'Cool' crystals: macromolecular cryocrystallography and radiation damage

Macromolecular crystals commonly suffer rapid radiation damage during room temperature X-ray data collection. Therefore, data are now routinely collected with the sample held at around 100K, significantly reducing secondary radiation damage, and usually resulting in higher resolution and better quality data. At synchrotron sources, the frequent observation of radiation damage even at cryotemperatures has prompted the development of exciting new experiments aimed at characterising and reducing this damage, and using it for structure determination and enzymatic studies. Current research into cryotechniques seeks to understand the basic physical and chemical processes involved in flash-cooling and radiation damage, which should eventually enable the rational optimisation of cryoprotocols.     

Approaching the molecular structure of ribosomes

Fifteen forms of three-dimensional crystals and three forms of two-dimensional sheets from ribosomal particles have been grown. In all cases only biologically active particles could be crystallized, the crystalline material retaining its integrity and biological activity for months. Cryastallographic data have been collected from crystals of 50 S ribosomal subunits, using synchrotron radiation, at temperatures between 19 and -180 degree C. Although at around 0 degrees C in the synchrotron X-ray beam the crystals rapidly lose their high-resolution reflections, at cryo-temperatures hardly any radiation damage occurs over long periods, and a complete set of diffraction data to about 6 A resolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50 S ribosomal subunits from a mutant of Bacillus stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with the native form. Models of the entire 70 S ribosome and of the 50 S subunit have been reconstructed from two-dimensional sheets at 47 and 30 A, respectively. These models demonstrate the overall shape of the particles, the contact areas between large and small subunits, the space where protein biosynthesis may take place and a tunnel through the 50 S subunit which could provide a path for the nascent polypeptide chain.     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

The road to the crystal structure of the cytochrome bc 1 complex from the anoxigenic, photosynthetic bacterium Rhodobacter sphaeroides

The advantages of using bacterial systems to study the mechanism and function of cytochrome bc (1) complexes do not extend readily to their structural investigations. High quality crystals of bacterial complexes have been difficult to obtain despite the enzymes' smaller sizes and simpler subunit compositions compared to their mitochondrial counterparts. In the course of the structure determination of the bc (1) complex from R. sphaeroides, we observed that the growth of only low quality crystals correlated with low activity and stability of the purified complex, which was mitigated in part by introducing a double mutations to the enzyme. The S287R(cyt b)/V135S(ISP) mutant shows 40% increase in electron transfer activity and displays a 4.3 degrees C increase in thermal stability over wild-type enzyme. The amino acid histidine was found important in maintaining structural integrity of the bacterial complex, while the respiratory inhibitors such as stigmatellin are required for immobilization of the iron-sulfur protein extrinsic domain. Crystal quality of the R. sphaeroides bc (1) complex can be improved further by the presence of strontium ions yielding crystals that diffracted X-rays to better than 2.3 A resolution. The improved crystal quality can be understood in terms of participation of strontium ions in molecular packing arrangement in crystal.     

Strategies for crystallization and structure determination of very large macromolecular assemblies

High-resolution structures of macromolecular assemblies are pivotal for our understanding of their biological functions in fundamental cellular processes. In the field of X-ray crystallography, recent methodological and instrumental advances have led to the structure determinations of macromolecular assemblies of increased size and complexity, such as those of ribosomal complexes, RNA polymerases, and large multifunctional enzymes. These advances include the use of robotic screening techniques that maximize the chances of obtaining well-diffracting crystals of large complexes through the fine sampling of crystallization space. Sophisticated crystal optimization and cryoprotection techniques and the use of highly brilliant X-ray beams from third-generation synchrotron light sources now allow data collection from weakly diffracting crystals with large asymmetric units. Combined approaches are used to derive phase information, including phases calculated from electron microscopy (EM) models, heavy atom clusters, and density modification protocols. New crystallographic software tools prove valuable for structure determination and model refinement of large macromolecular complexes.     

How does radiation damage in protein crystals depend on X-ray dose?

