Gleb Bourenkov

Graz University of Technology, Gratz, Styria, Austria

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Publications (103)

  • [Show abstract] [Hide abstract] ABSTRACT: The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics. © 2016; American Association for the Advancement of Science. All rights reserved.
    Article · Aug 2016 · Science
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    Ulrich Zander · Gleb Bourenkov · Alexander N Popov · [...] · Gordon A Leonard
    [Show abstract] [Hide abstract] ABSTRACT: Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.
    Full-text Article · Nov 2015 · Acta Crystallographica Section D Biological Crystallography
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    [Show abstract] [Hide abstract] ABSTRACT: Whether long-range quantum coherent states could exist in biological systems, and beyond low-temperature regimes where quantum physics is known to be applicable, has been the subject to debate for decades. It was proposed by Fröhlich that vibrational modes within protein molecules can order and condense into a lowest-frequency vibrational mode in a process similar to Bose-Einstein condensation, and thus that macroscopic coherence could potentially be observed in biological systems. Despite the prediction of these so-called Fröhlich condensates almost five decades ago, experimental evidence thereof has been lacking. Here, we present the first experimental observation of Fröhlich condensation in a protein structure. To that end, and to overcome the challenges associated with probing low-frequency molecular vibrations in proteins (which has hampered understanding of their role in proteins' function), we combined terahertz techniques with a highly sensitive X-ray crystallographic method to visualize low-frequency vibrational modes in the protein structure of hen-egg white lysozyme. We found that 0.4 THz electromagnetic radiation induces non-thermal changes in electron density. In particular, we observed a local increase of electron density in a long α-helix motif consistent with a subtle longitudinal compression of the helix. These observed electron density changes occur at a low absorption rate indicating that thermalization of terahertz photons happens on a micro- to milli-second time scale, which is much slower than the expected nanosecond time scale due to damping of delocalized low frequency vibrations. Our analyses show that the micro- to milli-second lifetime of the vibration can only be explained by Fröhlich condensation, a phenomenon predicted almost half a century ago, yet never experimentally confirmed.
    Full-text Article · Sep 2015 · Structural Dynamics
  • Michele Cianci · Gleb Bourenkov · Johanna Kallio · [...] · Thomas Schneider
    Article · Aug 2015 · Acta Crystallographica Section A: Foundations and Advances
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    Igor Melnikov · Gleb P. Bourenkov · Olof Svensson · Alexander N. Popov
    Full-text Article · Aug 2015 · Acta Crystallographica Section A: Foundations and Advances
  • Article · Aug 2015 · Acta Crystallographica Section A: Foundations and Advances
  • Thomas R. Schneider · Gleb Bourenkov · Cornelius Gati · [...] · Florent Cipriani
    Article · Aug 2015 · Acta Crystallographica Section A: Foundations and Advances
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    Valeriya R Samygina · Alexey V Sokolov · Gleb Bourenkov · [...] · Dmitri I Svergun
    Full-text Dataset · Apr 2015
  • [Show abstract] [Hide abstract] ABSTRACT: Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.
    Article · Jan 2015 · ChemBioChem
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    [Show abstract] [Hide abstract] ABSTRACT: Peroxisomes are membrane-enclosed organelles in eukaryotic cells with important roles in lipid metabolism and the scavenging of reactive oxygen species. Peroxisomes are capable of carrying an unusually high load of proteins, which under appropriate nutrient conditions results in the in situ crystallization of peroxisomal proteins in several yeast species and vertebrate hepatocytes [1,2]. In the methylotrophic yeast H.polymorpha, the predominant peroxisomal protein alcohol/methanol oxidase (AO) oligomerizes into octameric assemblies with a molecular mass of 600 kDa that spontaneously form 200-500 nm crystallites within peroxisomes [1]. We exposed H.polymorpha cell suspensions containing peroxisome-confined AO crystallites to femtosecond X-ray pulses at the Coherent X-ray Imaging (CXI) experimental endstation at the Linac Coherent Light Source. Peak detection routines mining the resulting scattering profiles identified >5000 Bragg-sampled diffraction patterns, providing the proof of concept that background scattering from the cells does not deteriorate the signal-to-noise ratio to an extent precluding observation of diffraction from individual AO crystallites. Summation patterns assembled from the individual frames match low-resolution powder diffraction patterns from concentrated suspensions of purified peroxisomes collected at the P14 beamline at the PETRAIII synchrotron, confirming that the observed diffraction mainly results from Bragg scattering of peroxisomal crystallites. To the best of our knowledge our data are the first to report room temperature X-ray diffraction from functional protein crystals in their native cellular environment. Currently the maximum resolution achieved in the diffraction patterns is limited to 20-15 Å. Future work will need to address improved sample preparation protocols in order to assess whether diffraction to a resolution sufficient to permit structure solution can be obtained. Protein crystal formation in vivo has been observed under physiological or pathological conditions in a number of other systems [3]. We hope that our results will help to establish serial femtosecond X-ray diffraction (SFX) as a method for structural characterization of cellular structures with crystalline content and provide a proof of concept for using in situ crystallization of proteins as a means to generate nanocrystalline samples for SFX.
