Volume 2, Issue 4, July 2015
- SPECIAL TOPIC: BIOLOGY WITH X-RAY LASERS 2
- Invited Prespectives
2(2015); http://dx.doi.org/10.1063/1.4919740View Description Hide Description
Single-particle structure recovery without crystals or radiation damage is a revolutionary possibility offered by X-ray free-electron lasers, but it involves formidable experimental and data-analytical challenges. Many of these difficulties were encountered during the development of cryogenic electron microscopy of biological systems. Electron microscopy of biological entities has now reached a spatial resolution of about 0.3 nm, with a rapidly emerging capability to map discrete and continuous conformational changes and the energy landscapes of biomolecular machines. Nonetheless, single-particle imaging by X-ray free-electron lasers remains important for a range of applications, including the study of large “electron-opaque” objects and time-resolved examination of key biological processes at physiological temperatures. After summarizing the state of the art in the study of structure and conformations by cryogenic electron microscopy, we identify the primary opportunities and challenges facing X-ray-based single-particle approaches, and possible means for circumventing them.
- Invited Articles
2(2015); http://dx.doi.org/10.1063/1.4918726View Description Hide Description
Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.
2(2015); http://dx.doi.org/10.1063/1.4919301View Description Hide Description
The Single Particles, Clusters and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL is located behind the SASE1 undulator and aims to support imaging and structure determination of biological specimen between about 0.1 μm and 1 μm size. The instrument is designed to work at photon energies from 3 keV up to 16 keV. Here, we propose a cost-effective proof-of-principle experiment, aiming to demonstrate the actual feasibility of a single molecule diffraction experiment at the European XFEL. To this end, we assume self-seeding capabilities at SASE1 and we suggest to make use of the baseline European XFEL accelerator complex—with the addition of a slotted-foil setup—and of the SPB/SFX instrument. As a first step towards the realization of an actual experiment, we developed a complete package of computational tools for start-to-end simulations predicting its performance. Single biomolecule imaging capabilities at the European XFEL can be reached by exploiting special modes of operation of the accelerator complex and of the SASE1 undulator. The output peak power can be increased up to more than 1.5 TW, which allows to relax the requirements on the focusing efficiency of the optics and to reach the required fluence without changing the present design of the SPB/SFX instrument. Explicit simulations are presented using the 15-nm size RNA Polymerase II molecule as a case study. Noisy diffraction patterns were generated and they were processed to generate the 3D intensity distribution. We discuss requirements to the signal-to-background ratio needed to obtain a correct pattern orientation. When these are fulfilled, our results indicate that one can achieve diffraction without destruction with about 0.1 photons per Shannon pixel per shot at 4 Å resolution with 1013 photons in a 4 fs pulse at 4 keV photon energy and in a 0.3 μm focus, corresponding to a fluence of 1014 photons/μm2. We assume negligible structured background. At this signal level, one needs only about 30 000 diffraction patterns to recover full 3D information. At the highest repetition rate manageable by detectors at European XFEL, one will be able to accumulate these data within a fraction of an hour, even assuming a relatively low hit probability of about a percent.
Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse2(2015); http://dx.doi.org/10.1063/1.4919398View Description Hide Description
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.
Radiation damage in a micron-sized protein crystal studied via reciprocal space mapping and Bragg coherent diffractive imaging2(2015); http://dx.doi.org/10.1063/1.4919641View Description Hide Description
For laboratory and synchrotron based X-ray sources, radiation damage has posed a significant barrier to obtaining high-resolution structural data from biological macromolecules. The problem is particularly acute for micron-sized crystals where the weaker signal often necessitates the use of higher intensity beams to obtain the relevant data. Here, we employ a combination of techniques, including Bragg coherent diffractive imaging to characterise the radiation induced damage in a micron-sized protein crystal over time. The approach we adopt here could help screen for potential protein crystal candidates for measurement at X-ray free election laser sources.
Improvements in serial femtosecond crystallography of photosystem II by optimizing crystal uniformity using microseeding procedures2(2015); http://dx.doi.org/10.1063/1.4919741View Description Hide Description
In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 μm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5 Å, using crystals grown without the micro seeding approach, to 4.5 Å using crystals generated with the new method.
2(2015); http://dx.doi.org/10.1063/1.4919407View Description Hide Description
In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.