The SIBYLS beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory is a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline. Key features and capabilities are described along with implementation and performance.

The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world’s mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B4C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources.

The point-spread function (PSF) of a powdered-phosphor fiber-optic taper-coupled CCD area detector was measured, and an analytic expression for its functional form proposed. The ‘tails’ of this PSF were shown to be due almost entirely to the fiber-optic taper, with no contribution from the phosphor itself.

The point-spread function (PSF) of a fiber-optic taper-coupled CCD area detector was measured over five decades of intensity using a 20 µm X-ray beam and ∼2000-fold averaging. The ‘tails’ of the PSF clearly revealed that it is neither Gaussian nor Lorentzian, but instead resembles the solid angle subtended by a pixel at a point source of light held a small distance (∼27 µm) above the pixel plane. This converges to an inverse cube law far from the beam impact point. Further analysis revealed that the tails are dominated by the fiber-optic taper, with negligible contribution from the phosphor, suggesting that the PSF of all fiber-coupled CCD-type detectors is best described as a Moffat function.

A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.

A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single ‘still’ diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed.

Radiation damage considerations affecting data collection by more than a factor of two are summarized and damage avoidance strategies are suggested.

Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes an expanding literature, and, to the new breed of protein crystallographer who is not really interested in X-ray physics or radiation chemistry but just wants to solve a biologically relevant structure, the technical nature and breadth of this literature can be daunting. The purpose of this paper is to serve as a rough guide to radiation damage issues, and to provide references to the more exacting and detailed work. No attempt has been made to report precise numbers (a factor of two is considered satisfactory), and, since there are aspects of radiation damage that are demonstrably unpredictable, the ‘worst case scenario’ as well as the ‘average crystal’ are discussed in terms of the practicalities of data collection.

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.

Summary

Kv7.x (KCNQ) voltage-gated potassium channels form the cardiac and auditory IKs current and the neuronal M-current. The five Kv7 subtypes have distinct assembly preferences encoded by a C-terminal cytoplasmic assembly domain, the A-domain Tail. Here, we present the high-resolution structure of the Kv7.4 A-domain Tail together with biochemical experiments that show that the domain is a self-assembling, parallel, four-stranded coiled coil. Structural analysis and biochemical studies indicate conservation of the coiled coil in all Kv7 subtypes and that a limited set of interactions encode assembly specificity determinants. Kv7 mutations have prominent roles in arrhythmias, deafness, and epilepsy. The structure together with biochemical data indicate that A-domain Tail arrhythmia mutations cluster on the solvent-accessible surface of the subunit interface at a likely site of action for modulatory proteins. Together, the data provide a framework for understanding Kv7 assembly specificity and the molecular basis of a distinct set of Kv7 channelopathies.

Ubiquitin-like proteins (UBLs), such as NEDD8, are transferred to their targets by distinct, parallel, multi-enzyme cascades that involve the sequential action of E1, E2, and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8’s E2, Ubc12, selectively recruits NEDD8’s E1 to promote Ubc12~NEDD8 thioester formation. A peptide corresponding to Ubc12’s N-terminus (Ubc12N26) specifically binds and inhibits NEDD8’s E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3-Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s, explaining why this interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1–E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.

A formula for absolute scattering power is derived to include spot fading arising from radiation damage and the crystal volume needed to collect diffraction data to a given resolution is calculated.

In this work, classic intensity formulae were united with an empirical spot-fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per spot at a desired resolution over the radiation-damage-limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X-ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal-to-noise ratio of 2 at 2 Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2 µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34 µm. These represent 15-fold to 700-fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits.

Ubiquitin-like proteins (Ubls) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, forming a covalent E1~Ubl thioester intermediate, and generating a thioester-linked E2~Ubl product, which must be released for subsequent reactions. We report structural analysis of a trapped Ubl activation complex containing NEDD8’s heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently-associated for adenylation), a catalytically-inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2~NEDD8 thioester product. Thus, transferring the Ubl’s thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in Ubl cascades.

The radiation-induced disordering of selenomethionine (SeMet) side chains represents a significant impediment to protein structure solution. Not only does the increased B-factor of these sites result in a serious drop in phasing power, but some sites decay much faster than others in the same unit cell. These radiolabile SeMet side chains decay faster than high-order diffraction spots with dose, making it difficult to detect this kind of damage by inspection of the diffraction pattern. The selenium X-ray absorbance near-edge spectrum (XANES) from samples containing SeMet was found to change significantly after application of X-ray doses of 10–100 MGy. Most notably, the sharp ‘white line’ feature near the canonical Se edge disappears. The change was attributed to breakage of the Cγ—Se bond in SeMet. This spectral change was used as a probe to measure the decay rate of SeMet with X-ray dose in cryo-cooled samples. Two protein crystal types and 15 solutions containing free SeMet amino acid were examined. The damage rate was influenced by the chemical and physical condition of the sample, and the half-decaying dose for the selenium XANES signal ranged from 5 to 43 MGy. These decay rates were 34- to 3.8-fold higher than the rate at which the Se atoms interacted directly with X-ray photons, so the damage mechanism must be a secondary effect. Samples that cooled to a more crystalline state generally decayed faster than samples that cooled to an amorphous solid. The single exception was a protein crystal where a nanocrystalline cryoprotectant had a protective effect. Lowering the pH, especially with ascorbic or nitric acids, had a protective effect, and SeMet lifetime increased monotonically with decreasing sample temperature (down to 93 K). The SeMet lifetime in one protein crystal was the same as that of the free amino acid, and the longest SeMet lifetime measured was found in the other protein crystal type. This protection was found to arise from the folded structure of the protein molecule. A mechanism to explain observed decay rates involving the damaging species following the electric field lines around protein molecules is proposed.

Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by ∼100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the In vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

Accurate measurement of photon flux from an X-ray source is a parameter required to calculate the dose absorbed by a sample. The development of a model for determining the photon flux incident on pin diodes, and experiments to test this model, are described for incident energies between 4 and 18 keV used in macromolecular crystallography.

Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced.

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.