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ABSTRACT: Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of 100 nm were imaged by 300 kV cryo-EM on a Falcon direct electron detector. The images were taken close to focus and to the eye appeared devoid of contrast. Fourier transforms of the images revealed the reciprocal lattice up to 3 Å resolution in favourable cases and up to 4 Å resolution for about half the crystals. The reciprocal-lattice spots showed structure, indicating that the ordering of the crystals was not uniform. Data processing revealed details at higher than 2 Å resolution and indicated the presence of multiple mosaic blocks within the crystal which could be separately processed. The prospects for full three-dimensional structure determination by electron imaging of protein three-dimensional nanocrystals are discussed.
Acta crystallographica. Section D, Biological crystallography 05/2013; 69(Pt 5):852-9. · 12.67 Impact Factor
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ABSTRACT: Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a β-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.
Acta crystallographica. Section D, Biological crystallography 07/2012; 68(Pt 7):794-9. · 12.67 Impact Factor
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Zunfeng Liu,
Federica Galli,
Willem-Jan Waterreus,
Elisabeth Meulenbroek,
Roman I Koning,
Gerda E M Lamers,
René C L Olsthoorn,
Navraj Pannu,
Tjerk H Oosterkamp,
Abraham J Koster,
Remus T Dame, Jan Pieter Abrahams
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ABSTRACT: Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration.
ChemPhysChem 03/2012; 13(6):1569-75. · 3.41 Impact Factor
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01/2012; , ISBN: 978-953-307-610-2
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ABSTRACT: Three-dimensional nanocrystals can be studied by electron diffraction using transmission cryo-electron microscopy. For molecular structure determination of proteins, such nanosized crystalline samples are out of reach for traditional single-crystal X-ray crystallography. For the study of materials that are not sensitive to the electron beam, software has been developed for determining the crystal lattice and orientation parameters. These methods require radiation-hard materials that survive careful orienting of the crystals and measuring diffraction of one and the same crystal from different, but known directions. However, as such methods can only deal with well-oriented crystalline samples, a problem exists for three-dimensional (3D) crystals of proteins and other radiation sensitive materials that do not survive careful rotational alignment in the electron microscope. Here, we discuss our newly released software AMP that can deal with nonoriented diffraction patterns, and we discuss the progress of our new preprocessing program that uses autocorrelation patterns of diffraction images for lattice determination and indexing of 3D nanocrystals.
Microscopy and Microanalysis 11/2011; 17(6):879-85. · 3.01 Impact Factor
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ABSTRACT: For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single- or multiple-wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/).
Acta crystallographica. Section D, Biological crystallography 04/2011; 67(Pt 4):331-7. · 12.67 Impact Factor
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ABSTRACT: Growing X-ray grade crystals of a specific protein is a process of trial and error. Usually, hundreds or even thousands of conditions are screened in order to identify useful crystallization conditions. Heterogeneous nucleants have been shown to increase the success rate of crystallization trials, and (human) hair has previously been identified as a promising nucleant. Here, we describe and evaluate a method for preparing crystallization plates that are locally coated with fragments of human hair, allowing automated, high-throughput crystallization trials in a fashion that is entirely compatible with standard hanging or sitting drop crystallization techniques. We assessed the effect of these nucleants on the crystallization of 11 different proteins in more than 4000 crystallization trials. We found additional crystallization conditions for 10 out of 11 proteins when using the standard JCSG+ screen (96 different conditions). In total, 34 additional crystallization conditions could be identified (13.1% of the total number of successful crystallizations). The increase in crystallization conditions ranged between 33.3% (two additional conditions were identified for myoglobin on top of four homogeneous crystallizations) to 1.2% (we identified a single additional condition for insulin, which crystallized in 85 out of 96 conditions); the median increase in crystallization hits was 14%. On the basis of these numbers, we conclude that the inclusion of human hair fragments in high throughput crystallization screens may be beneficial. The method is inexpensive, straightforward with standard equipment and uses materials available in any crystallization lab. Furthermore, initial experiments with the crystallization of membrane proteins on hair show the technique may also be beneficial for growing membrane proteins.
