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1.
    
Radiation damage is still the most limiting factor in obtaining high‐resolution structures of macromolecules in crystallographic experiments at synchrotrons. With the advent of X‐ray free‐electron lasers (XFELs) that produce ultrashort and highly intense X‐ray pulses, it became possible to outrun most of the radiation‐damage processes occurring in the sample during exposure to XFEL radiation. Although this is generally the case, several experimental and theoretical studies have indicated that structures from XFELs may not always be radiation‐damage free. This is especially true when higher intensity pulses are used and protein molecules that contain heavy elements in their structures are studied. Here, the radiation‐damage mechanisms that occur in samples exposed to XFEL pulses are summarized, results that show indications of radiation damage are reviewed and methods that can partially overcome it are discussed.  相似文献   

2.
    
A liquid‐droplet injector has been developed that delivers pristine microcrystals to an X‐ray irradiation area for conducting serial femtosecond crystallography (SFX) with an X‐ray free‐electron laser (XFEL). By finely tuning the pulsed liquid droplets in time and space, a high hit rate of the XFEL pulses to microcrystals in the droplets was achieved for measurements using 5 µm tetragonal lysozyme crystals, which produced 4265 indexable diffraction images in about 30 min. The structure was determined at a resolution of 2.3 Å from <0.3 mg of protein. With further improvements such as reduction of the droplet size, liquid droplets have considerable potential as a crystal carrier for SFX with low sample consumption.  相似文献   

3.
    
During the last decade, the number of three‐dimensional structures solved by X‐ray crystallography has increased dramatically. By 2014, it had crossed the landmark of 100 000 biomolecular structures deposited in the Protein Data Bank. This tremendous increase in successfully crystallized proteins is primarily owing to improvements in cloning strategies, the automation of the crystallization process and new innovative approaches to monitor crystallization. However, these improvements are mainly restricted to soluble proteins, while the crystallization and structural analysis of membrane proteins or proteins that undergo major post‐translational modifications remains challenging. In addition, the need for relatively large crystals for conventional X‐ray crystallography usually prevents the analysis of dynamic processes within cells. Thus, the advent of high‐brilliance synchrotron and X‐ray free‐electron laser (XFEL) sources and the establishment of serial crystallography (SFX) have opened new avenues in structural analysis using crystals that were formerly unusable. The successful structure elucidation of cathepsin B, accomplished by the use of microcrystals obtained by in vivo crystallization in baculovirus‐infected Sf9 insect cells, clearly proved that crystals grown intracellularly are very well suited for X‐ray analysis. Here, methods by which in vivo crystals can be obtained, isolated and used for structural analysis by novel highly brilliant XFEL and synchrotron‐radiation sources are summarized and discussed.  相似文献   

4.
    
Recent advances in X‐ray free‐electron laser (XFEL) sources have permitted the study of protein dynamics. Femtosecond X‐ray pulses have allowed the visualization of intermediate states in enzyme catalysis. In this study, the growth of carbonic anhydrase II microcrystals (40–80 µm in length) suitable for the collection of XFEL diffraction data at the Pohang Accelerator Laboratory is demonstrated. The crystals diffracted to 1.7 Å resolution and were indexed in space group P21, with unit‐cell parameters a = 42.2, b = 41.2, c = 72.0 Å, β = 104.2°. These preliminary results provide the necessary framework for time‐resolved experiments to study carbonic anhydrase catalysis at XFEL beamlines.  相似文献   

5.
    
Recent experiments in serial femtosecond crystallography (SFX) have demonstrated the feasibility of obtaining structural information from nanoscale crystals using X‐ray free‐electron lasers (XFELs). However, millions of crystals are required to determine one reliable structure. Here, an improved integration algorithm for SFX data processing is reported. By evaluating the dimensions of each crystal and correcting for the geometric factors of single patterns, the effective diffraction intensities, as opposed to the directly measured single‐shot pattern diffraction intensities, can be merged to acquire more accurate integrated intensities which can be used for structure determination. This improvement enhances the quality of electron‐density maps and decreases the number of diffraction patterns that are needed to solve the crystal structure in SFX experiments.  相似文献   

6.
    