    Full-text Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
  • Cornelius Gati · Gleb Bourenkov · Marco Klinge · [...] · Lars Redecke
    [Show abstract] [Hide abstract] ABSTRACT: Protein crystallography continues to be one of the most frequently used techniques to obtain structural information of biomacromolecules to atomic resolution. Since protein crystals of delicate target systems are often limited in size, one of the main goals in the design of modern beamlines is the construction of highly intense X-ray beams with small focal size to obtain high resolution diffraction images of microcrystals. However, this development has led to the situation, that the full intensity of the beam can destroy a protein crystal within fractions of a second. Therefore often only a small number of diffraction patterns can be obtained from one single crystal. Here we describe the adaptation of the serial crystallography approach, which has first been developed at X-ray Free-Electron Lasers (Chapman et al. 2011) to the usage of a microfocus synchrotron beamline, using a standard cryogenic loop for sample delivery. We proved this concept with in vivo grown cathepsinB microcrystals (TbCatB, Koopmann et al. 2012, Redecke et al. 2013) (average of 9 μm3), a medically and pharmaceutically relevant protein, involved in the life cycle of T. brucei. In these experiments it was possible to show that serial crystallography enables the utilization and outcome of the above described bottlenecks and features of modern 3rd generation synchrotron microfocus beamlines. Our strategy exploits the combination of a micron-sized X-ray beam, high precision diffractometry and shutterless data acquisition with a pixel-array detector. By combining the data of 80 TbCatB crystals, it was possible to assemble a dataset to 3.0 Å resolution. The data allow the refinement of a structural model that is consistent with that previously obtained using FEL radiation, providing mutual validation.
    Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
  • Thomas R. Schneider · Gleb Bourenkov · Michele Cianci · [...] · Stefan Fiedler
    [Show abstract] [Hide abstract] ABSTRACT: Since 2012, EMBL Hamburg operates two new beamlines for macromolecular crystallography - P13 and P14 - at PETRA III at DESY (Hamburg, Germany). We exploit the high brilliance and the wide energy range offered by PETRA III to offer a wide range of conditions to fit the experimental conditions to the challenges posed by the samples. P13 provides high photon flux down to 4 keV. With a helium cone and a kappa goniostat, this allows optimized data collection for SAD phasing. Using adaptive mirrors, the focus size (H x V) can be adjusted between 30 x 20 μm^2 and 150 x 100 μm^2 to match the size of the sample. A MARVIN sample changer is in operation for rapid loading and unloading of samples. P14 offers a high photon flux (>10^12 ph/sec at 12 keV into 5 x 5 µm^2). The beamsize can be varied between 1 x 1.5 mm^2 (unfocused) and 5 x 5 µm^2 (fully focused) in less than a minute by moving the KB mirrors in and out of the beam. For small crystals, an MD3 vertical diffractometer with a sphere of confusion smaller than 100 nm offers excellent conditions. Both beamlines are equipped with PILATUS 6M-F detectors for shutter-less data collection and dedicated data processing computers. The beamlines are embedded into the 'Integrated Facility for Structural Biology' offering facilities for sample preparation and characterization, a laboratory specifically equipped for the preparation of heavy atom derivatives, and downstream facilities for data evaluation We will report about the status of the beamlines and describe typical experimental situations (small crystals, large unit cells, serial crystallography, low-energy phasing, small molecules and others).
    Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
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    Oleksandr Yefanov · Cornelius Gati · Gleb Bourenkov · [...] · Anton Barty
    [Show abstract] [Hide abstract] ABSTRACT: Serial crystallography using X-ray free-electron lasers enables the collection of tens of thousands of measurements from an equal number of individual crystals, each of which can be smaller than 1 µm in size. This manuscript describes an alternative way of handling diffraction data recorded by serial femtosecond crystallography, by mapping the diffracted intensities into three-dimensional reciprocal space rather than integrating each image in two dimensions as in the classical approach. We call this procedure 'three-dimensional merging'. This procedure retains information about asymmetry in Bragg peaks and diffracted intensities between Bragg spots. This intensity distribution can be used to extract reflection intensities for structure determination and opens up novel avenues for post-refinement, while observed intensity between Bragg peaks and peak asymmetry are of potential use in novel direct phasing strategies.
