Biomedical subjects

Raimond B G Ravelli

Publications and source records attributed to Raimond B G Ravelli.

At least 19 recordsLinked to original sources

PubMed2006

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.

Adenosine Diphosphate↗

PubMed2006

Plastic-embedded protein crystals.

Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plastic-embedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.

Acrylic Resins↗

PubMed2006

X-ray radiation-induced damage in DNA monitored by online Raman.

High-quality Raman spectra have been collected on single DNA crystals kept at 100 K in a flow of cold nitrogen gas prior, during and after macromolecular crystallography (MX) data collection. The non-symmetric bending vibration for C-Br bonds in 8-bromo-2'-deoxyguanosine has been unambiguously identified at 293 cm(-1) in the non-resonant Raman spectra. The breakage of this bond could be monitored by the continuous recording of Raman spectra during X-ray exposure, and its decay versus dose could be directly correlated with the loss of the bromide definition within the electron-density maps as determined with MX.

Computer Simulation↗

PubMed2006

C3D: a program for the automated centring of cryocooled crystals.

A method is presented to automatically locate a crystal and its holder for centring on a goniometer spindle and alignment with an X-ray beam. Here, a novel algorithm that has been developed and tested with the images of users' crystals saved in an annotated database is described. The algorithm improves on the difficult situations that are commonly observed and poorly handled by the first-generation crystal-centring algorithms. These include highly transparent crystals, bad cryocooling or lens effects arising from the geometry of the drop. Most crystals have polyhedral shapes and a number of straight edges, which yield useful information. In this method, crystal detection relies on a feature-scoring system in which line extraction has the highest weight. Here, the image processing and calculations implemented in the program C3D are described. This program is designed to operate with a client program that controls specific diffractometer hardware. In order to select the best detection conditions, C3D provides various functionalities adapted to various hardware configurations.

Automation↗

PubMed2006

Radiation damage in macromolecular cryocrystallography.

X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing.

Animals↗

PubMed2006

Crystal structure of the rabies virus nucleoprotein-RNA complex.

Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.

Amino Acid Sequence↗

PubMed2006

Is radiation damage dependent on the dose rate used during macromolecular crystallography data collection?

This paper focuses on the radiation-damage effects when applying the same total X-ray dose to protein crystals at different dose rates. These experiments have been performed on both a selenomethionated protein and on bovine trypsin using dose rates that span nearly two orders of magnitude. The results show no clear dose-rate effect on the global indicators of radiation damage, but a small measurable dose-rate effect could be found when studying specific radiation damage. It is hypothesized that this observed dose-rate effect relates to differences in the steady-state free-radical concentration.

Animals↗

PubMed2006

Phasing macromolecular structures with UV-induced structural changes.

Experimental phasing of macromolecular crystal structures relies on the accurate measurement of two or more sets of reflections from isomorphous crystals, where the scattering power of a few atoms is different for each set. Recently, it was demonstrated that X-ray-induced intensity differences can also contain phasing information, exploiting specific structural changes characteristic of X-ray damage. This method (radiation damage-induced phasing; RIP) has the advantage that it can be performed on a single crystal of the native macromolecule. However, a drawback is that X-rays introduce many small changes to both solvent and macromolecule. In this study, ultraviolet (UV) radiation has been used to induce specific changes in the macromolecule alone, leading to a larger contrast between radiation-susceptible and nonsusceptible sites. Unlike X-ray RIP, UV RIP does not require the use of a synchrotron. The method has been demonstrated for a series of macromolecules.

Animals↗

PubMed2005

Improving radiation-damage substructures for RIP.

Specific radiation damage can be used to solve macromolecular structures using the radiation-damage-induced phasing (RIP) method. The method has been investigated for six disulfide-containing test structures (elastase, insulin, lysozyme, ribonuclease A, trypsin and thaumatin) using data sets that were collected on a third-generation synchrotron undulator beamline with a highly attenuated beam. Each crystal was exposed to the unattenuated X-ray beam between the collection of a 'before' and an 'after' data set. The X-ray 'burn'-induced intensity differences ranged from 5 to 15%, depending on the protein investigated. X-ray-susceptible substructures were determined using the integrated direct and Patterson methods in SHELXD. The best substructures were found by downscaling the 'after' data set in SHELXC by a scale factor K, with optimal values ranging from 0.96 to 0.99. The initial substructures were improved through iteration with SHELXE by the addition of negatively occupied sites as well as a large number of relatively weak sites. The final substructures ranged from 40 to more than 300 sites, with strongest peaks as high as 57sigma. All structures except one could be solved: it was not possible to find the initial substructure for ribonuclease A, however, SHELXE iteration starting with the known five most susceptible sites gave excellent maps. Downscaling proved to be necessary for the solution of elastase, lysozyme and thaumatin and reduced the number of SHELXE iterations in the other cases. The combination of downscaling and substructure iteration provides important benefits for the phasing of macromolecular structures using radiation damage.

