J. Pines

High-resolution protein structure determination by serial femtosecond crystallography

Size Matters Less X-ray crystallography is a central research tool for uncovering the structures of proteins and other macromolecules. However, its applicability typically requires growth of large crystals, in part because a sufficient number of molecules must be present in the lattice for the sample to withstand x-ray—induced damage. Boutet et al. (p. 362, published online 31 May) now demonstrate that the intense x-ray pulses emitted by a free-electron laser source can yield data in few enough exposures to uncover the high-resolution structure of microcrystals. A powerful x-ray laser source can probe proteins in detail using much smaller crystals than previously required. 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.

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this “probe-before-destroy” approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kβ1,3 XES spectra of MnII and Mn2III,IV complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.

The CSPAD megapixel x-ray camera at LCLS

The Linear Coherent Light Source (LCLS), a free electron laser operating from 250eV to10keV at 120Hz, is opening windows on new science in biology, chemistry, and solid state, atomic, and plasma physics1,2. The FEL provides coherent x-rays in femtosecond pulses of unprecedented intensity. This allows the study of materials on up to 3 orders of magnitude shorter time scales than previously possible. Many experiments at the LCLS require a detector that can image scattered x-rays on a per-shot basis with high efficiency and excellent spatial resolution over a large solid angle and both good S/N (for single-photon counting) and large dynamic range (required for the new coherent x-ray diffractive imaging technique3). The Cornell-SLAC Pixel Array Detector (CSPAD) has been developed to meet these requirements. SLAC has built, characterized, and installed three full camera systems at the CXI and XPP hutches at LCLS. This paper describes the camera system and its characterization and performance.

X‐ray detectors at the Linac Coherent Light Source

This paper offers an overview of area detectors developed for use at the Linac Coherent Light Source (LCLS) with particular emphasis on their impact on science. The experimental needs leading to the development of second-generation cameras for LCLS are discussed and the new detector prototypes are presented.

The Cornell-SLAC pixel array detector at LCLS

The Cornell-SLAC pixel array detector (CSpad) is a general-purpose integrating hybrid pixel x-ray camera developed for use at the Linear Coherent Light Source (LCLS) x-ray free electron laser at the SLAC National Accelerator Laboratory (SLAC). The detector has a full well capacity of about 2.Sk photons in low-gain mode and a SIN of about 6 in high-gain mode. Its 2.3M pixels are read out at 120 Hz. The detector comprises 32 500μm silicon sensors bump-bonded to 64 185×194-pixel ASICs. The pixel size is 110μm. The water-cooled detector quadrants can be radially moved in-situ to vary the beam aperture. SLAC has built, calibrated, and optimized three complete camera systems based on a sensor and ASIC designed by Cornell. The camera is read out by a DAQ system which provides extensive online monitoring and prompt analysis capabilities. We have also built a dozen smaller cameras in a portable form-factor for use in confined spaces and for ease of development, testing, and deployment. Through 2012 user experiments have taken almost a petabyte of data with these detectors in a variety of applications. We have extensively tested the detector at synchrotrons and with an x-ray tube, in addition to commissioning tests at the LCLS, investigating linearity, cross-talk, homogeneity, and radiation hardness. The SLAC detector group is deploying improved support infrastructure and an updated ASIC and electronics based on this experience. This paper describes the instrument, its calibration and performance, and presents preliminary results from the updated camera.

Experience with the CSPAD during dedicated detector runs at LCLS

In-house developed cameras and other commercial detectors are typically tested with x-ray tubes and at synchrotron beamlines before being deployed and used for science experiments. In a prototyping phase, this is needed to understand and characterize the behavior of the detector. In a more advanced development phase, measurements with x-rays are required to characterize and calibrate the camera. Tests at synchrotron beamlines in actual experimental conditions are indeed a valuable source for detector developers. However, when all photons arrive at once, as for FELs, the response of the detector can be very different from that obtained with a synchrotron beam which behaves more like a CW (continuous) source. This behavior was already observed during users runs at LCLS and recently investigated during dedicated detector beamtime. The linearity of the response of the Cornell-SLAC Pixel Array Detector (CSPAD) was investigated. Results are presented and discussed.

Detector Development for the Linac Coherent Light Source

Since it began operations in 2009, the Linac Coherent Light Source (LCLS) has opened a new and dynamic frontier in terms of light sources and their associated science [1, 2]. An increase in brightness by a factor of a billion over pre-existing synchrotrons, in combination with ultra-brief pulses of coherent X-rays, is ushering in a new era in the photon sciences. Pulses with durations of 50 fs under standard conditions and below 10 fs with a reduced energy per bunch are possible. Over 1013 or 1012 X-rays per pulse can be generated at the upper and lower ends of the X-ray energy range of 285 eV to 9600 eV. One of the unique machine parameters is its strobe-like time structure, where single ultra-brief pulses are delivered at a repetition rate of 120 Hz. The above characteristics represent a singular environment in which to operate detectors and demand the development of a new class of high-frame-rate camera systems.

2nd generation cameras for LCLS and the new challenges of high repetition rates at LCLS-II

With the experience of the first years of operation of the Linac Coherent Light Source (LCLS), SLAC developed a 2nd generation camera system with improved features and performance. The first camera to be deployed is the ePix-One, a compact camera which is a 155 mm long box with a quadratic front face of 52×52 mm2 which will feature 4 ASICs, either the ePIX100 or the ePIX10k, bump-bonded with a single sensor offering 35 × 38 mm2 active area. Combined with the ePIX100 hybrid pixel module which features 50 μm pixels and is targeted for X-ray Photon Correlation Spectroscopy and as a detector in wavelength dispersive spectrometer setups this will result in a 0.5Mpixel camera. Whereas the 100 μm pixels of ePIX10k, targeted towards protein crystallography, imaging and pump probe experiments, will provide a camera of 135kpixel. The camera uses simple Peltier/water cooling in combination with dry nitrogen purge against condensation. The compact housing and the simple interface (26pin cable & optical fiber) eases deployment and gives experimenters more flexibility in utilizing the camera where needed. The current ePix cameras support full frame readout faster than 120Hz and ROI modes which can be read at up to 1kHz rate. Next developments will target larger cameras and higher frame rates for the upcoming LCLS II.

Studies of the ePix100 low-noise x-ray camera at SLAC

A new hybrid pixel array detector, the ePix100, has been developed at SLAC for tender and hard x-ray experiments at the Linac Coherent Light Source (LCLS). It is intended for low noise and good spatial resolution applications, particularly X-ray Photon Correlation Spectroscopy (XPCS) and in combination with wavelength dispersive spectrometers. The detector has 50 μm pixel size and less than 100 e- r.m.s. noise over the range of tested operating conditions. A series of measurements to validate its performance with x-rays was carried out at the Stanford Synchrotron Radiation Lightsource (SSRL) and LCLS. Results are here reported and discussed.

CSPAD upgrades at LCLS

The Cornell-SLAC Pixel Array Detector (CSPAD) is the workhorse for LCLS hard x-rays experiments. After deploying three 2.3Mpixel and many 140kpixel cameras, SLAC detector group has focused in improving the detector performances and optimizing its use in experiments. This was achieved first by improving thermo-mechanical assembly, PCB level electronics and firmware. The next step consisted in upgrading the ASIC. Continuous improvements in GUI and DAQ completed the evolution of the CSPAD systems, up to the current version V1.6, and made these cameras easy to use and optimize for a given experiment. More than 85% of the hard x-ray experiments scheduled in run 7 used one or multiple CSPAD cameras.