron beamlines and their evolution is reflected in their scientific output, and MX is now the method of choice for a variety of aims from ligand binding to structure determination of membrane proteins, viruses and ribosomes, resulting in a much deeper understanding of the machinery of life. A main driving force of beamline evolution have been advances in almost every aspect of the instrumentation comprising a synchrotron beamline. In this review we aim to provide an overview of the current status of instrumentation at modern MX experiments. The most critical optical components are discussed, as are aspects of endstation design, sample delivery, visualisation and positioning, the sample environment, beam shaping, detectors and data acquisition and processing.

Experimental setup for investigating silicon solid phase crystallization at high temperatures.

An experimental setup is presented to measure and interpret the solid phase crystallization of amorphous silicon thin films on glass at very high temperatures of about 800 °C. Molybdenum-SiO(2)-silicon film stacks were irradiated by a diode laser with a well-shaped top hat profile. From the relevant thermal and optical parameters of the system the temperature evolution can be calculated accurately. A time evolution of the laser power was applied which leads to a temperature constant in time in the center of the sample. Such a process will allow the observation and interpretation of solid phase crystallization in terms of nucleation and growth in further work.

Canadian macromolecular crystallography facility: a suite of fully automated beamlines.

The Canadian light source is a 2.9 GeV national synchrotron radiation facility located on the University of Saskatchewan campus in Saskatoon. The small-gap in-vacuum undulator illuminated beamline, 08ID-1, together with the bending magnet beamline, 08B1-1, constitute the Canadian Macromolecular Crystallography Facility (CMCF). The CMCF provides service to more than 50 Principal Investigators in Canada and the United States. Up to 25% of the beam time is devoted to commercial users and the general user program is guaranteed up to 55% of the useful beam time through a peer-review process. CMCF staff provides "Mail-In" crystallography service to users with the highest scored proposals. Both beamlines are equipped with very robust end-stations including on-axis visualization systems, Rayonix 300 CCD series detectors and Stanford-type robotic sample auto-mounters. MxDC, an in-house developed beamline control system, is integrated with a data processing module, AutoProcess, allowing full automation of data collection and data processing with minimal human intervention. Sample management and remote monitoring of experiments is enabled through interaction with a Laboratory Information Management System developed at the facility.

Quasi-nonvolatile storage in Ru-doped Bi12SiO20 crystals by two-wavelength holography.

Prolonged read-out process of a hologram recorded at near infrared with simultaneous green light exposure is measured in Ru-doped Bi12SiO20 crystal. The experimental results are confirmed by numerical simulations, suggesting two different traps involved in the space-charge transport mechanism. In addition, quasi-permanent holographic recording of image with fast updating speed by using two-wavelength recording is demonstrated.

Determination of crystallographic orientation of large grain metals with surface acoustic waves.

A previously described laser ultrasonic technique known as spatially resolved acoustic spectroscopy (SRAS) can be used to image surface microstructure, using the local surface acoustic wave (SAW) velocity as a contrast mechanism. It is shown here that measuring the SAW velocity in multiple directions can be used to determine the crystallographic orientation of grains. The orientations are determined by fitting experimentally measured velocities to theoretical velocities. Using this technique the orientations of 12 nickel and 3 aluminum single crystal samples have been measured, and these are compared with x-ray Laue backreflection (LBR) measurements with good agreement. The root mean square difference between SRAS and LBR measurements in terms of an R-value is less than 4.1°. The influence of systematic errors in the SAW velocity determination due to instrument miscalibration, which affects the accurate determination of the planes, is discussed. SRAS has great potential for complementary measurements or even for replacing established orientation determination and imaging techniques.

First experiences with semi-autonomous robotic harvesting of protein crystals.

The demonstration unit of the Universal Micromanipulation Robot (UMR) capable of semi-autonomous protein crystal harvesting has been tested and evaluated by independent users. We report the status and capabilities of the present unit scheduled for deployment in a high-throughput protein crystallization center. We discuss operational aspects as well as novel features such as micro-crystal handling and drip-cryoprotection, and we extrapolate towards the design of a fully autonomous, integrated system capable of reliable crystal harvesting. The positive to enthusiastic feedback from the participants in an evaluation workshop indicates that genuine demand exists and the effort and resources to develop autonomous protein crystal harvesting robotics are justified.

Generation of hybrid polarization-orbital angular momentum entangled states.

Hybrid entangled states exhibit entanglement between different degrees of freedom of a particle pair and thus could be useful for asymmetric optical quantum network where the communication channels are characterized by different properties. We report the first experimental realization of hybrid polarization-orbital angular momentum (OAM) entangled states by adopting a spontaneous parametric down conversion source of polarization entangled states and a polarization-OAM transferrer. The generated quantum states have been characterized through quantum state tomography. Finally, the violation of Bell's inequalities with the hybrid two photon system has been observed.

