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1.  Monitoring pH-Triggered Drug Release from Radioluminescent Nanocapsules with X-Ray Excited Optical Luminescence 
ACS nano  2013;7(2):1178-1187.
One of the greatest challenges in cancer therapy is to develop methods to deliver chemotherapy agents to tumor cells while reducing systemic toxicity to non-cancerous cells. A promising approach to localizing drug release is to employ drug-loaded nanoparticles with coatings that release the drugs only in the presence of specific triggers found in the target cells such as pH, enzymes, or light. However, many parameters affect the nanoparticle distribution and drug release rate and it is difficult to quantify drug release in situ. In this work, we show proof of principle for a “smart” radioluminescent nanocapsule with X-ray excited optical luminescence (XEOL) spectrum that changes during release of the optically absorbing chemotherapy drug, doxorubicin. XEOL provides an almost background-free luminescent signal for measuring drug release from particles irradiated by a narrow X-ray beam. We study in vitro pH triggered release rates of doxorubicin from nanocapsules coated with a pH responsive polyelectrolyte multilayer using HPLC and XEOL spectroscopy. The doxorubicin was loaded to over 5 % by weight, and released from the capsule with a time constant in vitro of ~ 36 days at pH 7.4, and 21.4 hr at pH 5.0, respectively. The Gd2O2S:Eu nanocapsules are also paramagnetic at room temperature with similar magnetic susceptibility and similarly good MRI T2 relaxivities to Gd2O3, but the sulfur increases the radioluminescence intensity and shifts the spectrum. Empty nanocapsules did not affect cell viability up to concentrations of at least 250 μ/ml. These empty nanocapsules accumulated in a mouse liver and spleen following tail vein injection, and could be observed in vivo using XEOL. The particles are synthesized with a versatile template synthesis technique which allows for control of particle size and shape. The XEOL analysis technique opens the door to non-invasive quantification of drug release as a function of nanoparticle size, shape, surface chemistry and tissue type.
doi:10.1021/nn304369m
PMCID: PMC3612352  PMID: 23281651
pH triggered drug release; release monitoring; radioluminescent nanocapsules; theranostics
2.  Real Time Observation of X-ray-Induced Surface Modification Using Simultaneous XANES and XEOL-XANES 
Analytical Chemistry  2013;85(20):9556-9563.
In experiments preliminary to the design of an X-ray-excited optical luminescence (XEOL)-based chemical mapping tool we have used X-ray micro (4.5 × 5.2 μm) and macro (1 × 6 mm) beams with similar total fluxes to assess the effects of a high flux density beam of X-rays at energies close to an absorption edge on inorganic surfaces in air. The near surface composition of corroded cupreous alloys was analyzed using parallel X-ray and optical photoemission channels to collect X-ray absorption near-edge structure (XANES) data at the Cu K edge. The X-ray fluorescence channel is characteristic of the composition averages over several micrometers into the surface, whereas the optical channel is surface specific to about 200 nm. While the X-ray fluorescence data were mostly insensitive to the X-ray dose, the XEOL-XANES data from the microbeam showed significant dose-dependent changes to the superficial region, including surface cleaning, changes in the oxidation state of the copper, and destruction of surface compounds responsible for pre-edge fluorescence or phosphorescence in the visible. In one case, there was evidence that the lead phase in a bronze had melted. Conversely, data from the macrobeam were stable over several hours. Apart from localized heating effects, the microbeam damage is probably associated with the O3 loading of the surface and increased reaction rate with atmospheric water vapor.
doi:10.1021/ac401646q
PMCID: PMC3805311  PMID: 24044633
3.  Development of an online UV–visible microspectrophotometer for a macromolecular crystallography beamline 
Journal of Synchrotron Radiation  2013;20(Pt 6):948-952.
An online UV–visible microspectrophotometer has been developed for the macromolecular crystallography beamline at SPring-8. Details of this spectrophotometer are reported.
