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.     

X-ray diffraction photographs of chicken gizzard G-actin.DNase I complex crystals taken with synchrotron radiation

X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 degrees oscillation range were recorded on a single film up to 3.5 A resolution.     

The impact of AlphaFold Protein Structure Database on the fields of life sciences

Arguably, 2020 was the year of high-accuracy protein structure predictions, with AlphaFold 2.0 achieving previously unseen accuracy in the Critical Assessment of Protein Structure Prediction (CASP). In 2021, DeepMind and EMBL-EBI developed the AlphaFold Protein Structure Database to make an unprecedented number of reliable protein structure predictions easily accessible to the broad scientific community. We provide a brief overview and describe the latest developments in the AlphaFold database. We highlight how the fields of data services, bioinformatics, structural biology, and drug discovery are directly affected by the influx of protein structure data. We also show examples of cutting-edge research that took advantage of the AlphaFold database. It is apparent that connections between various fields through protein structures are now possible, but the amount of data poses new challenges. Finally, we give an outlook regarding the future direction of the database, both in terms of data sets and new functionalities.     

Protein purification and preliminary crystallographic analysis of human Lyn tyrosine kinase

Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2 Å with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.     

Protein crystal microspectrophotometry

Single crystal microspectrophotometry has emerged as a valuable technique for monitoring molecular events that take place within protein crystals, thus tightly coupling structure to function. Absorption and fluorescence spectra, ligand binding affinities and kinetic constants can be determined, allowing i) the definition of the experimental conditions for X-ray crystallography experiments and their interpretation, ii) the assessment of whether crystal lattice forces have altered conformational equilibria, iii) the comparison with data obtained in solution. Microspectrophotometric measurements using oriented crystals and linearly polarized light are carried out usually off-line with respect to X-ray data collection and are aimed at an in- depth characterization of protein function in the crystal, leading to robust structure-function relationships. The power of this approach is highlighted by reporting a few case studies, including hemoglobins, pyridoxal 5'-phosphate-dependent enzymes and acetylcholinesterases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Crystallization and preliminary X-ray crystallographic analysis of PAS factor from Vibrio vulnificus

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor-diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a=56.1, b=74.4, c=80.0 A, a=b=g=90 degrees. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was 1.9 A using synchrotron X-ray irradiation at beam line BL-18B at the Photon Factory, Japan.     

X-ray fluorescence analysis using synchrotron radiation at the photon factory

X-ray fluorescence experiments at the Photon Factory in Japan are described. An energy-dispersive X-ray fluorescence system was combined with various excitation modes, i.e., a continuum and a monochromatic excitation, which consist of a crystal monochromator or a wide band pass monochromator. These excitation modes provide a wide range of band width and photon flux of excitation beams. Minimum detection limits obtained for a thin sample were less than 0.1 ppm and 0.1 pg when there was no line interference. Advantages of using monochromatic excitation are discussed, with emphasis on the possibility of chemical state analysis. Grazing incidence X-ray fluorescence is a technique very appropriate to synchrotron radiation characteristics. Near-surface analysis and trace analysis of solution samples placed on a total reflection support were made. Future plans are discussed, including microbeam analysis, tomography, X-ray excited optical fluorescence, and applications of insertion devices (undulator and wiggler).     

Protein Circular Dichroism Data Bank (PCDDB): data bank and website design

The Protein Circular Dichroism Data Bank (PCDDB) is a new deposition data bank for validated circular dichroism spectra of biomacromolecules. Its aim is to be a resource for the structural biology and bioinformatics communities, providing open access and archiving facilities for circular dichroism and synchrotron radiation circular dichroism spectra. It is named in parallel with the Protein Data Bank (PDB), a long-existing valuable reference data bank for protein crystal and NMR structures. In this article, we discuss the design of the data bank structure and the deposition website located at http://pcddb.cryst.bbk.ac.uk. Our aim is to produce a flexible and comprehensive archive, which enables user-friendly spectral deposition and searching. In the case of a protein whose crystal structure and sequence are known, the PCDDB entry will be linked to the appropriate PDB and sequence data bank files, respectively. It is anticipated that the PCDDB will provide a readily accessible biophysical catalogue of information on folded proteins that may be of value in structural genomics programs, for quality control and archiving in industrial and academic labs, as a resource for programs developing spectroscopic structural analysis methods, and in bioinformatics studies.     

Evaluating protein structures determined by structural genomics consortia

Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X-ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely-used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283-291), MolProbity (Lovell et al., Proteins 2003;50:437-450), Verify3D (Luthy et al., Nature 1992;356:83-85), ProsaII (Sippl, Proteins 1993;17: 355-362), the PDB validation software, and various structure-validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness-of-fit between structures and experimental data, and knowledge-based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site-specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5-year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher-resolution X-ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this "structure quality score gap" between NMR and X-ray crystal structures are discussed.     

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.     

Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones

Background  

The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia colihost cells.     

Protein crystallography microdiffraction

Protein microdiffraction using monochromatic beams is becoming a routine tool at third-generation synchrotron radiation sources. Beam sizes have reached the scale of about 5 microm, with illuminated crystal volumes of approximately 500 microm3, as shown for the case of bovine rhodopsin, which was refined to a resolution of 2.6 A. Progress in X-ray optical systems and instrumentation will enable the method to be extended to smaller beams and smaller crystal volumes.     