Animals↗

PubMed2005

Structural basis for the regulation of tubulin by vinblastine.

Vinblastine is one of several tubulin-targeting Vinca alkaloids that have been responsible for many chemotherapeutic successes since their introduction in the clinic as antitumour drugs. In contrast with the two other classes of small tubulin-binding molecules (Taxol and colchicine), the binding site of vinblastine is largely unknown and the molecular mechanism of this drug has remained elusive. Here we report the X-ray structure of vinblastine bound to tubulin in a complex with the RB3 protein stathmin-like domain (RB3-SLD). Vinblastine introduces a wedge at the interface of two tubulin molecules and thus interferes with tubulin assembly. Together with electron microscopical and biochemical data, the structure explains vinblastine-induced tubulin self-association into spiral aggregates at the expense of microtubule growth. It also shows that vinblastine and the amino-terminal part of RB3-SLD binding sites share a hydrophobic groove on the alpha-tubulin surface that is located at an intermolecular contact in microtubules. This is an attractive target for drugs designed to perturb microtubule dynamics by interfacial interference, for which tubulin seems ideally suited because of its propensity to self-associate.

Binding Sites↗

PubMed2005

Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage.

The X-ray susceptibility of the lysine-pyridoxal-5'-phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 A resolution, complemented by on-line spectroscopic studies. X-rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal-5'-phosphate and the Lys residue. Analysis of the "undamaged" structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X-rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 x 10(6) Ggamma. Our data provide new insights into the enzymatic activation of pyridoxal-5'-phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.

Bacillus↗

PubMed2005

Parameters affecting the X-ray dose absorbed by macromolecular crystals.

The lifetime of a macromolecular crystal in an X-ray beam is assumed to be limited by the absorbed dose. This dose, expressed in Gray (Gy = J kg(-1)), is a function of a number of parameters: the absorption coefficients of the constituent atoms of the crystal, the number of molecules per asymmetric unit, the beam energy, flux, size and profile, the crystal size, and the total irradiation time. The effects of these variables on the predicted absorbed dose, calculated using the program RADDOSE, are discussed and are illustrated with reference to the irradiation of a selenomethionine protein crystal of unknown structure. The results of RADDOSE can and will in the future be used to inform the data collection procedure as it sets a theoretical upper limit on the total exposure time at a certain X-ray source. However, as illustrated with an example for which the experimental data are compared with prediction, the actual lifetime of a crystal could become shorter in those cases where specific damage breaks down crucial crystal contacts.

Archaeal Proteins↗

PubMed2005

Phasing in the presence of radiation damage.

In the accurate estimation of small signals, redundancy of observations is often seen as an essential tool for the experimenter. This is particularly true during macromolecular structure determination by single-wavelength anomalous dispersion (SAD), where the exploitable signal can be less than a few percent. At the most intense undulator synchrotron beamlines, the effect of radiation damage can be such that all usable signal is obscured. Here the magnitude of this effect in experiments performed at the Se K-edge is quantified. Six successive data sets were collected on the same crystal, interspersed with two exposures to the X-ray beam during which data were not collected. It is shown that the very first data set has excellent phasing statistics, whereas these statistics degrade for the later data sets. Merging several data sets into one, highly redundant, data set only gave moderate improvements as a result of the presence of radiation damage. Part of the damage could be corrected for using a linear interpolation scheme. Interpolation of the data to a low-dose as well as to a high-dose data set allowed us to combine the SAD method with the radiation-damage induced phasing (RIP) technique, which further improved the experimental phases, especially after density modification. Some recommendations are given on how to mitigate the effect of radiation damage during structure determination.