Is radiation damage to cryopreserved protein crystals strictly proportional to accumulated dose at the high-flux density of beams from undulators at third-generation synchrotron sources? The answer is "yes," for overall damage to several different kinds of protein crystals at flux densities up to 10(15) ph/sec/mm(2) (APS beamline 19-ID). We find that, at 12 keV (1 A wavelength), about ten absorbed photons are sufficient to "kill" a unit cell. As this corresponds to about one elastically scattered photon, each unit cell can contribute only about one photon to total Bragg diffraction. The smallest crystal that can yield a full data set to 3.5 A resolution has a diameter of about 20 microm (100 A unit cell).     

Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside

The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.     

Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae

Pilus fibers are long protein filaments on many pathogenic bacteria that participate in attachment to host cells. Although the self-assembling protein pilin is the major structural component of the Neisseria gonorrhoeae pilus fiber, several other proteins co-purified with pilin through the repeated solubilization-reassociation steps of the biochemical purification. Pilin solubilized in the nondenaturing detergent n-octyl-beta-D-glucopyranoside remained an aggregate of about 100 kDa at pH 9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly involves electrostatic interactions of lysine, tyrosine, or other side chains with high pKa values. Pilin dimers and aggregates of higher molecular mass were partially stable even in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Removal of pilus-associated proteins and stabilization of pilin multimers permitted the reproducible crystallization of pilin. Three-dimensional needle- and plate-shaped crystals of purified N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40% polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell dimensions a = 126.4, b = 121.2, c = 26.7 A and Vm = 2.84 A3/dalton for one molecule per asymmetric unit. The best crystals diffracted to 2.4 A resolution using synchrotron radiation, were stable to x-ray damage, and appear suitable for determination of the atomic structure. This approach of stabilizing and crystallizing an intermediate assembly state may be useful for other fiber-forming proteins, which have previously not been successfully crystallized in forms that diffract to atomic resolution.     

Three dimensional structure of bacterial pili

Crystallographic and associated biochemical and structural studies are in progress on the fiber-forming pilin proteins of the gonococcal pilus. Preparative scale purification procedures have been developed for the gonococcal pilin protein, which appear generally applicable to bacterial pilins. For three gonococcal pilin protein strains, we have obtained both reassembled pilus fibers and three-dimensional crystals. One needle-shaped crystal form of gonococcal C30 pilin diffracts beyond 3 Å resolution using synchrotron x-ray radiation. A diffraction data set to 3.5 Å resolution has been collected on these needle-shaped crystals (lattice spacings a=125.4(3) b=120.4(3), c=26.61(4) Å) in which the packing arrangement of the pilin subunits appears to resemble that seen in the pilus fibers using electron microscopy. X-ray diffraction data confirm our proposed model for the overall polypeptide fold of a pilin subunit, which is an antiparallel 4- helix bundle similar to tobacco mosaic virus coat protein and myohemerythrin.     

Crystallization and preliminary x-ray analysis of complexes of porcine pancreatic elastase with two natural inhibitors

Two different natural protease inhibitors, the squash inhibitor MCEI III and the third domain of turkey ovomucoid inhibitor OMTKY3, were crystallized in complexes with porcine pancreatic elastase (PPE). About 700 conditions were screened altogether. Crystals of the complex between MCEI III and PPE were grown in citrate buffer with and without ammonium acetate. X-ray diffraction data were collected to 1.9 angstroms resolution at room temperature using synchrotron radiation. The crystals belong to space group P2(1), with unit-cell parameters a = 49.17, b = 44.59, c = 67.08 angstroms, beta = 110.97 degrees. Crystals of the OMTKY3/PPE complex were obtained in the presence of ammonium sulfate, MES buffer and polyethylene glycol monomethylether (PEG). These crystals of this complex diffracted to 2.1 angstroms resolution and belongs to space group I222, with unit-cell parameters a = 84.58, b = 84.61, c = 89.92 angstroms and diffracted to 2.2 angstroms resolution. The diffraction data were collected using a conventional rotating anode X-ray generator at room temperature. In both cases the presence of inhibitor in the crystals was confirmed by crystallography.     