Measurement of the UV–visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV–visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury–xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.
doi:10.1107/S0909049513022887
PMCID: PMC3795562  PMID: 24121346
UV–visible spectroscopy; protein crystallography; radiation damage; microspectroscopy; SPring-8
4.  XANES measurements of the rate of radiation damage to selenomethionine side chains 
Journal of synchrotron radiation  2006;14(Pt 1):51-72.
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.
doi:10.1107/S0909049506048898
PMCID: PMC2806430  PMID: 17211072
X-ray dose; protective solutes; pH and temperature effects; protein crystal structure; mechanism of radiation damage
5.  To scavenge or not to scavenge, that is STILL the question 
Journal of Synchrotron Radiation  2012;20(Pt 1):23-36.
The reasons for the conflicting results on the efficacy of scavengers in macromolecular crystallography are examined in the light of some new results.
An extensive radiation chemistry literature would suggest that the addition of certain radical scavengers might mitigate the effects of radiation damage during protein crystallography diffraction data collection. However, attempts to demonstrate and quantify such an amelioration and its dose dependence have not yielded consistent results, either at room temperature (RT) or 100 K. Here the information thus far available is summarized and reasons for this lack of quantitative success are identified. Firstly, several different metrics have been used to monitor and quantify the rate of damage, and, as shown here, these can give results which are in conflict regarding scavenger efficacy. In addition, significant variation in results from data collected from crystals treated in nominally the same way has been observed. Secondly, typical crystallization conditions contain substantial concentrations of chemical species which already interact strongly with some of the X-ray-induced radicals that the added scavengers are intended to intercept. These interactions are probed here by the complementary technique of on-line microspectrophotometry carried out on solutions and crystals held both at 100 K and RT, the latter enabled by the use of a beamline-mounted humidifying device. With the help of computational chemistry, attempts are made to assign some of the characteristic spectral features observed experimentally. A further source of uncertainty undoubtedly lies in the challenge of reliably measuring the parameters necessary for the accurate calculation of the absorbed dose (e.g. crystal size and shape, beam profile) and its distribution within the volume of the crystal (an issue addressed in detail in another article in this issue). While microspectrophotometry reveals that the production of various species can be quenched by the addition of scavengers, it is less clear that this observation can be translated into a significant gain in crystal dose tolerance for macromolecular crystallographers.
doi:10.1107/S0909049512046237
PMCID: PMC3526919  PMID: 23254653
radiation damage; crystallization buffers; radicals; scavengers; lithium nitrate
6.  Colouring cryo-cooled crystals: online microspectrophotometry 
Journal of Synchrotron Radiation  2009;16(Pt 2):163-172.
A portable and readily aligned online microspectrophotometer that can be easily installed on macromolecular crystallography beamlines is described. It allows measurement of the spectral characteristics of macromolecular crystals prior, during, and after the X-ray diffraction experiment.
X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200–1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.