Bacterial Proteins↗

PubMed2005

X-ray crystallographic and NMR studies of the third KH domain of hnRNP K in complex with single-stranded nucleic acids.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K is implicated in multiple functions in the regulation of gene expression and acts as a hub at the intersection of signaling pathways and processes involving nucleic acids. Central to its function is its ability to bind both ssDNA and ssRNA via its KH (hnRNP K homology) domains. We determined crystal structures of hnRNP K KH3 domain complexed with 15-mer and 6-mer (CTC(4)) ssDNAs at 2.4 and 1.8 A resolution, respectively, and show that the KH3 domain binds specifically to both TCCC and CCCC sequences. In parallel, we used NMR to compare the binding affinity and mode of interaction of the KH3 domain with several ssRNA ligands and CTC(4) ssDNA. Based on a structure alignment of the KH3-CTC(4) complex with known structures of other KH domains in complex with ssRNA, we discuss recognition of tetranucleotide sequences by KH domains.

Amino Acid Sequence↗

PubMed2004

Expression, crystallization and crystallographic analysis of DegS, a stress sensor of the bacterial periplasm.

Regulated proteolysis is a key event in transmembrane signalling between intercellular compartments. In Escherichia coli, a protein DegS has been identified as being a periplasmic stress sensor for unfolded or misfolded outer membrane proteins (OMPs). Activation of DegS initiates a proteolytic cascade which results in the transcription of periplasmic genes under sigmaE control, most importantly chaperones and proteases. DegS has been cloned and expressed as full-length protein and in an N-terminally truncated form. Both proteins were tested for crystallization and two forms of well diffracting crystals of the truncated form were obtained. Crystals of form I diffract to 3.5 A and belong to space group P2(1)3, while crystals of form II diffract to 2.2 A and belong to space group I23. Crystals of form II were soaked with a consensus peptide representing the C-termini of outer membrane proteins and data to 2.4 A resolution were collected. Molecular-replacement trials using a homologous protease domain indicate the presence of two molecules in the asymmetric unit of crystal form I. The correctness of the molecular-replacement solution was verified by identifying radiation-damage-induced structural changes.

Crystallization↗

PubMed2004

Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes.

Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.

Amino Acid Sequence↗

PubMed2004

Crystallization, microPIXE and preliminary crystallographic analysis of the complex between the third KH domain of hnRNP K and single-stranded DNA.

hnRNP K is one of the major proteins found in hnRNP particles which are ribonucleoprotein complexes containing proteins and pre-mRNA. hnRNP K contains hnRNP K homology (KH) domains which bind both CT-rich single-stranded DNA (ssDNA) and CU-rich ssRNA. Co-crystallization of the third KH domain of human hnRNP K with a 15-mer ssDNA gave rod-shaped crystals belonging to the trigonal space group P3(1)21 (unit-cell parameters a = 54.0, c = 149.7 A) and diffracting to 2.4 A resolution. MicroPIXE (proton-induced X-ray emission) experiments showed that the crystals contained the complex and that the phosphorus to sulfur atomic ratio was consistent with the asymmetric unit containing three KH3 domains per 15-mer ssDNA. This was confirmed by structure solution by molecular replacement.

Cloning, Molecular↗

PubMed2004

Initial events in the photocycle of photoactive yellow protein.

The light-induced isomerization of a double bond is the key event that allows the conversion of light energy into a structural change in photoactive proteins for many light-mediated biological processes, such as vision, photosynthesis, photomorphogenesis, and photo movement. Cofactors such as retinals, linear tetrapyrroles, and 4-hydroxy-cinnamic acid have been selected by nature that provide the essential double bond to transduce the light signal into a conformational change and eventually, a physiological response. Here we report the first events after light excitation of the latter chromophore, containing a single ethylene double bond, in a low temperature crystallographic study of the photoactive yellow protein. We measured experimental phases to overcome possible model bias, corrected for minimized radiation damage, and measured absorption spectra of crystals to analyze the photoproducts formed. The data show a mechanism for the light activation of photoactive yellow protein, where the energy to drive the remainder of the conformational changes is stored in a slightly strained but fully cis-chromophore configuration. In addition, our data indicate a role for backbone rearrangements during the very early structural events.

Bacterial Proteins↗