A crystallographic and spectroscopic study on the effect of X-ray radiation on the crystal structure of Melanocarpus albomyces laccase

Laccases (p-diphenol dioxygen oxidoreductases) belong to the family of blue multicopper oxidases, which catalyse the four-electron reduction of dioxygen to water concomitantly through the oxidation of substrate molecules. Blue multicopper oxidases have four coppers, a copper (T1) forming a mononuclear site and a cluster of three coppers (T2, T3, and T3') forming a trinuclear site. Because X-rays are known to liberate electrons during data collection and may thus affect the oxidation state of metals, we have investigated the effect of X-ray radiation upon the crystal structure of a recombinant laccase from Melanocarpus albomyces through the use of crystallography and crystal absorption spectroscopy. Two data sets with different strategies, a low and a high-dose data set, were collected at synchrotron. We have observed earlier that the trinuclear site had an elongated electron density amidst coppers, suggesting dioxygen binding. The low-dose synchrotron structure showed similar elongated electron density, but the high-dose X-ray radiation removed the bulk of this density. Therefore, X-ray radiation could alter the active site of laccase from M. albomyces. Absorption spectra of the crystals (320, 420, and 590nm) during X-ray radiation were measured at a home laboratory. Spectra clearly showed how that the band at 590nm had vanished, resulting from the T1 copper being reduced, during the long X-ray measurements. The crystal colour changed from blue to colourless. Absorptions at 320 and 420nm seemed to be rather permanent. The absorption at 320nm is due to the T3 coppers and it is proposed that absorption at 420nm is due to the T2 copper when dioxygen or a reaction intermediate is close to this copper.     

ISPyB: an information management system for synchrotron macromolecular crystallography

MOTIVATION: Individual research groups now analyze thousands of samples per year at synchrotron macromolecular crystallography (MX) resources. The efficient management of experimental data is thus essential if the best possible experiments are to be performed and the best possible data used in downstream processes in structure determination pipelines. Information System for Protein crystallography Beamlines (ISPyB), a Laboratory Information Management System (LIMS) with an underlying data model allowing for the integration of analyses down-stream of the data collection experiment was developed to facilitate such data management. RESULTS: ISPyB is now a multisite, generic LIMS for synchrotron-based MX experiments. Its initial functionality has been enhanced to include improved sample tracking and reporting of experimental protocols, the direct ranking of the diffraction characteristics of individual samples and the archiving of raw data and results from ancillary experiments and post-experiment data processing protocols. This latter feature paves the way for ISPyB to play a central role in future macromolecular structure solution pipelines and validates the application of the approach used in ISPyB to other experimental techniques, such as biological solution Small Angle X-ray Scattering and spectroscopy, which have similar sample tracking and data handling requirements.     

Synthesis,crystallization and preliminary crystallographic analysis of a 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with a substrate

A 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with its substrate was synthesized, purified and crystallized by the hanging-drop method using 0.8 M sodium potassium tartrate as a precipitant. A data set to 1.21 Å resolution was collected from a monocrystal at 100 K using synchrotron radiation on a beamline BL14.1 at BESSY. The crystal belonged to the P2₁ group with unit-cell a = 49.42, b = 24.69, c = 50.23, β = 118.48.     

3D complex: a structural classification of protein complexes

Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.     

Preparation and X-ray crystallographic analysis of rubredoxin crystals from Desulfovibrio gigas to beyond ultra-high 0.68 A resolution

Rubredoxin (D.g. Rd), a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas, has been crystallized using the hanging-drop vapor diffusion method and macroseeding method. Rubredoxin crystals diffract to an ultra-high resolution 0.68 A using synchrotron radiation X-ray, and belong to the space group P2(1) with unit-cell parameters a=19.44 A, b=41.24 A, c=24.10 A, and beta=108.46 degrees. The data set of single-wavelength anomalous dispersion signal of iron in the native crystal was also collected for ab initio structure re-determination. Preliminary analysis indicates that there is one monomer with a [Fe-4S] cluster in each asymmetric unit. The crystal structure at this ultra-high resolution will reveal the details of its biological function. The crystal character and data collection strategy for ultra-high resolution will also be discussed.     

Protein structure determination using a database of interatomic distance probabilities

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available.     

Atomic structure of a CK2alpha human kinase by microfocus diffraction of extra-small microcrystals grown with nanobiofilm template

Extra-small microcrystals of a human kinase CK2alpha were obtained for the first time by the optimization of a recent protein crystallization method based on highly packed protein nanofilm template. Protein crystal induction and growth appear indeed optimal at high surface pressure of the film template yielding high protein orientation and packing. The resulting extra-small CK2alpha microcrystals (of about 20 microm in diameter) was subsequently used for synchrotron radiation diffraction data collection, which proves possible by means of the Microfocus Beamline at the ESRF Synchrotron in Grenoble. The quality of the resulting crystal diffraction patterns and of its resulting atomic structure at 2.4 A resolution proves the unique validity of the above two combined frontier technologies in defining a new approach to structural proteomics capable to solve the atomic structure of proteins so far never been crystallized and of pharmaceutical relevance. Physical explanation in terms of template dipole moments and possibility of generalization of this method to the wide class of proteins not yet crystallized are finally discussed. The structure of our CK2alpha mutant is in the Protein Data Bank (PDB ID Code 1NA7, deposited on 27 November 2002).