03/2011;
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Jan-Pieter Abrahams,
Rolf Apweiler,
Rudi Balling,
Michela G Bertero,
Janusz M Bujnicki,
Naomi E Chayen,
Patrick Chène,
Gary L Corthals,
Tomasz Dyląg,
Friedrich Förster, [......],
Cristiano Migliorini,
Andrea Musacchio,
Marjetka Podobnik,
Gebhard F X Schertler,
Gideon Schreiber,
Titia K Sixma,
August B Smit,
David Stuart,
Dmitri I Svergun,
Michael J Taussig
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ABSTRACT: The "4D Biology Workshop for Health and Disease", held on 16-17th of March 2010 in Brussels, aimed at finding the best organising principles for large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughput research and large-scale data gathering in biological and medical science. Major conclusions of the workshop include the following. (i) Development of new technologies and approaches to data analysis is crucial. Biophysical methods should be developed that span a broad range of time/spatial resolution and characterise structures and kinetics of interactions. Mathematics, physics, computational and engineering tools need to be used more in biology and new tools need to be developed. (ii) Database efforts need to focus on improved definitions of ontologies and standards so that system-scale data and associated metadata can be understood and shared efficiently. (iii) Research infrastructures should play a key role in fostering multidisciplinary research, maximising knowledge exchange between disciplines and facilitating access to diverse technologies. (iv) Understanding disease on a molecular level is crucial. System approaches may represent a new paradigm in the search for biomarkers and new targets in human disease. (v) Appropriate education and training should be provided to help efficient exchange of knowledge between theoreticians, experimental biologists and clinicians. These conclusions provide a strong basis for creating major possibilities in advancing research and clinical applications towards personalised medicine.
New Biotechnology 10/2010; 28(4):291-3. · 2.76 Impact Factor
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Advanced Functional Materials 07/2010; 20(17):2857 - 2865. · 10.18 Impact Factor
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Zunfeng Liu,
Federica Galli,
Kjeld G. H. Janssen,
Linhua Jiang,
Heiko J. van der Linden,
Daniël C. de Geus,
Patrick Voskamp,
Maxim E. Kuil,
René C. L. Olsthoorn,
Tjerk. H. Oosterkamp,
Thomas Hankemeier, Jan Pieter Abrahams
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ABSTRACT: A novel method is described for preparing single walled carbon nanotube (SWNT)−streptavidin complexes via the biotin−streptavidin recognition. The complex shows stability in 18 days, strong biotin recognition capability, and excellent loading capacity (about 1 streptavidin tetramer per 20 nm of SWNT). Capturing biotinylated DNA, fluorophores, and Au nanoparticles (NPs) on the SWNT−streptavidin complexes demonstrates their usefulness as a docking matrix, for instance for electron microscopy studies, a technique requiring a virtually electron transparent support.
02/2010;
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ABSTRACT: The typical resolution of three-dimensional reconstruction by cryo-EM single particle analysis is now being pushed up to and beyond the nanometer scale. Correction of the contrast transfer function (CTF) of electron microscopic images is essential for achieving such a high resolution. Various correction methods exist and are employed in popular reconstruction software packages. Here, we present a novel approximation method that corrects the amplitude modulation introduced by the contrast transfer function by convoluting the images with a piecewise continuous function. Our new approach can easily be implemented and incorporated into other packages. The implemented method yielded higher resolution reconstructions with data sets from both highly symmetric and asymmetric structures. It is an efficient alternative correction method that allows quick convergence of the 3D reconstruction and has a high tolerance for noisy images, thus easing a bottleneck in practical reconstruction of macromolecules.
