Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
Add more filters










Publication year range
1.
ACS Nano ; 17(24): 24802-24813, 2023 Dec 26.
Article in English | MEDLINE | ID: mdl-37890869

ABSTRACT

Structural characterization is crucial to understanding protein function. Compared with X-ray diffraction methods, electron crystallography can be performed on nanometer-sized crystals and can provide additional information from the resulting Coulomb potential map. Whereas electron crystallography has successfully resolved three-dimensional structures of vitrified protein crystals, its widespread use as a structural biology tool has been limited. One main reason is the fragility of such crystals. Protein crystals can be easily damaged by mechanical stress, change in temperature, or buffer conditions as well as by electron irradiation. This work demonstrates a methodology to preserve these nanocrystals in their natural environment at room temperature for electron diffraction experiments as an alternative to existing cryogenic techniques. Lysozyme crystals in their crystallization solution are hermetically sealed via graphene-coated grids, and their radiation damage is minimized by employing a low-dose data collection strategy in combination with a hybrid-pixel direct electron detector. Diffraction patterns with reflections of up to 3 Å are obtained and successfully indexed using a template-matching algorithm. These results demonstrate the feasibility of in situ protein electron diffraction. The method described will also be applicable to structural studies of hydrated nanocrystals important in many research and technological developments.


Subject(s)
Electrons , Proteins , Temperature , Proteins/chemistry , Crystallography, X-Ray , X-Ray Diffraction
2.
J Vis Exp ; (170)2021 04 11.
Article in English | MEDLINE | ID: mdl-33900284

ABSTRACT

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.


Subject(s)
Lab-On-A-Chip Devices , Muramidase/chemistry , Crystallization , Crystallography, X-Ray , Microdialysis , X-Ray Diffraction
3.
J Vis Exp ; (169)2021 03 13.
Article in English | MEDLINE | ID: mdl-33779594

ABSTRACT

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand. In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.


Subject(s)
Crystallization/methods , Macromolecular Substances/chemistry , Neutron Diffraction/methods , Neutrons , Proteins/chemistry , Crystallography, X-Ray , Humans
4.
J Appl Crystallogr ; 53(Pt 3): 686-698, 2020 Jun 01.
Article in English | MEDLINE | ID: mdl-32684884

ABSTRACT

A rational way to find the appropriate conditions to grow crystal samples for bio-crystallography is to determine the crystallization phase diagram, which allows precise control of the parameters affecting the crystal growth process. First, the nucleation is induced at supersaturated conditions close to the solubility boundary between the nucleation and metastable regions. Then, crystal growth is further achieved in the metastable zone - which is the optimal location for slow and ordered crystal expansion - by modulation of specific physical parameters. Recently, a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant-concentration phase diagrams has been constructed. Here, it is demonstrated that a thorough knowledge of the phase diagram is vital in any crystallization experiment. The relevance of the selection of the starting position and the kinetic pathway undertaken in controlling most of the final properties of the synthesized crystals is shown. The rational crystallization optimization strategies developed and presented here allow tailoring of crystal size and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.

5.
Methods Enzymol ; 634: 21-46, 2020.
Article in English | MEDLINE | ID: mdl-32093834

ABSTRACT

The use of neutron protein crystallography (NPX) is expanding rapidly, with most structures determined in the last decade. This growth is stimulated by a number of developments, spanning from the building of new NPX beamlines to the availability of improved software for structure refinement. The main bottleneck preventing structural biologists from adding NPX to the suite of methods commonly used is the large volume of the individual crystals required for a successful experiment. A survey of deposited NPX structures in the Protein Data Bank shows that about two-thirds came from crystals prepared using vapor diffusion, while batch and dialysis-based methods all-together contribute to most of the remaining one-third. This chapter explains the underlying principles of these protein crystallization methods and provides practical examples that may help others to successfully prepare large crystals for NPX.


Subject(s)
Neutrons , Proteins , Crystallization , Crystallography , Crystallography, X-Ray , Diffusion
6.
Lab Chip ; 20(2): 296-310, 2020 01 21.
Article in English | MEDLINE | ID: mdl-31804643

ABSTRACT

This paper reports a versatile microfluidic chip developed for on-chip crystallization of proteins through the dialysis method and in situ X-ray diffraction experiments. A microfabrication process enabling the integration of regenerated cellulose dialysis membranes between two layers of the microchip is thoroughly described. We also describe a rational approach for optimizing on-chip protein crystallization via chemical composition and temperature control, allowing the crystal size, number and quality to be tailored. Combining optically transparent microfluidics and dialysis provides both precise control over the experiment and reversible exploration of the crystallization conditions. In addition, the materials composing the microfluidic chip were tested for their transparency to X-rays in order to assess their compatibility for in situ diffraction data collection. Background scattering was evaluated using a synchrotron X-ray source and the background noise generated by our microfluidic device was compared to that produced by commercial crystallization plates used for diffraction experiments at room temperature. Once crystals of 3 model proteins (lysozyme, IspE, and insulin) were grown on-chip, the microchip was mounted onto the beamline and partial diffraction data sets were collected in situ from several isomorphous crystals and were merged to a complete data set for structure determination. We therefore propose a robust and inexpensive way to fabricate microchips that cover the whole pipeline from crystal growth to the beam and does not require any handling of the protein crystals prior to the diffraction experiment, allowing the collection of crystallographic data at room temperature for solving the three-dimensional structure of the proteins under study. The results presented here allow serial crystallography experiments on synchrotrons and X-ray lasers under dynamically controllable sample conditions to be observed using the developed microchips.