doi:10.1107/S0909049509001629
PMCID: PMC2651762  PMID: 19240328
radiation damage; macromolecular crystallography; online microspectrophotometry
7.  Femtosecond X-ray protein nanocrystallography 
Chapman, Henry N. | Fromme, Petra | Barty, Anton | White, Thomas A. | Kirian, Richard A. | Aquila, Andrew | Hunter, Mark S. | Schulz, Joachim | DePonte, Daniel P. | Weierstall, Uwe | Doak, R. Bruce | Maia, Filipe R. N. C. | Martin, Andrew V. | Schlichting, Ilme | Lomb, Lukas | Coppola, Nicola | Shoeman, Robert L. | Epp, Sascha W. | Hartmann, Robert | Rolles, Daniel | Rudenko, Artem | Foucar, Lutz | Kimmel, Nils | Weidenspointner, Georg | Holl, Peter | Liang, Mengning | Barthelmess, Miriam | Caleman, Carl | Boutet, Sébastien | Bogan, Michael J. | Krzywinski, Jacek | Bostedt, Christoph | Bajt, Saša | Gumprecht, Lars | Rudek, Benedikt | Erk, Benjamin | Schmidt, Carlo | Hömke, André | Reich, Christian | Pietschner, Daniel | Strüder, Lothar | Hauser, Günter | Gorke, Hubert | Ullrich, Joachim | Herrmann, Sven | Schaller, Gerhard | Schopper, Florian | Soltau, Heike | Kühnel, Kai-Uwe | Messerschmidt, Marc | Bozek, John D. | Hau-Riege, Stefan P. | Frank, Matthias | Hampton, Christina Y. | Sierra, Raymond G. | Starodub, Dmitri | Williams, Garth J. | Hajdu, Janos | Timneanu, Nicusor | Seibert, M. Marvin | Andreasson, Jakob | Rocker, Andrea | Jönsson, Olof | Svenda, Martin | Stern, Stephan | Nass, Karol | Andritschke, Robert | Schröter, Claus-Dieter | Krasniqi, Faton | Bott, Mario | Schmidt, Kevin E. | Wang, Xiaoyu | Grotjohann, Ingo | Holton, James M. | Barends, Thomas R. M. | Neutze, Richard | Marchesini, Stefano | Fromme, Raimund | Schorb, Sebastian | Rupp, Daniela | Adolph, Marcus | Gorkhover, Tais | Andersson, Inger | Hirsemann, Helmut | Potdevin, Guillaume | Graafsma, Heinz | Nilsson, Björn | Spence, John C. H.
Nature  2011;470(7332):73-77.
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 recorded1-3. 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 Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. 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 processes6. 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.
doi:10.1038/nature09750
PMCID: PMC3429598  PMID: 21293373
8.  Femtosecond X-ray diffraction from two-dimensional protein crystals 
Iucrj  2014;1(Pt 2):95-100.
Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented.
X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
doi:10.1107/S2052252514001444
PMCID: PMC4062087  PMID: 25075325
two-dimensional protein crystal; femtosecond crystallography; single layer X-ray diffraction; membrane protein
9.  All-diamond optical assemblies for a beam-multiplexing X-ray monochromator at the Linac Coherent Light Source 
Journal of Applied Crystallography  2014;47(Pt 4):1329-1336.
All-diamond optical assemblies holding state-of-the-art type IIa diamond crystals enable the construction of a beam-multiplexing X-ray double-crystal monochromator for hard X-ray free-electron lasers. Details on the design, fabrication and X-ray diffraction characterization of the assemblies are reported.
A double-crystal diamond (111) monochromator recently implemented at the Linac Coherent Light Source (LCLS) enables splitting of the primary X-ray beam into a pink (transmitted) and a monochromatic (reflected) branch. The first monochromator crystal, with a thickness of ∼100 µm, provides sufficient X-ray transmittance to enable simultaneous operation of two beamlines. This article reports the design, fabrication and X-ray characterization of the first and second (300 µm-thick) crystals utilized in the monochromator and the optical assemblies holding these crystals. Each crystal plate has a region of about 5 × 2 mm with low defect concentration, sufficient for use in X-ray optics at the LCLS. The optical assemblies holding the crystals were designed to provide mounting on a rigid substrate and to minimize mounting-induced crystal strain. The induced strain was evaluated using double-crystal X-ray topography and was found to be small over the 5 × 2 mm working regions of the crystals.
doi:10.1107/S1600576714013028
PMCID: PMC4119950  PMID: 25242912
diamond; X-ray monochromators; beam multiplexing
10.  Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals 
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.
doi:10.1107/S0108767310050981
PMCID: PMC3066792  PMID: 21325716
nanocrystals; femtosecond diffraction; free-electron lasers; Monte Carlo methods; protein microdiffraction
11.  Crystallization and preliminary X-ray diffraction analysis of the dimerization domain of the tumour suppressor ING4 
The N-terminal domain of ING4 was overexpressed in Escherichia coli and purified to homogeneity. Crystallization experiments yielded crystals that were suitable for high-resolution X-ray diffraction analysis.
