Radiation damage to protein specimens from electron beam imaging and diffraction: a mini‐review of anti‐damage approaches,with special reference to synchrotron X‐ray crystallography

Recent research progress using X‐ray cryo‐crystallography with the photon beams from third‐generation synchrotron sources has resulted in recognition that this intense radiation commonly damages protein samples even when they are held at 100 K. Other structural biologists examining thin protein crystals or single particle specimens encounter similar radiation damage problems during electron diffraction and imaging, but have developed some effective countermeasures. The aim of this concise review is to examine whether analogous approaches can be utilized to alleviate the X‐ray radiation damage problem in synchrotron macromolecular crystallography. The critical discussion of this question is preceded by presentation of background material on modern technical procedures with electron beam instruments using 300–400 kV accelerating voltage, low‐dose exposures for data recording, and protection of protein specimens by cryogenic cooling; these practical approaches to dealing with electron radiation damage currently permit best resolution levels of 6 Å (0.6 nm) for single particle specimens, and of 1.9 Å for two‐dimensional membrane protein crystals. Final determination of the potential effectiveness and practical value of using such new or unconventional ideas will necessitate showing, by experimental testing, that these produce significantly improved protection of three‐dimensional protein crystals during synchrotron X‐ray diffraction.     

Energy resolution of the CdTe‐XPAD detector: calibration and potential for Laue diffraction measurements on protein crystals

The XPAD3S‐CdTe, a CdTe photon‐counting pixel array detector, has been used to measure the energy and the intensity of the white‐beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy‐resolved X‐ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending‐magnet beamline METROLOGIE at Synchrotron SOLEIL. The X‐ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging‐plate detector and then measured with both the XPAD3S‐CdTe and the XPAD3S‐Si, a silicon photon‐counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit‐cell parameter determination and the measurement of high‐quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X‐ray diffraction opened up by this type of technology are discussed.     

A first low‐resolution difference Fourier map of phosphorus in a membrane protein from near‐edge anomalous diffraction

Crystal diffraction of three membrane proteins (cytochrome bc₁ complex, sarcoplasmic reticulum Ca²⁺ ATPase, ADP‐ATP carrier) and of one nucleoprotein complex (leucyl tRNA synthetase bound to tRNAleu, leuRS:tRNAleu) was tested at wavelengths near the X‐ray K‐absorption edge of phosphorus using a new set‐up for soft X‐ray diffraction at the beamline ID01 of the ESRF. The best result was obtained from crystals of Ca²⁺ ATPase [adenosin‐5′‐(β,γ‐methylene) triphosphate complex] which diffracted out to 7 Å resolution. Data were recorded at a wavelength at which the real resonant scattering factor of phosphorus reaches the extreme value of ?20 electron units. The positions of the four triphosphates of the monoclinic unit cell of the ATPase have been obtained from a difference Fourier synthesis based on a limited set of anomalous diffraction data.     

A new aspect of specific radiation damage: hydrogen abstraction from organic molecules

Radiation damage is one of the major impediments in obtaining high‐resolution structural information utilizing ionizing radiation. From electron microscopy it is known that electron irradiation of biological samples results in the formation of molecular hydrogen. In the present work radiation‐induced structural changes of the polypeptide cyclosporine A were observed at a temperature of 100 K. Bond length changes are thought to be due to radiation‐induced hydrogen abstraction which chemically modifies the molecules in an irreversible way. The resulting formation of molecular hydrogen might explain the observed increase of the crystal mosaicity, which has also been reported in many previous radiation damage studies.     

Automated sample‐scanning methods for radiation damage mitigation and diffraction‐based centering of macromolecular crystals

Automated scanning capabilities have been added to the data acquisition software, JBluIce‐EPICS, at the National Institute of General Medical Sciences and the National Cancer Institute Collaborative Access Team (GM/CA CAT) at the Advanced Photon Source. A `raster' feature enables sample centering via diffraction scanning over two‐dimensional grids of simple rectangular or complex polygonal shape. The feature is used to locate crystals that are optically invisible owing to their small size or are visually obfuscated owing to properties of the sample mount. The raster feature is also used to identify the best‐diffracting regions of large inhomogeneous crystals. Low‐dose diffraction images taken at grid positions are automatically processed in real time to provide a quick quality ranking of potential data‐collection sites. A `vector collect' feature mitigates the effects of radiation damage by scanning the sample along a user‐defined three‐dimensional vector during data collection to maximize the use of the crystal volume and the quality of the collected data. These features are integrated into the JBluIce‐EPICS data acquisition software developed at GM/CA CAT where they are used in combination with a robust mini‐beam of rapidly changeable diameter from 5 µm to 20 µm. The powerful software–hardware combination is being applied to challenging problems in structural biology.     

