Czech Infrastructure for Intergrative Structural Biology
The Core Facility of High Field NMR Spectroscopy provides access to NMR spectrometers in the range of proton frequencies from 500 MHz to 950 MHz. The equipment is suited mainly to the studies of structure, dynamics and interactions of biomolecules, i.e. proteins, nucleic acids and carbohydrates. However, the instrumentation is flexible enough to cover also various research needs in material science, organic and inorganic chemistry, biochemistry, biology and biophysics.
Expertise: • Studies of biomolecules and other bio-objects at the molecular level using scanning probe techniques, i.e. atomic force microscopy (AFM) and related techniques (STM, SNOM, electrochemical variants), including combinations with optical microscopy (inverted, fluorescence, confocal) • Production and bioconjugation of nanoparticles • Advanced methods - nanolithograpy, nanomechanical manipulations, ink-jet based deposition
In analytical ultracentrifugation (AUC), molecules are characterized directly in solution, often under biologically relevant conditions. In contrast to many other methods, there are no complications caused by interactions with matrices or surfaces. Also, no immobilization or labeling is necessary for the analysis. Analytical ultracentrifugation is considered to be one of the most accurate methods for determination of molar mass of the molecule. Since it is a first-principle method, no calibration is required to determine the mass. AUC is a non-destructive technique which is applicable to particles with molar masses ranging from several hundreds of Da (small peptides) to hundreds of MDa (viruses).
VP-DSC calorimeter measures heat changes that occur in the sample (biomolecule solution) during a controlled increase or decrease in temperature, on the basis of a temperature difference between the sample and the reference material. It is a valuable technique for the study of samples in solution providing fast and accurate determination of the transition midpoint Tm - when 50 % of the biomolecule are unfolded. In a typical arrangement of ITC (Isothermal Titration Calorimetry), the titrant, also referred as the ligand, is injected into the sample cell containing the macromolecule sample solution. The calorimetric measurement can be done over a range of biologically relevant conditions (temperature, salt, pH, etc.).
Circular dichroism spectroscopy measures differences in the absorption on left and right - handed polarised lite that arrise from the structural assymetry (presence in chiral atoms in the molecule). CD spectroscopy is used to investigate the secondary structure of proteins α-helices, β-strands and randome coils can be identified in the fare UV region where thay give rise to characteristic shape and magnitude of the spectrum.
Biomacromolecular Crystallization can be used for (mainly together with X-ray diffraction): Determination of 3D structure – no principal size limit is applied (both NaCl and ribosome were crystallized and their structure solved). Identification of conserved and flexible regions. Analysis of structural changes caused by mutations. Identification of ligand interaction, architecture and number of binding sites. Determination of oligomeric state of the protein. Protein identification and purification (not commonly used).
Light scattering is caused by the interaction of light with dispersed particles (typically in solution), while organized particles (typically in crystal) result in diffraction phenomenon. The intensity of the scattered light depends on the size and the shape of the interacting particles. The experiment using visible light may be performed in two different modes: dynamic and static light scattering. Dynamic light scattering (DLS) measurement analyses the time variance in the scattered light. Static light scattering (SLS) measurement depends on the analysis of light scattering at various angles with respect to the incident beam.
Key equipment: High-end electron microscope for high-resolution cryoEM and cryo-tomography FEI Titan Krios (80 - 300 kV) equipped with an energy filter and a direct detector camera. Conventional cryoEM microscope FEI F20 (200 kV) equipped with a CCD camera and a dual beam FIB/SEM instrument (Versa3D) for micromachining of thin lamellas of vitrified cells usable for electron tomography.
The Monolith systems measure equilibrium binding constants for a variety of molecules - thus allows to measure wide range of interactions from ion fragment binding up to interactions of large complexes (liposomes and ribosomes). This method allows to detect changes in hydration shell, charge or size of molecules and thus detect biomolecular interaction. Volume of buffer is kept constant in whole dilution series so the observed alterations in the thermophoretic depletion or enrichment can come only from changes in the size, charge or solvation entropy of the Auorescently labeled molecule caused by binding of non-labeled molecule to the labeled one.
SPR systems exploit the phenomenon of surface plasmon resonance to monitor the interaction between molecules in a real time. One of the interactants is immobilized on the sensor chip surface, while the other is passed over that surface in solution. Applications of SPR include biotherapeutic and drug discovery research, as well as protein activity and stability analysis. SPR is suitable also for characterization of membranes, lipids, nucleic acids and micellar systems. SPR system represents one platform for characterization of biomolecular interactions - kinetics, affinity, specificity, concentration and thermodynamics.