Inhibitor of growth protein 4 (ING4) belongs to the ING family of tumour suppressors and is involved in chromatin remodelling, in growth arrest and, in cooperation with p53, in senescence and apoptosis. Whereas the structure and histone H3-binding properties of the C-terminal PHD domains of the ING proteins are known, no structural information is available for the N-terminal domains. This domain contains a putative oligomerization site rich in helical structure in the ING2–5 members of the family. The N-terminal domain of ING4 was overexpressed in Escherichia coli and purified to homogeneity. Crystallization experiments yielded crystals that were suitable for high-resolution X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 129.7, b = 188.3, c = 62.7 Å. The self-rotation function and the Matthews coefficient suggested the presence of three protein dimers per asymmetric unit. The crystals diffracted to a resolution of 2.3 Å using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).
doi:10.1107/S1744309110010080
PMCID: PMC2864694  PMID: 20445261
ING4; tumour suppressors; dimerization domain
12.  Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin 
A purified blue-light-absorbing proteorhodopsin D97N mutant protein (BPR_D97N) has been crystallized using the vapour-diffusion method.
Proteorhodopsins (PRs), seven-transmembrane chromoproteins with retinal as a chromophore, are light-driven proton pumps. To elucidate the light-driven proton-pumping mechanism of PRs, a pET28a vector containing the blue-light-absorbing proteorhodopsin (BPR) gene was constructed and the protein was overexpressed in Escherichia coli. The protein was purified by immobilized metal-ion affinity chromatography (IMAC). The purified BPR D97N mutant protein (BPR_D97N) was crystallized using the vapour-diffusion method. Preliminary X-ray diffraction data analysis showed that the crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 161.6, b = 168.6, c = 64.7 Å. A complete data set was collected to 3.3 Å resolution using synchrotron radiation on beamline X06 of the Swiss Light Source (SLS). Molecular replacement was unsuccessful. To solve the structure of BPR_D97N by experimental phasing, selenomethionine-substituted protein crystals were prepared. These crystals diffracted to 3.0 Å resolution and a complete data set was collected on beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contained five molecules in the asymmetric unit, with a V M of 3.26 Å3 Da−1 and a solvent content of 62.3%.
doi:10.1107/S1744309111043612
PMCID: PMC3310530  PMID: 22442222
proteorhodopsins; BPR
13.  Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA 
I-Dmo-I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis. The enzyme was cloned and overexpressed in Escherichia coli. Crystallization experiments of I-Dmo-I in complex with its DNA target in the presence of Ca2+ and Mg2+ yielded crystals that were suitable for X-ray diffraction analysis.
Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of base pairs. The availability of these enzymes has opened novel perspectives for genome engineering in a wide range of fields, including gene therapy, by taking advantage of the homologous gene-targeting enhancement induced by a double-strand break. I-Dmo-I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis. The enzyme was cloned and overexpressed in Escherichia coli. Crystallization experiments of I-Dmo-I in complex with its DNA target in the presence of Ca2+ and Mg2+ yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 106.75, b = 70.18, c = 106.85 Å, α = γ = 90, β = 119.93°. The self-rotation function and the Matthews coefficient suggested the presence of three protein–DNA complexes per asymmetric unit. The crystals diffracted to a resolution limit of 2.6 Å using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).
doi:10.1107/S1744309107049706
PMCID: PMC2344106  PMID: 18084082
homing endonucleases; I-Dmo-I
14.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain 
Crystals of DDX3 RNA helicase domain have been obtained in a monoclinic form that diffract to 2.2 Å resolution using synchrotron radiation at the ID14-1 ESRF beamline.
DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P21, with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 Å, α = γ = 90, β = 101.02°, and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).
doi:10.1107/S1744309107006434
PMCID: PMC2330201  PMID: 17401195
DDX3; RNA helicases
15.  Revealing low-dose radiation damage using single-crystal spectroscopy 
Journal of Synchrotron Radiation  2011;18(Pt 3):367-373.
