Czech Infrastructure for Intergrative Structural Biology
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.
NMR (Nuclear Magnetic Resonance) spectroscopy is a key technology for
research in modern life sciences allowing detailed investigation of
biomolecular structure and dynamics at the atomic level, both in
solutions and in solid state. The successful application of NMR in
biology requires multidisciplinary approach combining knowledge in
biochemistry, molecular biology, quantum physics, electronics, data
analysis, and computational chemistry. The high-end instrumentation and
the team of experienced researchers ensures expert services, user
training, and the cost-effective use of resources. Benefits include access to state-of-the-art high-field
NMR instrumentation and support in processing, analysis and
interpretation of the experimental data. External user projects are selected by peer review on the basis of scientific merit, technical
suitability and feasibility. The Centre also offers training
enabling non-specialists to develop the necessary skills.
The platform provides instruments and services leading to the biophysical characterization of (bio)molecules and the sample quality control using analytical ultracentrifugation (AUC), dynamic light scattering (DLS), differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF).
The platform provides instruments and services leading to the characterization of biomolecular interactions (determination of affinity, kinetic and thermodynamic parameters) using isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST) and analytical ultracetrifugation (AUC).
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 100 up to 1000000, 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.
Range of applicable particle sizes for SAXS: from 2 to 100 nm.
The diffraction of X-rays in single crystal samples is the most important and – if an appropriate sample is available – also the fastest methodology currently available for the determination of the atomic structures of molecules and/or macromolecules and their complexes.
On the other hand, SAXS is a technique capable of determining structural characteristics such as mono dispersity or aggregation, oligomeric state, low resolution three-dimensional shape or even quaternary structure not from a crystal but from a solution of (bio) macromolecules.
The centralized organization of expensive instrumentation for SAXS and single crystal studies and highly trained staff allow the cost-effective use of resources and obtaining of experimental results in rapid responses to the demands of even untrained users.
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
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
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