Ultramicroscopy 02/2010; 110(4):350-8. · 2.47 Impact Factor
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ABSTRACT: Unit-cell determination is the first step towards the structure solution of an unknown crystal form. Standard procedures for unit-cell determination cannot cope with data collections that consist of single diffraction patterns of multiple crystals, each with an unknown orientation. However, for beam-sensitive nanocrystals these are often the only data that can be obtained. An algorithm for unit-cell determination that uses randomly oriented electron-diffraction patterns with unknown angular relationships is presented here. The algorithm determined the unit cells of mineral, pharmaceutical and protein nanocrystals in orthorhombic high- and low-symmetry space groups, allowing (well oriented) patterns to be indexed.
Acta crystallographica. Section D, Biological crystallography 08/2009; 65(Pt 7):625-32. · 12.67 Impact Factor
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ABSTRACT: The resolution gap between macromolecular crystallography and electron microscopy continues to decrease. Recent advances in specimen preparation, instrumentation and computational power have allowed accurate structure determination of larger macromolecular complexes by crystallography and/or by electron microscopy on cryovitrified samples. New possibilities in structural biology have opened up and new challenges are faced to further reduce the resolution gap. A workshop at the Lorentz Center, Leiden, The Netherlands, which took place in May 2008, was organized to push further the limits of both complementary techniques through improved computational methods.
Acta crystallographica. Section D, Biological crystallography 08/2009; 65(Pt 7):623-4. · 12.67 Impact Factor
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ABSTRACT: Mammalian microtubule plus-end tracking proteins (+TIPs) specifically associate with the ends of growing microtubules. +TIPs are involved in many cellular processes, including mitosis, cell migration and neurite extension. Navigators are mammalian homologues of the C. elegans unc-53 protein, an ATPase that has been linked to the migration and outgrowth of muscles, axons and excretory canals. Here we show that all three mammalian Navigators are +TIPs, consistent with a previous study on Navigator 1 (NAV1) (Martinez-Lopez et al., Mol Cell Neurosci 2005;28:599-612). Overexpression of GFP-tagged Navigators causes displacement of CAP_GLY-motif containing +TIPs, such as CLIP-170, from microtubule ends, suggesting that the Navigator-binding sites on microtubule ends overlap with those of the CAP_GLY-motif proteins. In interphase cells, mammalian Navigators also prominently localize to centrosomes, a localization that does not depend on an intact microtubule network. Fluorescence recovery after photobleaching (FRAP) experiments indicate that NAV1 associates with intracellular structures other than microtubules or centrosomes. Expression of GFP-tagged Navigators induces the formation of neurite-like extensions in non-neuronal cells, showing that Navigators can dominantly alter cytoskeletal behavior. For NAV1 this function depends on its ATPase activity; it is not achieved by a classical type of MT bundling and stabilization. Combined our data suggest that Navigators are +TIPs that can reorganize the cytoskeleton to guide cell shape changes. Our data are consistent with a role for Navigators in neurite outgrowth.
Cell Motility and the Cytoskeleton 05/2009; 66(10):824-38. · 4.19 Impact Factor
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ABSTRACT: UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5' to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post-translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X-ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal-coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.
Protein Science 02/2009; 18(3):549-58. · 2.80 Impact Factor
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ABSTRACT: Chlorite dismutase (Cld) is a key enzyme of perchlorate and chlorate respiration. This heme-based protein reduces the toxic compound chlorite into the innocuous chloride anion in a very efficient way while producing molecular oxygen. A sequence comparison between Cld homologues shows a highly conserved family. The crystal structure of Azospira oryzae strain GR-1 Cld is reported to 2.1 A resolution. The structure reveals a hexameric organization of the Cld, while each monomer exhibits a ferredoxin-like fold. The six subunits are organized in a ring structure with a maximal diameter of 9 nm and an inner diameter of 2 nm. The heme active-site pocket is solvent accessible both from the inside and the outside of the ring. Moreover, a second anion binding site that could accommodate the assumed reaction intermediate ClO(-) for further transformation has been identified near the active site. The environment of the heme cofactor was investigated with electron paramagnetic resonance spectroscopy. Apart from the high-spin ferric signal of the five-coordinate resting-state enzyme, two low-spin signals were found corresponding to six-coordinate species. The current crystal structure confirms and complements a recently proposed catalytic mechanism that proceeds via a ferryl species and a ClO(-) anion. Our structural data exclude cooperativity between the iron centers.