Data on the rapid reduction of haem proteins in the X-ray beam at synchrotron sources are presented. The use of single-crystal spectroscopy to detect these changes and their implication for diffraction data collection from oxidized species is also discussed.
The structural information and functional insight obtained from X-ray crystallography can be enhanced by the use of complementary spectroscopies. Here the information that can be obtained from spectroscopic methods commonly used in conjunction with X-ray crystallography and best-practice single-crystal UV-Vis absorption data collection are briefly reviewed. Using data collected with the in situ system at the Swiss Light Source, the time and dose scales of low-dose X-ray-induced radiation damage and solvated electron generation in metalloproteins at 100 K are investigated. The effect of dose rate on these scales is also discussed.
doi:10.1107/S0909049511004250
PMCID: PMC3083913  PMID: 21525644
macromolecular crystallography; single-crystal microspectrophotometry; radiation damage; myoglobin; cytochrome c
16.  Crystallization and preliminary X-ray diffraction studies on the human Plk1 Polo-box domain in complex with an unphosphorylated and a phosphorylated target peptide from Cdc25C 
Crystals of the human Plk1 Polo-box domain in complex with a Cdc25C target peptide in an unphosphorylated and a phosphorylated state have been obtained in orthorhombic and monoclinic forms that diffract to 2.1 and 2.85 Å, respectively, using synchrotron radiation.
Polo-like kinase (Plk1) is crucial for cell-cycle progression via mitosis. Members of the Polo-like kinase family are characterized by the presence of a C-terminal domain termed the Polo-box domain (PBD) in addition to the N-terminal kinase domain. The PBD of Plk1 was cloned and overexpressed in Escherichia coli. Crystallization experiments of the protein in complex with an unphosphorylated and a phosphorylated target peptide from Cdc25C yield crystals suitable for X-­ray diffraction analysis. Crystals of the PBD in complex with the phosphorylated peptide belong to the orthorhombic space group P212121, with unit-cell parameters a = 38.23, b = 67.35, c = 88.25 Å, α = γ = β = 90°, and contain one molecule per asymmetric unit. Crystals of the PBD in complex with the unphosphorylated peptide belong to the monoclinic space group P21, with unit-cell parameters a = 40.18, b = 49.17, c = 56.23 Å, α = γ = 90, β = 109.48°, and contain one molecule per asymmetric unit. The crystals diffracted to resolution limits of 2.1 and 2.85 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS), respectively.
doi:10.1107/S1744309106007494
PMCID: PMC2222578  PMID: 16582488
Polo-like kinase; Polo-box domain; Cdc25C
17.  Synchrotron radiation: micrometer-sized x-ray beams as fine tools for macromolecular crystallography 
HFSP Journal  2008;2(6):302-306.
Structural data play a central role in understanding biological function at the molecular level. At present, the majority of high-resolution structural data about biological macromolecules and their complexes originates from crystallography. In crystal structure determination, the major hurdle to overcome is the production of crystals of sufficient size and quality. High-flux x-ray beams with diameters of a few micrometers or less help to alleviate this problem as small beams allow the use of small crystals or scanning of large crystals for regions of acceptable diffraction. Using sophisticated x-ray optics and mechanics with submicrometer precision, Riekel et al.[Acta Crystallogr., Sect. D: Biol. Crystallogr., 64, 158–166 (2008)], have recently demonstrated that an x-ray beam of 1 μm can be used to determine the crystal structure of a protein to a resolution of 1.5 Å. The smallest volume from which usable diffraction data were collected amounted to 20 μm3, corresponding to not more than 2×108 unit cells. In a diffraction volume of micrometer dimensions, radiation damage is expected to be reduced with respect to large volumes as a significant fraction of the photoelectrons produced by the incident radiation escapes from the diffracting volume before dissipating their energy. The possibility to make use of small and∕or inhomogeneous crystals in combination with a possible reduction in radiation damage due to size effects has the potential to make many more systems amenable to crystal structure analysis.
doi:10.2976/1.2982661
PMCID: PMC2645582  PMID: 19436492
18.  Assessment of radiation damage behaviour in a large collection of empirically optimized datasets highlights the importance of unmeasured complicating effects 
Journal of Synchrotron Radiation  2011;18(Pt 3):387-397.