X-ray Diffraction and Bio-SAXS core facility is equipped with top-class instruments for diffraction experiments with single crystal samples focused on the determination of the 3-D structure of (macro) molecules down to atomic resolution and for small angle X-ray scattering (SAXS) experiments with isotropically scattering samples focused on determination of the shape and size of macromolecules or nanoparticles. The range of applicable molecular mass for diffraction methods: from 102 up to 106, where the lower value covers molecules significant for nanotechnology, materials science or pharmacology and the upper limit covers biomacromolecules such as nucleic acids, proteins and their complexes.
CIISB - Czech Infrastructure for Integrative Structural Biology is formed by two Centers of Excellence for Structural Biology constructed within the projects CEITEC – Central European Institute of Technology, Brno and BIOCEV - Biotechnology and Biomedicine Centre, Vestec, Prague-West. CEITEC and BIOCEV have been financed from the EU Structural Funds through the Operational Program Research and Development for Innovation, priority axis 1 – European Centers of Excellence, which is managed by the Ministry of Education, Youth and Sports of the Czech Republic. The Czech structural biology community is represented by the Czech Society for Structural Biology (CSSB), which is forming a national link to INSTRUCT. CIISB affiliation with INSTRUCT contributes to the development of human resources in research, attracts qualified national and international researchers, and enables efficient dissemination of knowledge and expertise within INSTRUCT, as well as efficient use of the infrastructure.
CEITEC is a scientific centre of excellence in the fields of life sciences, advanced materials, and technologies, which aims to establish itself as a recognized centre for basic as well as applied research. A consortium of partners includes the most prominent universities and research institutes in Brno, Czech Republic. CEITEC Core Faciilties for Integrative Structural Biology are part of the Structural Biology Program comprising research groups focusing on molecular biology and biochemical and structural characterization of cellular processes, including RNA, studies of the ribosome, understanding the structure-function relationships of proteins in pathogenic bacteria, fungi, and viruses, and on the acquisition of knowledge necessary for the development of biosensors for nanomechanical detection of clinical markers using their in vivo/in situ detection in real time.
Head of Core Facility: Assoc. Prof. RNDr. Radovan Fiala, CSc.
Key Equipment: NMR spectrometer for high-resolution spectroscopy in liquids (600 MHz, 700 MHz, 850 MHz and 950 MHz) • NMR spectrometer for high-resolution spectroscopy in liquids and solids (500 MHz and 700 MHz)
Expertise: High-resolution NMR of proteins, nucleic acids, and their complexes in liquids • Structure elucidation from NMR data at atomic resolution • Studies of bio-macromolecular dynamics using NMR, computational chemistry based studies of biomacromolecules
Open-access: Measurement of NMR spectra at magnetic fields ranging from 11.75 T to 22.32 T (corresponding to proton frequencies 500 MHz to 950 MHz) • Consultation of choice and setup of multidimensional experiments, data processing, and evaluation strategies • Measurements of complete sets of very small residual dipolar couplings (RDCs) and evaluation of their accurate values supported by in-house software • Cross-validation of RDCs in nucleic acid bases, peptide planes, and protein secondary structure elements • 3D structure calculations, validation of structure/restraints • Measurement, evaluation, and interpretation of relaxation data describing intramolecular motions • ab-initio calculation of NMR parameters, molecular dynamics simulations
Head of Core Facility: Jiří Nováček, Ph.D.
Key equipment: High-end electron microscope for high-resolution cryoEM and cryo-tomography FEI Titan Krios (80 - 300 kV) equipped with an energy filter and a direct detector camera • Conventional cryoEM microscope FEI F20 (200 kV) equipped with a CCD camera and a dual beam FIB/SEM instrument (Versa3D) for micromachining of thin lamellas of vitrified cells usable for electron tomography • Equipment for sample preparation • Vitrification robot Vitrobot Mark IV
Expertise: Studies, at the highest currently possible level, of cellular structures and organelles, protein complexes and various phenomena at the macromolecular level by 3D imaging
Open-access: Advanced and routine application of cellular electron tomography • Application of correlative methods integrating light/fluorescence microscopy with high resolution cryo-electron microscopy techniques • Single particle analysis • Preliminary negative stain/RT- and advanced cryo-electron microscopy methods • Assistance in integration of biochemical data into structural models using computational approaches • Supporting techniques - preparation of EM samples (negative stain and frozen hydrated), utilization of image processing tools, consultations, design of experiments, evaluation and interpretation of data, training
Head of Core Facility: Assoc. Prof. RNDr. Jaromír Marek, Ph.D.