Journal of Molecular Biology 02/2009; 387(1):192-206. · 4.00 Impact Factor
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ABSTRACT: Lewis X trisaccharides normally function as essential cell-cell interaction mediators. However, oligomers of Lewis X trisaccharides expressed by the parasite Schistosoma mansoni seem to be related to its evasion of the immune response of its human host. Here we show that monoclonal antibody 54-5C10-A, which is used to diagnose schistosomiasis in humans, interacts with oligomers of at least three Lewis X trisaccharides, but not with monomeric Lewis X. We describe the sequence and the 2.5 A crystal structure of its Fab fragment and infer a possible mode of binding of the polymeric Lewis X from docking studies. Our studies indicate a radically different mode of binding compared to Fab 291-2G3-A, which is specific for monomeric Lewis X, thus providing a structural explanation of the diagnostic success of 54-5C10-A.
Proteins Structure Function and Bioinformatics 01/2009; 76(2):439-47. · 3.39 Impact Factor
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ABSTRACT: When heat shock prematurely dissociates a translating bacterial ribosome, its 50S subunit is prevented from reinitiating protein synthesis by tRNA covalently linked to the unfinished protein chain that remains threaded through the exit tunnel. Hsp15, a highly upregulated bacterial heat shock protein, reactivates such dead-end complexes. Here, we show with cryo-electron microscopy reconstructions and functional assays that Hsp15 translocates the tRNA moiety from the A site to the P site of stalled 50S subunits. By stabilizing the tRNA in the P site, Hsp15 indirectly frees up the A site, allowing a release factor to land there and cleave off the tRNA. Such a release factor must be stop codon independent, suggesting a possible role for a poorly characterized class of putative release factors that are upregulated by cellular stress, lack a codon recognition domain and are conserved in eukaryotes.
Journal of Molecular Biology 12/2008; 386(5):1357-67. · 4.00 Impact Factor
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ABSTRACT: The plus ends of microtubules (MTs) alternate between phases of growth, pause, and shrinkage, a process called "dynamic instability." Cryo-EM of in vitro-assembled MTs indicates that the dynamic state of the plus end corresponds with a particular MT plus-end conformation. Frayed ("ram's horn like"), blunt, and sheet conformations are associated with shrinking, pausing, and elongating plus ends, respectively. A number of new conformations have recently been found in situ but their dynamic states remained to be confirmed. Here, we investigated the dynamics of MT plus ends in the peripheral area of interphase mouse fibroblasts (3T3s) using electron microscopical and tomographical analysis of cryo-fixed, freeze-substituted, and flat-embedded sections. We identified nine morphologically distinct plus-end conformations. The frequency of these conformations correlates with their proximity to the cell border, indicating that the dynamic status of a plus end is influenced by features present in the periphery. Shifting dynamic instability toward depolymerization with nocodazole enabled us to address the dynamic status of these conformations. We suggest a new transition path from growth to shrinkage via the so-called sheet-frayed and flared ends, and we present a kinetic model that describes the chronology of events taking place in nocodazole-induced MT depolymerization.
Molecular biology of the cell 08/2008; 19(7):3138-46. · 5.98 Impact Factor
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ABSTRACT: Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (>2 kU mg(-1)) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P2(1)2(1)2 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 A. The crystals diffracted X-rays to 2.1 A resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2008; 64(Pt 8):730-2. · 0.51 Impact Factor