Monitoring and validating active site redox states in protein crystals

High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Towards atomic resolution with crystals grown in gel: the case of thaumatin seen at room temperature

One reason for introducing a gel in the crystallization medium of proteins is its ability to reduce convection in solution. This can lead to better nucleation and growth conditions, and to crystals having enhanced diffraction properties. We report here the X-ray characterization at room temperature of high-quality crystals of the intensely sweet thaumatin prepared in a sodium tartrate solution gelified with 0.15% (m/v) agarose. Using a synchrotron radiation, these crystals diffracted to a previously unachieved resolution. A diffraction dataset was collected from four crystals at a resolution of 1.2 A with a R(sym) of 3.6% and a completeness of 99%. Refinement was carried out to a final crystallographic R-factor of 12.0%. The quality of the electron density map allowed for the observation of fine structural details in the protein and its solvation shell. Crystallization in gel might be used more generally to improve the quality of macromolecular crystals. Advantages provided by the gelified medium in the frame of structural studies are emphasized.     

Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin

The crystal structure of mistletoe lectin I (ML-I) isolated from the European mistletoe Viscum album in complex with the most active phytohormone zeatin has been analyzed and refined to 2.54 A resolution. X-ray suitable crystals of ML-I were obtained by the counter-diffusion method using the Gel-Tube R crystallization kit (GT-R) onboard the Russian Service Module on the international space station ISS. High quality hexagonal bipyramidal crystals were grown during 3 months under microgravity conditions. Selected crystals were soaked in a saturated solution of zeatin and subsequently diffraction data were collected applying synchrotron radiation. A distinct F(o)-F(c) electron density has been found inside a binding pocket located in subunit B of ML-I and has been interpreted as a single zeatin molecule. The structure was refined to investigate the zeatin-ML-I interactions in detail. The results demonstrate the ability of mistletoe to protect itself from the host transpiration regulation by absorbing the most active host plant hormones as part of a defense mechanism.     

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.     

Crystallization of halorhodopsin from Halobacterium sp. shark

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.     

Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.

The major signalling entity of the receptors for the haemopoietic cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is the shared beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the beta(c) subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N-terminal sequence. N-glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X-ray crystallographic studies, following mutagenesis to remove one of the N-glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 A and 103.5 A, diffracted to a resolution of 2.9 A using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.     

自由电子激光在生物学中的应用

同步辐射的发展和应用已经极大的推动了自然科学包括生物学的巨大发展,其中结构生物学更是离不开X射线衍射分析,小角散射等。X射线自由电子激光(XFEL)相比同步辐射具有更高强度,完全相干等特点,被称为第四代光源。科学家已经利用XFEL实现了尺度约为1微米的蛋白质晶体的高分辨率结构解析,并且也实现了单颗粒的病毒的低分辨重构。未来,XFEL将会为生物学的发展打开一扇新的大门。     

Molecular structure of flavocytochrome b2 at 2.4 A resolution

The crystal structure of flavocytochrome b2 has been solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering. Area detector data from native and two heavy-atom derivative crystals were used. The phases were refined by the B.C. Wang phase-filtering procedure utilizing the 67% (v/v) solvent content of the crystals. A molecular model was built first on a minimap and then on computer graphics from a combination of maps both averaged and not averaged about the molecular symmetry axis. The structure was extended to 2.4 A resolution using film data recorded at a synchrotron and refined by the Hendrickson-Konnert procedure. The molecule, a tetramer of Mr 230,000, is located on a crystallographic 2-fold axis and possesses local 4-fold symmetry. Each subunit is composed of two domains, one binding a heme and the other an FMN prosthetic group. In subunit 1, both the cystochrome and the flavin-binding domain are visible in the electron density map. In subunit 2 the cytochrome domain is disordered. However, in the latter, a molecule of pyruvate, the product of the enzymatic reaction, is bound at the active site. The cytochrome domain consists of residues 1 to 99 and is folded in a fashion similar to the homologous soluble fragment of cytochrome b5. The flavin binding domain contains a parallel beta 8 alpha 8 barrel structure and is composed of residues 100 to 486. The remaining 25 residues form a tail that wraps around the molecular 4-fold axis and is in contact with each remaining subunit. The FMN moiety, which is located at the C-terminal end of the central beta-barrel, is mostly sequestered from solvent; it forms hydrogen bond interactions with main- and side-chain atoms from six of the eight beta-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.