    Full-text Article · Jul 2014 · Philosophical Transactions of The Royal Society B Biological Sciences
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    Cornelius Gati · Gleb Bourenkov · Marco Klinge · [...] · Lars Redecke
    [Show abstract] [Hide abstract] ABSTRACT: Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm 3 , a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.
    Full-text Article · Mar 2014
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    Cornelius Gati · Gleb Bourenkov · Marco Klinge · [...] · Lars Redecke
    Full-text Dataset · Feb 2014
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    Dataset: Figure S6
    Valeriya R. Samygina · Alexey V. Sokolov · Gleb Bourenkov · [...] · Dmitri I. Svergun
    [Show abstract] [Hide abstract] ABSTRACT: Elements of β-sheets stabilization in Cp structure. Elements of β-sheets stabilization in Cp structure, related to Discussion. (A–D) Conservative disulfide bridges in the Cp structure. Cp molecule is shown in cartoon representation, cysteins are shown by sticks. (E) Elements stabilizing the beginning and the end of the loop between domains 5 and 6. Disulfide bridge between Cys881 and Cys855 and H-bond network. (F) 2Fo–Fc electron density map countered at 1σ level for residues forming H-bond networks in the end of the domain 5-domain 6 loop. (TIF)
    Full-text Dataset · Jul 2013
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    Dataset: Figure S5
    Valeriya R. Samygina · Alexey V. Sokolov · Gleb Bourenkov · [...] · Dmitri I. Svergun
    [Show abstract] [Hide abstract] ABSTRACT: Sequence alignment of five homology protein fragments: human Cp, rat Cp, human hephaestin, human coagulation factor V and coagulation factor VIII. Sequence alignment of five homology protein fragments: human Cp, rat Cp, human hephaestin, human coagulation factor V and coagulation factor VIII, related to Discussion. Only fragments containing disulfide bridges are shown. Cysteins are shown in green. Conservative metal-binding residues are shown in red; half-conservative metal-binding residues are shown in magenta. Disulfide bridges are shown by green dash-lines. Numbers of cystein residues are shown in green (Cp numbering). (TIF)
    Full-text Dataset · Jul 2013
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    Dataset: Figure S1
    Valeriya R. Samygina · Alexey V. Sokolov · Gleb Bourenkov · [...] · Dmitri I. Svergun
    [Show abstract] [Hide abstract] ABSTRACT: Overall structure organization of Cp and Lf. Right view is rotated by 90 degrees around horizontal axis. The molecule is represented as cartoon; its six domains are marked in Roman numerals and colored as follows: domain I in light blue, domain II in blue, domain III in pale cyan, domain IV in purple, domain V in dark sky blue and domain VI in cyan. Copper ions are shown as orange spheres. (B) Structural organization of Lf. Lf molecule is shown in cartoon representation. Domain 1 of N-lobe is shown in chocolate, domain 2 of N-lobe is shown in red, domain 1 of C-lobe is shown in pink and domain 2 of C-lobe is shown in dark pink. Two inter-lobe helices are shown in grey. Ferrous ions are shown as black spheres. (TIF)
    Full-text Dataset · Jul 2013
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    Dataset: Figure S2
    Valeriya R. Samygina · Alexey V. Sokolov · Gleb Bourenkov · [...] · Dmitri I. Svergun
    [Show abstract] [Hide abstract] ABSTRACT: Contacts between symmetrical molecules of individual proteins in Cp-Mpo complex. (A) Overall view of contacts between two symmetrical molecules of Mpo which forms a crystallographic dimer. Symmetrical molecule is shown by light green and labeled with *. (B) Electron density map 2Fo-Fc at 1σ level for two symmetrical sugar chains is shown. (C,D) Contact between Cps molecules related by 2-fold axes. Symmetrical subunit is shown in cyan and labeled with *. (TIF)
    Full-text Dataset · Jul 2013
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    Dataset: Figure S4
    Valeriya R. Samygina · Alexey V. Sokolov · Gleb Bourenkov · [...] · Dmitri I. Svergun
    [Show abstract] [Hide abstract] ABSTRACT: Possible changing of loop tracing during Cp-Mpo complex formation in order to avoid steric clashes. Possible changing of loop tracing during Cp-Mpo complex formation in order to avoid steric clashes, related to Discussion. (A) Superposition of Cp and Cp-Mpo structure. Proteins are shown in cartoon representation. Free Cp is shown in dark blue, loop 885–892 is shown in yellow; Cp in Cp-Mpo complex is shown in pale cyan, loop 885–892 is shown in dark red. Area of interaction between Cp an Mpo is marked by dashed red oval. (B) 2Fo–Fc electron density map shown at 1 σ level for visible residues of interdomain loop 885–892. (TIF)
    Full-text Dataset · Jul 2013

Publication Stats

5k Citations

Institutions

  • 2005
    • Graz University of Technology
      Gratz, Styria, Austria
  • 2000
    • Max Planck Society
      München, Bavaria, Germany
    • Max Planck Research Unit for Structural Molecular Biology at DESY
      Hamburg, Hamburg, Germany