从2009年诺贝尔化学奖看同步辐射装置和技术在生物化学研究中的重要作用

文章介绍了获得2009年诺贝尔化学奖的原核细胞核糖体结构解析的历程.在这个历时20年的探索过程中,同步辐射装置起到了重要的作用.同步辐射这种大型的科学装置为前沿的科学研究提供了不可缺少的支撑,文章通过核糖体结构解析的过程,阐述了这种支撑所具有的明显的地域特征.     

从2009年诺贝尔化学奖看同步辐射装置和技术在生物化学研究中的重要作用

文章介绍了获得2009年诺贝尔化学奖的原核细胞核糖体结构解析的历程.在这个历时20年的探索过程中,同步辐射装置起到了重要的作用.同步辐射这种大型的科学装置为前沿的科学研究提供了不可缺少的支撑,文章通过核糖体结构解析的过程,阐述了这种支撑所具有的明显的地域特征.     

用直接法提高抗生蛋白链菌素电子显微像的分辨率

将直接法相位外推运用于抗生蛋白链菌素的二维电子衍射数据，相位外推所需的结构因子振幅和初始相位分别来自电子衍射数据和相应的电子显微像。利用最大熵原理识别法和团簇分析方法从多重解中推导出最佳解，最终使电子显微像的分辨率由3 ?提高到2.5 ?，图像质量得到显著改善。     

Energy dependence of site‐specific radiation damage in protein crystals

It is important to consider radiation damage to crystals caused by data collection when solving structures and critical when determining protein function, which can often depend on very subtle structural characteristics. In this study the rate of damage to specific sites in protein crystals cooled at 100 K is found to depend on the energy of the incident X‐ray beam. Several lysozyme crystals were each subjected to 3–26 MGy of cumulative X‐ray exposure by collecting multiple data sets from each crystal at either 9 keV or 14 keV. The integrated electron density surrounding each S atom in the structure was calculated for each data set and the change in electron density was evaluated as a function of dose at the two energies. The rate of electron density decrease per cubic Å per MGy was determined to be greater at 14 keV than at 9 keV for cysteine sulfurs involved in disulphide bridges; no statistically significant differences in the decay rates were found for methionine sulfurs. These preliminary results imply that it might be possible to minimize certain types of specific radiation damage by an appropriate choice of energy. Further experiments studying a variety of photolabile sites over a wider range of energies are needed to confirm this conclusion.     

Effective scavenging at cryotemperatures: further increasing the dose tolerance of protein crystals

The rate of radiation damage to macromolecular crystals at both room temperature and 100 K has previously been shown to be reduced by the use of certain radical scavengers. Here the effects of sodium nitrate, an electron scavenger, are investigated at 100 K. For sodium nitrate at a concentration of 0.5 M in chicken egg‐white lysozyme crystals, the dose tolerance is increased by a factor of two as judged from the global damage parameters, and no specific structural damage to the disulfide bonds is seen until the dose is greatly in excess (more than a factor of five) of the value at which damage appears in electron density maps derived from a scavenger‐free crystal. In the electron density maps, ordered nitrate ions adjacent to the disulfide bonds are seen to lose an O atom, and appear to protect the disulfide bonds. In addition, results reinforcing previous reports on the effectiveness of ascorbate are presented. The mechanisms of action of both scavengers in the crystalline environment are elucidated.     

Dissociation of the light-harvesting membrane protein complex I from Rhodobacter sphaeroides under high hydrostatic pressure

The light-harvesting complex 1 (LH1) from Rhodobacter sphaeroides is an excellent model system for investigating the stability of oligomeric membrane proteins under high hydrostatic pressure. The currently investigated LH1 forms a 16-meric ring structure of B825 subunits. B825 is a heterodimer of transmembrane α- and β-polypeptide chains, which non-covalently binds two bacteriochlorophyll a molecules. These pigment molecules were used as intrinsic spectroscopic sensors to follow the dissociation reaction. Our results demonstrate that the LH1 dissociates into B825 subunits through an intermediary tetrameric unit B845. The dissociation mechanism depends on pressure. At ～200–500?MPa the dissociation corresponds to a pseudo-first-order reaction, characterised by the apparent reaction rate at atmospheric pressure k₀?=?3·10^?5?s^?1, activation volume ΔV^??=??4?mL/mol, and free energy of activation ΔG^??=?26?kJ/mol. Below 200?MPa and above 500?MPa, the reaction is more complex, including further dissociation of B825 into monomers B777.     