This article provides a step‐by‐step guide to the use of the CrystFEL software for processing serial crystallography data from an X‐ray free‐electron laser or a synchrotron light source. Whereas previous papers have described the theory and algorithms and their rationale, this paper describes the steps to be performed from a user perspective, including command‐line examples.  相似文献   

7.
    
X‐ray crystallography promises direct insights into electron‐density changes that lead to and arise from structural changes such as electron and proton transfer and the formation, rupture and isomerization of chemical bonds. The ultrashort pulses of hard X‐rays produced by free‐electron lasers present an exciting opportunity for capturing ultrafast structural events in biological macromolecules within femtoseconds after photoexcitation. However, shot‐to‐shot fluctuations, which are inherent to the very process of self‐amplified spontaneous emission (SASE) that generates the ultrashort X‐ray pulses, are a major source of noise that may conceal signals from structural changes. Here, a new approach is proposed to angularly split a single SASE pulse and to produce a temporal delay of picoseconds between the split pulses. These split pulses will allow the probing of two distinct states before and after photoexcitation triggered by a laser pulse between the split X‐ray pulses. The split pulses originate from a single SASE pulse and share many common properties; thus, noise arising from shot‐to‐shot fluctuations is self‐canceling. The unambiguous interpretation of ultrafast structural changes would require diffraction data at atomic resolution, as these changes may or may not involve any atomic displacement. This approach, in combination with the strategy of serial crystallography, offers a solution to study ultrafast dynamics of light‐initiated biochemical reactions or biological processes at atomic resolution.  相似文献   

8.
    
In principle, serial femtosecond crystallography (SFX) could yield data sets that are completely free of the effects caused by slow, radiation‐induced chemical reactions, for example, oxygen additions, responsible for radiation damage. However, experimental evidence is presented here that SFX data sets obtained by techniques that expose different parts of the same specimen to single pulses of radiation do not have this property, even if the specimen in question is frozen. The diffraction image of each such crystal obtained with the first pulse of radiation is certain to represent the structure of a protein that has not been modified chemically, but all of the images obtained subsequently from the same crystal will represent structures that have been modified to a lesser or greater extent by oxygen additions because of the rapid diffusion of oxygenic free radicals through the specimen. The higher the level of oxygen additions a crystal suffers during data collection, the poorer the statistical quality of data set obtained from it will, and the higher the free R‐factors of the resulting structural model.  相似文献   

9.
    
Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.  相似文献   

10.
    
The additions of oxygen and peroxide to residues that result when proteins are exposed to the free radicals produced using the Fenton reaction or X‐rays have been studied for over a century. Nevertheless little is known about the impact these modifications have on protein crystal structures. Here evidence is presented that both kinds of modifications occur in protein crystals on a significant scale during the collection of X‐ray diffraction data. For example, at least 538 of the 5,351 residues of protein molecules in the crystal used to obtain the structure for photosystem II described by the PDB accession number 3ARC became oxygenated during data collection.  相似文献   

11.
    
The high peak brilliance and femtosecond pulse duration of X‐ray free‐electron lasers (XFELs) provide new scientific opportunities for experiments in physics, chemistry and biology. In structural biology, one of the major applications is serial femtosecond crystallography. The intense XFEL pulse results in the destruction of any exposed microcrystal, making serial data collection mandatory. This requires a high‐throughput serial approach to sample delivery. To this end, a number of such sample‐delivery techniques have been developed, some of which have been ported to synchrotron sources, where they allow convenient low‐dose data collection at room temperature. Here, the current sample‐delivery techniques used at XFEL and synchrotron sources are reviewed, with an emphasis on liquid injection and high‐viscosity extrusion, including their application for time‐resolved experiments. The challenges associated with sample delivery at megahertz repetition‐rate XFELs are also outlined.  相似文献   

12.
    
A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X‐ray crystallographic studies, has been developed. The technology described here is compatible both with X‐ray and neutron cryo‐crystallography. The mounts have low density and low background scattering for both neutrons and X‐rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom‐design the mount to specific crystal sizes and large‐scale production.  相似文献   

13.
    