Subject(s)
Insulin/chemistry , Lab-On-A-Chip Devices , Muramidase/chemistry , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Agrobacterium tumefaciens/enzymology , Crystallization , Muramidase/metabolism , Particle Size , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Surface Properties , X-Ray Diffraction
7.
J Appl Crystallogr ; 49(Pt 3): 806-813, 2016 Jun 01.
Article in English | MEDLINE | ID: mdl-27275137

ABSTRACT

Many instrumentation developments in crystallization have concentrated on massive parallelization assays and reduction of sample volume per experiment to find initial crystallization conditions. Yet improving the size and diffraction quality of the crystals for diffraction studies often requires decoupling of crystal nucleation and growth. This in turn requires the control of variables such as precipitant and protein concentration, equilibration rate, and temperature, which are all difficult parameters to control in the existing setups. The success of the temperature-controlled batch method, originally developed to grow very large crystals for neutron crystallography, demonstrated that the rational optimization of crystal growth has potential in structural biology. A temperature-controlled dialysis button has been developed for our previous device, and a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant concentration phase diagrams has been constructed. The presented approach differs from the current paradigm, since it involves serial instead of parallel experiments, exploring multiple crystallization conditions with the same protein sample. The sample is not consumed in the experiment and the conditions can be changed in a reversible fashion, using dialysis with a flowing precipitant reservoir as well as precise temperature control. The control software allows visualization of the crystals, as well as control of the temperature and composition of the crystallization solution. The rational crystallization optimization strategies presented here allow tailoring of crystal size, morphology and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.

8.
PLoS One ; 9(1): e86651, 2014.
Article in English | MEDLINE | ID: mdl-24466188

ABSTRACT

Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10-T57-H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.


Subject(s)
Aspergillus flavus/enzymology , Neutrons , Protons , Urate Oxidase/chemistry , Xanthines/metabolism , Catalysis , Crystallography , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Urate Oxidase/metabolism , Xanthines/chemistry
9.
Biochemistry ; 46(11): 2930-7, 2007 Mar 20.
Article in English | MEDLINE | ID: mdl-17319692

ABSTRACT

Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. The rate-limiting step of this catalysis is the transfer of a proton between the Zn-bound solvent molecule and residue His64. In order to fully characterize the active site structural features implicated in the proton transfer mechanism, the refined X-ray crystal structure of uncomplexed wild type HCA II to 1.05 A resolution with an Rcryst value of 12.0% and an Rfree value of 15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecular proton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solvent network, the unique positioning of solvent molecule W2, and the significance of the dual conformation of His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculations of the Zn-bound hydroxyl/His64+ (charged) and the Zn-bound water/His64 (uncharged) HCA II states. A comparison of the crystallographic anisotropic atomic thermal parameters and MD simulation root-mean-square fluctuation values show excellent agreement in the atomic motion observed between the two methods. It is also interesting that the observed active site solvent positions in the crystal structure are also the most probable positions of the solvent during the MD simulations. On the basis of the comparative study of the MD simulation results, the HCA II crystal structure observed is most likely in the Zn-bound water/His64 state. This conclusion is based on the following observations: His64 is mainly (80%) orientated in an inward conformation; electron density omit maps infer that His64 is not charged in an either inward or outward conformation; and the Zn-bound solvent is most likely a water molecule.


Subject(s)
Carbonic Anhydrase II/chemistry , Protons , Amino Acid Sequence , Computational Biology , Computer Simulation , Crystallization , Crystallography, X-Ray , Histidine/chemistry , Humans , Models, Molecular
10.
Article in English | MEDLINE | ID: mdl-16511248

ABSTRACT

Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH-) and the proton-shuttling residue His64. This distance (approximately 7.5 A) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (approximately 1.0 A) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 A resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.


Subject(s)
Carbonic Anhydrase II/chemistry , Binding Sites , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Carbonic Anhydrase II/biosynthesis , Carbonic Anhydrase II/genetics , Crystallography, X-Ray , Deuterium , Humans , Protein Conformation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
11.
Article in English | MEDLINE | ID: mdl-16511303

ABSTRACT

A C-terminal fragment of the Epstein-Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 A resolution using synchrotron radiation (lambda = 0.976 A). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 A, beta = 103.9 degrees.


Subject(s)
DNA-Binding Proteins/biosynthesis , Herpesvirus 4, Human/chemistry , Trans-Activators/biosynthesis , Viral Proteins/biosynthesis , Crystallization/methods , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Escherichia coli/metabolism , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/isolation & purification , Peptide Fragments/chemistry , Protein Structure, Quaternary , Trans-Activators/chemistry , Trans-Activators/isolation & purification , Viral Proteins/chemistry , Viral Proteins/isolation & purification
12.
Article in English | MEDLINE | ID: mdl-16511330

ABSTRACT

Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 A resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 A) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.