Interpretation of protein structure and dynamics from the statistics of the open and closed times measured in a single ion-channel protein

Ion channels are proteins in the lipid cell membrane. They spontaneously fluctuate between conformational shapes that are open or closed to the passage of ions. The ionic currents through an individual channel can be resolved by the patch clamp technique. Thus, the time sequence of open and closed conformational states can be measured in one channel molecule. The probability density function of the dwell times in the open and closed states displays scaling functions that may arise from: (1) a large number of conformational substates having a continuous distribution of activation energy barriers, (2) time-dependent changes in the energy barriers between states, or (3) local interactions that constrain local structures which interact hierarchically to form global structure.     

Ultraviolet resonance Raman spectroscopy of a β‐sheet peptide: a model for membrane protein folding

The partitioning of a hydrophobic hexapeptide, N‐acetyl‐tryptophan‐pentaleucine (AcWL5), into self‐associated β‐sheets within a vesicle membrane was studied as a model for integral membrane protein folding and insertion via vibrational and electronic spectroscopy. Ultraviolet resonance Raman spectroscopy allows selective examination of the structures of amino acid side chains and the peptide backbone and provides information about local environment and molecular conformation. The secondary structure of AcWL5 within a vesicle membrane was investigated using 207.5‐nm excitation and found to consist of β‐sheets, in agreement with previous studies. The β‐sheet peptide shows enhanced Raman scattering cross‐sections for all amide modes as well as extensive hydrogen‐bonding networks. Tryptophan vibrational structure was probed using 230‐nm excitation. Increases in Raman cross‐sections of tryptophan modes W1, W3, W7, W10, W16, W17, and W18 of membrane‐incorporated AcWL5 are primarily attributed to greater resonance enhancement with the B_b electronic transition. The W17 mode, however, undergoes a much greater enhancement than is expected for a simple resonance effect, and this observation is discussed in terms of hydrogen bonding of the indole ring in a hydrophobic environment. The observed tryptophan mode frequencies and intensities overall support a hydrophobic environment for the indole ring within a vesicle, and these results have implications for the location of tryptophan in membrane protein systems. Copyright © 2009 John Wiley & Sons, Ltd.     

Binding of regulatory protein omega from Streptococcus pyogenes plasmid pSM19035 to direct and inverted 7‐base pair repeats of operator DNA

pSM19035‐encoded dimeric regulator omega (ω₂) belongs to the MetJ/Arc family of ribbon–helix–helix DNA binding proteins. ω₂ binds to a set of unspaced 7‐base pair repeat units with sequence 5′‐^A/_TATCAC^A/_T‐3′. Protein ω₂ and its variant ω₂ΔN18, lacking the first 18 N‐terminal amino acids, bind poorly to one heptad DNA, but the affinity to DNA markedly increases with two and more heptads organized in direct or inverted orientation. Raman difference spectra indicate that ω₂ and ω₂ΔN18 bind to operator DNA in the major groove and contact thymine, adenine and guanine residues. The mode of ω₂ or ω₂ΔN18 interaction with operator DNA is not affected by the orientation and number of heptads. The Raman data of ω₂T29A, an ω₂ variant with Ala instead of Thr at position 29 of the β‐sheet, show a sequence‐independent binding mode to DNA, which differs from the binding mode of wt ω₂ protein to its cognate site. The Raman data strongly support the notion that the 18 N‐terminal amino acids are not required for ω₂ activity, whereas Thr29 plays an essential role for operator DNA binding. The data suggest the formation of a hydrogen bond between Thr29 of wild‐type ω₂ to one of the bases in the central 5′‐TCA‐3′/5′‐TGA‐3′ stretch of the 7‐bp binding site. Copyright © 2006 John Wiley & Sons, Ltd.     