A retrospective analysis of radiation damage behaviour in a statistically significant number of real-life datasets is presented, in order to gauge the importance of the complications not yet measured or rigorously evaluated in current experiments, and the challenges that remain before radiation damage can be considered a problem solved in practice.
The radiation damage behaviour in 43 datasets of 34 different proteins collected over a year was examined, in order to gauge the reliability of decay metrics in practical situations, and to assess how these datasets, optimized only empirically for decay, would have benefited from the precise and automatic prediction of decay now possible with the programs RADDOSE [Murray, Garman & Ravelli (2004 ▶). J. Appl. Cryst. 37, 513–522] and BEST [Bourenkov & Popov (2010 ▶). Acta Cryst. D66, 409–419]. The results indicate that in routine practice the diffraction experiment is not yet characterized well enough to support such precise predictions, as these depend fundamentally on three interrelated variables which cannot yet be determined robustly and practically: the flux density distribution of the beam; the exact crystal volume; the sensitivity of the crystal to dose. The former two are not satisfactorily approximated from typical beamline information such as nominal beam size and transmission, or two-dimensional images of the beam and crystal; the discrepancies are particularly marked when using microfocus beams (<20 µm). Empirically monitoring decay with the dataset scaling B factor (Bourenkov & Popov, 2010 ▶) appears more robust but is complicated by anisotropic and/or low-resolution diffraction. These observations serve to delineate the challenges, scientific and logistic, that remain to be addressed if tools for managing radiation damage in practical data collection are to be conveniently robust enough to be useful in real time.
doi:10.1107/S0909049511008235
PMCID: PMC3083914  PMID: 21525647
radiation damage; data collection; strategy; beamline software; datasets
19.  Three-dimensional electron crystallography of protein microcrystals 
eLife  2013;2:e01345.
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.
DOI: http://dx.doi.org/10.7554/eLife.01345.001
eLife digest
X-ray crystallography has been used to work out the atomic structure of a large number of proteins. In a typical X-ray crystallography experiment, a beam of X-rays is directed at a protein crystal, which scatters some of the X-ray photons to produce a diffraction pattern. The crystal is then rotated through a small angle and another diffraction pattern is recorded. Finally, after this process has been repeated enough times, it is possible to work backwards from the diffraction patterns to figure out the structure of the protein.
The crystals used for X-ray crystallography must be large to withstand the damage caused by repeated exposure to the X-ray beam. However, some proteins do not form crystals at all, and others only form small crystals. It is possible to overcome this problem by using extremely short pulses of X-rays, but this requires a very large number of small crystals and ultrashort X-ray pulses are only available at a handful of research centers around the world. There is, therefore, a need for other approaches that can determine the structure of proteins that only form small crystals.
Electron crystallography is similar to X-ray crystallography in that a protein crystal scatters a beam to produce a diffraction pattern. However, the interactions between the electrons in the beam and the crystal are much stronger than those between the X-ray photons and the crystal. This means that meaningful amounts of data can be collected from much smaller crystals. However, it is normally only possible to collect one diffraction pattern from each crystal because of beam induced damage. Researchers have developed methods to merge the diffraction patterns produced by hundreds of small crystals, but to date these techniques have only worked with very thin two-dimensional crystals that contain only one layer of the protein of interest.