Key Equipment: Rigaku HighFlux HomeLab™ robotized macromolecular diffraction system with ACTOR sample changer optimized for work at Cu-Kα wavelength • Rigaku HighFlux HomeLab™ universal, dual wavelength (Mo-Kα and Cu-Kα) diffractometer • Rigaku BioSAXS-1000 SAXS camera for small angle X-ray scattering from solutions of biological macromolecules
Expertise: Basic characterization of solutions of biological macromolecules by SAXS • Determination of a low resolution 3-D shape of biological macromolecules by SAXS • Testing of the diffraction quality of protein crystals, derivatives, etc. prior to data collection • Collection of diffraction data from crystals of biological macromolecules at home source • Data collection and solving of the crystal structures with non-biological single crystals
Open-access: Screening for the best diffracting protein crystal • In-house diffraction experiments with macromolecular crystal samples • Organization and coordination of diffraction experiment at the large scale diffraction facilities abroad • Complex multi-step experiments as e.g. SAD/MAD diffraction measurement with Se-Met derivatives; data collection and/or solving of structures of "small molecules" (with Mr < 5.103) • Supporting techniques - preparation of "special" samples, e.g. large scale production of Se-Met proteins • Consultations focused on interpretation of macromolecular experimental data/solving of protein structures
Head of Core Facility: Prof. RNDr. Michaela Wimmerová, Ph.D.
Key Equipment: Crystallization robot Mosquito and Tecan Evo 150 • Automated Desktop UV Minstrel + Gallery Hotel 160 for inspection of screening plates • Leica microscope with polarizing filter • Temperature optimizer for crystallization TG40 for 5 different temperatures • 96-well UVP screening plates and 24-well optimization plates • SPR BiaCore 3000 • Calorimeters AutoITC200, VP-ITC, VP-DSC• Analytical ultracentrifuge ProteomLab XLI • CD spectrometer Jasco 850 equipped with fluorescence detector • DLS Dynapro Plate Reader, DelsaMax
Expertise: High throughput screening and optimization of conditions for an ideal growth of perfect protein crystal(s) • Studies of physical properties of the molecules • Characterization of (bio)molecular interactions in a real time using mainly biosensor and calorimetry-based methods
Open-access: Crystallization of biomolecules and their complexes • Structure characterization of biomolecules • Basic characterization of physical properties of the molecules (analytical ultracentrifugation, dynamic light scattering, CD spectroscopy, differential scanning calorimetry) • Studying of thermodynamics and/or kinetics of interactions (isothermal titration calorimetry, surface plasmon resonance, CD spectroscopy, analytical ultracentrifugation)
Head of Core Facility: Assoc. Prof. RNDr. Petr Skládal, CSc.
Key equipment: Scanning probe microscope - Ntegra Vita / Solaris (NTMDT) • Atomic force microscope NanoWizzard3 (JPK) • ForceRobot 300 (JPK) • Automated system SolverNEXT (NTMDT) • Ink-jet based deposition system S3 (Scienion)
Expertise: Studies of biomolecules and other bio-objects at the molecular lever using scanning probe techniques, i.e. atomic force microscopy (AFM) and related techniques (STM, SNOM, electrochemical variants), including combinations with optical microscopy (inverted, fluorescence, confocal) • Production and bioconjugation of nanoparticles • Advanced methods - nanolithograpy, nanomechanical manipulations, ink-jet based deposition
Open-access: Preparation of samples for AFM • Atomic force microscopic (AFM) imaging in contact and non-contact modes in dry and wet conditions, using bare and functionalised scanning tips for bioforce and biointeraction studies • Advanced scanning techniques - combination of AFM with inverted optical microscopy, fluorescence and confocal microscopy, electrochemistry, near-field optical microscopy (SNOM) • Nanomanipulations, nanolitography, nanopatterning and nanodeposition of biomolecules
Head of Core Facility: Assoc. Prof. RNDr. Zbyněk Zdráhal, Dr.
Key equipment: High-resolution mass spectrometer (FTMS) with Orbitrap • Hybrid mass spectrometer (e.g. Q-LIT) with quadrupole and linear ion trap and liquid chromatography system • MALDI-TOF/TOF mass spectrometer
Expertise: All steps of proteomic analysis - protein isolation, separation of protein mixtures, protein characterization by mass spectrometry and bioinformatics data processing.
Open-access: Separation of protein mixtures • intact protein analysis • Peptide/protein profiling (e.g. microorganism characterization) • Protein identification • Characterization of protein modifications • Protein quantification • Data processing - image analysis of 2D gels, interpretation of mass spectrometric data • Teaching courses, hands-on courses • Consulting services