Chemical and Structural Properties of Drug–protein Nanocomposites Prepared by Pulsed Laser Deposition from Conjugated Targets

Nanostructured indomethacin (IM) – bovine serum albumin (BSA) composite particulates below 50 nm were obtained by pulsed laser deposition (PLD) with 1064 nm IR beam. IM and BSA powders were coground for up to 210 min to prepare conjugated targets for PLD with varying structural states. The ablation threshold was determined to be around 1.8 J/cm², from in situ monitoring of the deposited amount by a QCM sensor. The crystallinity of IM was preserved after PLD at 5 J/cm² and decreased with increasing the laser fluence. The non-crystalline state of IM in the target after prolonged cogrinding with BSA, was also preserved in the deposited film. FT-IR absorption bands due to amide I of BSA became sharper while preserving the peak top position after cogrinding with IM. Conversely, we observed broadening of the amide I bands by PLD. Preservation and change in the chemical and structural states of the conjugates after PLD is discussed in terms of the secondary structure of BSA and crystallinity of IM.     

Design and construction of a compact end-station at NSRRC for circular-dichroism spectra in the vacuum-ultraviolet region

A synchrotron‐radiation‐based circular‐dichroism end‐station has been implemented at beamline BL04B at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan for biological research. The design and performance of this compact end‐station for measuring circular‐dichroism spectra in the vacuum‐ultraviolet region are described. The linearly polarized light from the beamline is converted to modulated circularly polarized light with a LiF photoelastic modulator to provide a usable wavelength region of 130–330 nm. The light spot at the sample position is 5 mm × 5 mm at a slit width of 300 µm and provides a flux greater than 1 × 10¹¹ photons s^?1 (0.1% bandwidth)^?1. A vacuum‐compatible cell made of two CaF₂ windows has a variable path length from 1.3 µm to 1 mm and a temperature range of 253–363 K. Measured CD spectra of (1S)‐(+)‐10‐camphorsulfonic acid and proteins demonstrated the ability of this system to extend the wavelength down to 172 nm in aqueous solution and 153 nm in hexafluoro‐2‐propanol.     

Ultrasound-assisted immersion freezing reduces the structure and gel property deterioration of myofibrillar protein from chicken breast

The effects of air freezing (AF), immersion freezing (IF) and ultrasound-assisted immersion freezing (UF) at different power levels (125, 165, 205 and 245 W) on the structure and gel properties of the myofibrillar protein (MP) of chicken breast were investigated. UF at 165 W (UF-165) had no obvious negative impact on the primary structure of the MP and effectively reduced the change in the secondary and tertiary structure. In addition, UF-165 significantly reduced the losses in the elastic modulus (G′), gel strength, and gel water holding capacity (P < 0.05). According to low field nuclear magnetic resonance analysis, the T₂₁ and T₂₂ of the UF-165 MP gels were shorter than those of the AF and IF samples, which meant that the UF-165 reduced the mobility of the immobilized water and free water in MP gel. A scanning electron microscopy analysis showed that the appropriate ultrasonic power promoted the formation of a compact and homogeneous protein gel network. These results suggested that the appropriate ultrasonic power maintained the MP structure and reduced the loss of gel quality.     

Effect of acoustical damping with a porous absorptive layer in the cavity to reduce the structure-borne sound radiation from a double-leaf structure

Structure-borne sound radiation from a double-leaf structure with a porous absorptive layer in the cavity is studied theoretically as well as experimentally. The study is for establishing a countermeasure to reduce the structure-borne noise radiated from an interior leaf into rooms and for clarifying its reduction effect. The sound field radiated from a double-leaf elastic plate with layers of arbitrary media in the cavity set into vibration by a point force excitation is theoretically analyzed. The effect of the bulk vibration of an absorptive layer is also considered by a simple model into the present theory. Radiation reduction of an inner-layer derived from the theory is experimentally validated. Parametric studies reveal that increasing the ratio of an absorptive layer thickness to the cavity depth is effective to reduce the structure-borne sound radiation but high flow resistivity of the absorbent material is not necessarily required. A practical equation to predict the mass-air-mass resonance frequency for absorbent cavity case is given in a simple form.     

Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings

Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using high-resolution NMR methods when the molecules are dissolved in dilute liquid crystalline media. The resulting couplings can in principle be used to constrain the relative orientation of molecular fragments in macromolecular systems to build a complete structure. However, determination of relative fragment orientations based on a single set of residual dipolar couplings is inherently hindered by the multi-valued nature of the angular dependence of the dipolar interaction. Even with unlimited dipolar data, this gives rise to a fourfold degeneracy in fragment orientations. In this Communication, we demonstrate a procedure based on an order tensor analysis that completely removes this degeneracy by combining residual dipolar coupling measurements from two alignment media. Application is demonstrated on (15)N-(1)H residual dipolar coupling data acquired on the protein zinc rubredoxin from Clostridium pasteurianum dissolved in two different bicelle media.