Subject(s)
Urate Oxidase/chemistry , Xanthines/chemistry , Binding Sites , Crystallization/methods , Deuterium Oxide , Neutron Diffraction/methods , Recombinant Proteins/chemistry , Saccharomyces cerevisiae/metabolism
13.
Mol Immunol ; 39(7-8): 383-94, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12413689

ABSTRACT

C1 is the multimolecular protease that triggers activation of the classical pathway of complement, a major element of antimicrobial host defense also involved in immune tolerance and various pathologies. This 790,000 Da complex is formed from the association of a recognition protein, C1q, and a catalytic subunit, the Ca2+-dependent tetramer C1s-C1r-C1r-C1s comprising two copies of each of the modular proteases C1r and C1s. Early studies mainly based on biochemical analysis and electron microscopy of C1 and its isolated components have allowed for characterization of their domain structure and led to a low-resolution model of the C1 complex in which the elongated C1s-C1r-C1r-C1s tetramer folds into a more compact, "8-shaped" conformation upon interaction with C1q. A major strategy used over the past years has been to dissect the C1 proteins into modular segments to characterize their function and solve their structure by either X-ray crystallography or nuclear magnetic resonance spectroscopy (NMR). The purpose of this review is to focus on this information, with particular emphasis on the architecture of the C1 complex and the mechanisms underlying its activation and proteolytic activity.


Subject(s)
Complement Activation , Complement C1/chemistry , Animals , Catalytic Domain , Complement C1/physiology , Complement C1q/chemistry , Crystallography, X-Ray , Epidermal Growth Factor/chemistry , Humans , Magnetic Resonance Spectroscopy , Protein Structure, Secondary , Serine Endopeptidases/chemistry
14.
Structure ; 10(11): 1509-19, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12429092

ABSTRACT

C1r is the serine protease (SP) that mediates autoactivation of C1, the complex that triggers the classical complement pathway. We have determined the crystal structure of two fragments from the human C1r catalytic domain, each encompassing the second complement control protein (CCP2) module and the SP domain. The wild-type species has an active structure, whereas the S637A mutant is a zymogen. The structures reveal a restricted hinge flexibility of the CCP2-SP interface, and both are characterized by the unique alpha-helical conformation of loop E. The zymogen activation domain exhibits high mobility, and the active structure shows a restricted access to most substrate binding subsites. Further implications relevant to the C1r self-activation process are derived from protein-protein interactions in the crystals.


Subject(s)
Complement Activation , Complement C1r/chemistry , Enzyme Precursors/chemistry , Binding Sites , Catalytic Domain , Complement C1r/metabolism , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Substrate Specificity
15.
Immunobiology ; 205(4-5): 365-82, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12396000

ABSTRACT

C1r, the enzyme responsible for intrinsic activation of the C1 complex of complement, is a modular serine protease featuring an overall structural organization homologous to those of C1s and the mannan-binding lectin-associated serine proteases (MASPs). This review will initially summarize current information on the structure and function of C1r, with particular emphasis on the three-dimensional structure of its catalytic domain, which provides new insights into the activation mechanism of C1. The second part of this review will focus on recent discoveries dealing with a truncated, C1r-related protein, and the occurrence in the mouse of two isoforms, C1rA and C1rB, exhibiting tissue-specific expression patterns.


Subject(s)
Complement C1r/chemistry , Complement C1r/genetics , Complement C1r/physiology , Complement Pathway, Classical/physiology , Animals , Complement C1s/chemistry , Complement C1s/physiology , Enzyme Activation , Humans , Mice , Protein Isoforms , Protein Structure, Secondary , Structure-Activity Relationship
16.
EMBO J ; 21(3): 231-9, 2002 Feb 01.
Article in English | MEDLINE | ID: mdl-11823416

ABSTRACT

C1r is the modular serine protease (SP) that mediates autolytic activation of C1, the macromolecular complex that triggers the classical pathway of complement. The crystal structure of a mutated, proenzyme form of the catalytic domain of human C1r, comprising the first and second complement control protein modules (CCP1, CCP2) and the SP domain has been solved and refined to 2.9 A resolution. The domain associates as a homodimer with an elongated head-to-tail structure featuring a central opening and involving interactions between the CCP1 module of one monomer and the SP domain of its counterpart. Consequently, the catalytic site of one monomer and the cleavage site of the other are located at opposite ends of the dimer. The structure reveals unusual features in the SP domain and provides strong support for the hypothesis that C1r activation in C1 is triggered by a mechanical stress caused by target recognition that disrupts the CCP1-SP interfaces and allows formation of transient states involving important conformational changes.


Subject(s)
Complement C1/metabolism , Complement C1r/chemistry , Amino Acid Sequence , Catalysis , Complement C1/chemistry , Complement C1r/genetics , Complement C1r/metabolism , Complement Pathway, Classical , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Stress, Mechanical , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...