Evaluation of imaging plates as recording medium for images of negatively stained single particles and electron diffraction patterns of two-dimensional crystals.

We evaluated imaging plates (IPs) and the DITABIS Micron scanner for their use in recording images of negatively stained single-particle specimens and electron diffraction patterns of two-dimensional crystals. We first established the optimal imaging and read-out conditions for images of negatively stained single-particle specimens using the signal-to-noise ratio of the images as the evaluation criterion. We found that images were best recorded on IPs at a magnification of 67,000x, read out with a gain setting of 20,000 and a laser power setting of 30% with subsequent binning over 2 x 2 pixels. Our results show that for images of negatively stained specimens, for which the resolution is limited to approximately 20 A, IPs are a good alternative to EM film. We also compared IPs with a 2K x 2K Gatan charge-coupled device (CCD) camera for their use in recording electron diffraction patterns of sugar-embedded two-dimensional crystals. Diffraction patterns of aquaporin-0 recorded on IPs and with the CCD camera showed reflections beyond 3 A and had similar R(Friedel) as well as R(merge) values. IPs can thus be used to collect diffraction patterns, but CCD cameras are more convenient and remain the best option for recording electron diffraction patterns.

[Beginning of use of a new biological neutron diffractometer (iBIX) in J-PARC].

Ibaraki Prefectural Government together with Ibaraki University and Japan Atomic Energy Agency (JAEA) has almost finished constructing a time-of-flight (TOF) neutron diffractometer for biological macromolecules for industrial use at J-PARC, IBARAKI Biological Crystal Diffractometer (iBIX). Since 2009, Ibaraki University has been asked to operate this machine in order for users to do experiments by Ibaraki Prefecture. The diffractometer is designed to cover sample crystals which have their cell edges up to around 150 A. It is expected to measure more than 100 samples per year if they have 2 mm(3) in crystal volume, and to measure even around 0.1 mm(3) in crystal volume of biological samples. The efficiency of iBIX is also expected about 100 times larger than those of the present high performance diffractometers at JRR-3 in JAEA when 1MW power realizes in J-PARC. Since December 2008, iBIX has been open to users and several proteins and organic compounds were tested under 20 kW proton power of J-PARC. It was found that one of their proteins was diffracted up to 1.4 A in d-spacing, which was nearly comparable resolution to that of BIX-3 in JRR-3 when used the same crystal as at iBIX for reasonable exposure time. In May 2009, 14 detector units were set up. By the end of fiscal year 2009, the basic part of data reduction software will be finished and an equipment blowing low temperature gas to the sample will be installed with the cooperation of JAEA.

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Membrane Protein Preparation for Serial Crystallography Using High-Viscosity Injectors: Rhodopsin as an Example.

Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned.

Terahertz birefringence for orientation analysis.

A terahertz time-domain spectrometer is employed to study different birefringent samples. We develop a method based on the temporal waveform and the impulse response of a sample to map the anisotropy of their inner structure. To validate our algorithm, we study the polarization-affecting structure of various classes of materials such as crystals, plastics, and natural products. Among all samples we observe the largest birefringence for a rutile crystal with Deltan=3.3 at 1 THz.

Virus Structures by X-Ray Free-Electron Lasers.

Until recently X-ray crystallography has been the standard technique for virus structure determinations. Available X-ray sources have continuously improved over the decades, leading to the realization of X-ray free-electron lasers (XFELs). They provide high-intensity femtosecond X-ray pulses, which allow for new kinds of experiments by making use of the diffraction-before-destruction principle. By overcoming classical dose constraints, they at least in principle allow researchers to perform X-ray virus structure determination for single particles at room temperature. Simultaneously, the availability of XFELs led to the development of the method of serial femtosecond crystallography, where a crystal structure is determined from the measurement of hundreds to thousands of microcrystals. In the case of virus crystallography this method does not require freezing of the crystals and allows researchers to perform experiments under non-equilibrium conditions (e.g., by laser-induced temperature jumps or rapid chemical mixing), which is currently not possible with electron microscopy.

Slow light in a dielectric waveguide with negative-refractive-index photonic crystal cladding.

A slow light waveguide made of a dielectric slab inserted in a two-dimensional photonic crystal with a negative effective refractive index is proposed and numerically studied. The waveguide may possess modes with zero group velocity, and its frequency varies with the thickness of the waveguide. A linearly tapered left-handed photonic crystal waveguide is also proposed and studied. It is shown that the so-called 'trapped rainbow' proposed by Tsakmakidis, Boardman, and Hess [1] is difficult to realize due to a coupling of forward- and backward-propagating modes near zero group velocity. However, different frequency components of a broadband excitation can still be separated through partial accumulation at waveguide sections of different thicknesses.