The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP‐1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester‐peptide segment was synthesized using the sulfonamide safety‐catch linker and 9‐fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full‐length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP‐1 cells, as expected. CCL2 was crystallized and the structure was determined by X‐ray diffraction at 1.9‐Å resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9‐Thr10‐Cys11, which form an intersubunit antiparallel β‐sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety‐catch linker and NCL. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 350–359, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com  相似文献   

14.
    
μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP‐μNS(448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP‐μNS(448–605) baculovirus formed rod‐shaped microcrystals (5–15 µm in length) which were reconstituted in high‐viscosity media (LCP and agarose) and investigated by serial femtosecond X‐ray diffraction using viscous jets at an X‐ray free‐electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit‐cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron‐density maps were obtained. While more diffraction data are required to solve the structure of μNS(448–605), the current experimental strategy, which couples high‐viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.  相似文献   

15.
    
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16.
    
Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol α, Pol δ and Pol . The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol α. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol α reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B–CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.  相似文献   

17.
    
Apoptosis is a key innate defence mechanism to eliminate virally infected cells. To counteract premature host‐cell apoptosis, poxviruses have evolved numerous molecular strategies, including the use of Bcl‐2 proteins, to ensure their own survival. Here, it is reported that the Deerpox virus inhibitor of apoptosis, DPV022, only engages a highly restricted set of death‐inducing Bcl‐2 proteins, including Bim, Bax and Bak, with modest affinities. Structural analysis reveals that DPV022 adopts a Bcl‐2 fold with a dimeric domain‐swapped topology and binds pro‐death Bcl‐2 proteins via two conserved ligand‐binding grooves found on opposite sides of the dimer. Structures of DPV022 bound to Bim, Bak and Bax BH3 domains reveal that a partial obstruction of the binding groove is likely to be responsible for the modest affinities of DPV022 for BH3 domains. These findings reveal that domain‐swapped dimeric Bcl‐2 folds are not unusual and may be found more widely in viruses. Furthermore, the modest affinities of DPV022 for pro‐death Bcl‐2 proteins suggest that two distinct classes of anti‐apoptotic viral Bcl‐2 proteins exist: those that are monomeric and tightly bind a range of death‐inducing Bcl‐2 proteins, and others such as DPV022 that are dimeric and only bind a very limited number of death‐inducing Bcl‐2 proteins with modest affinities.  相似文献   

18.
    
In serial crystallography, a very incomplete partial data set is obtained from each diffraction experiment (a `snapshot'). In some space groups, an indexing ambiguity exists which requires that the indexing mode of each snapshot needs to be established with respect to a reference data set. In the absence of such re‐indexing information, crystallographers have thus far resorted to a straight merging of all snapshots, yielding a perfectly twinned data set of higher symmetry which is poorly suited for structure solution and refinement. Here, two algorithms have been designed for assembling complete data sets by clustering those snapshots that are indexed in the same way, and they have been tested using 15 445 snapshots from photosystem I [Chapman et al. (2011), Nature (London), 470 , 73–77] and with noisy model data. The results of the clustering are unambiguous and enabled the construction of complete data sets in the correct space group P63 instead of (twinned) P6322 that researchers have been forced to use previously in such cases of indexing ambiguity. The algorithms thus extend the applicability and reach of serial crystallography.  相似文献   

19.
Walkiewicz K  Davlieva M  Wu G  Shamoo Y 《Proteins》2011,79(7):2335-2340
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20.
    
Recent research has identified a potential role of the hyaluronic acid receptor stabilin‐2 (Stab2) in cancer metastasis. Stab2 belongs to a group of scavenger receptors and is responsible for the clearance of more than ten ligands, including hyaluronic acid (HA). In vivo experiments on mice have shown that the absence of Stab2, or its blocking by an antibody, effectively opposes cancer metastasis, which is accompanied by an increase in the level of circulating HA. Knowledge of ligand recognition and signal transduction by Stab2 is limited and no three‐dimensional structures of any protein fragments of this receptor have been solved to date. Here, a high‐resolution X‐ray structure of the seventh FAS1 domain of Stab2 is reported. This structure provides the first insight into the Stab2 structure.  相似文献   

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