Now Shi et al. report a new approach to electron crystallography that works with very small three-dimensional crystals. Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. The normal ‘low-dose’ electron beam in one of these microscopes would normally damage the crystal after a single diffraction pattern had been collected. However, Shi et al. realized that it was possible to obtain diffraction patterns without severely damaging the crystal if they dramatically reduced the normal low-dose electron beam. By reducing the electron dose by a factor of 200, it was possible to collect up to 90 diffraction patterns from the same, very small, three-dimensional crystal, and then—similar to what happens in X-ray crystallography—work backwards to figure out the structure of the protein. Shi et al. demonstrated the feasibility of the MicroED approach by using it to determine the structure of lysozyme, which is widely used as a test protein in crystallography, with a resolution of 2.9 Å. This proof-of principle study paves the way for crystallographers to study protein that cannot be studied with existing techniques.
DOI: http://dx.doi.org/10.7554/eLife.01345.002
doi:10.7554/eLife.01345
PMCID: PMC3831942  PMID: 24252878
electron crystallography; electron diffraction; electron cryomicroscopy (cryo-EM); microED; protein structure; microcrystals; None
20.  Towards protein-crystal centering using second-harmonic generation (SHG) microscopy 
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-­ray diffraction of protein crystals has been explored.
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-­ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-­ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.
doi:10.1107/S0907444913002746
PMCID: PMC3640472  PMID: 23633594
second-harmonic generation microscopy; crystal centering; imaging
21.  Can radiation damage to protein crystals be reduced using small-molecule compounds? 
Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal.
Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystalliz­ation or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.
doi:10.1107/S0907444911032835
PMCID: PMC3176623  PMID: 21931220
radiation damage; scavengers; radioprotection; global damage; site-specific damage
22.  Super-resolution biomolecular crystallography with low-resolution data 
Nature  2010;464(7292):1218-1222.
X-ray diffraction plays a pivotal role in understanding of biological systems by revealing atomic structures of proteins, nucleic acids, and their complexes, with much recent interest in very large assemblies like the ribosome. Since crystals of such large assemblies often diffract weakly (resolution worse than 4 Å), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, while others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex1. Determining such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution below 5 Å generally exceed the number of degrees of freedom. Here we introduce a new method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with Rfree determines the optimum deformation and influence of the homology model. For test cases at 3.5 – 5 Å resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model coordinate accuracy, the definition of secondary structure, and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the PDB, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to studying weakly diffracting crystals using X-ray micro-diffraction2 as well as data from new X-ray light sources3. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to sub-nanometer resolution4,5, it can use similar tools.
doi:10.1038/nature08892
PMCID: PMC2859093  PMID: 20376006
X-ray crystallography; homology modeling; cross-validation; Rfree value; refinement
23.  Chromophore Protonation State Controls Photoswitching of the Fluoroprotein asFP595 
PLoS Computational Biology  2008;4(3):e1000034.
Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.
Author Summary
Proteins whose fluorescence can be reversibly switched on and off hold great promise for applications in high-resolution optical microscopy and nanotechnology. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements of their properties requires a detailed understanding of the molecular switching mechanism. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 by atomistic molecular dynamics simulations. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Our results show that a tight coupling between trans-cis isomerization of the chromophore and proton transfer is essential for the function of asFP595. The structural similarity between asFP595 and other fluoroproteins suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.
doi:10.1371/journal.pcbi.1000034
PMCID: PMC2274881  PMID: 18369426
24.  Toward structure determination using membrane-protein nanocrystals and microcrystals 
Methods (San Diego, Calif.)  2011;55(4):387-404.
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (~ 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-Å wavelength, Bragg peaks were recorded to 8.5-Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [Chapman H.N., et al. Femtosecond X-ray protein nanocrystallography, Nature 470 (2011) 73–81]. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
doi:10.1016/j.ymeth.2011.12.006
PMCID: PMC3414265  PMID: 22197730
membrane proteins; structure determination; femtosecond nanocrystallography; protein nanocrystals; X-ray crystallography; XFEL
25.  Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging 
A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material.
The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.
doi:10.1107/S0907444913011359
PMCID: PMC3689528  PMID: 23793151
membrane proteins; lipid cubic phase; microradiography; microtomography

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