Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University in Vestec
Analysis of intact proteins; protein identification (incl. protein complexes, de novo sequencing); identification of protein modifications; absolute and relative protein quantification
Classical and robotic crystallogenesis with remote experiment monitoring in three dedicated laboratories with strictly controlled temperature regimes with full backup. Automated screening of variation of crystallization conditions and its effects. Anaerobic crystallization and manipulation of biomacromolecules.
All services at CMS (Centre of Molecular Structure of BIOCEV) are available both to unexperienced users and to experienced users (that do not require the assistance of the scientist in charge). Biophysical core research facilities offer a range of services, including investigations of biomolecular interactions, of structure, stability and conformation of DNA and proteins, determination of hydrodynamic radii, zeta potential and electrophoretic mobility of molecules, together with crystallization screens.
Calorimetric measurement of protein-ligand interaction (Standard titration method, Single injection method) Competitive-based measurement - low or high affinity interactions Data evaluation - thermodynamic parameters determination using curve fitting models: One set of binding site, Two sets of binding sites Eventuality of manual data evaluation using fitting models: Sequential binding sites, Competitive binding, Dissociation
The imaging of biomolecules, cells and other biological structures and objects is realized in aqueous solution or in the dry state. The carrier materials for sample deposition range from ultra flat mica slides suitable for atomic resolution to highly oriented graphite, gold, silicon, glass and polymers as polystyrene petri dishes. Different scanning tips with appropriate sharpness and cantilevers with a wide range of force constants.
Bruker D8 Venture diffractometer with a high-flux liquid Gallium X-ray source MetalJet D2, Photon II detector and Kappa goniometer. The diffractometer is used (at CMS) for X-ray diffraction of biomolecular crystals. ISX stage for D8 Venture – motorized stage for in-situ X-ray diffraction experiments, enabling screening of diffraction properties in crystallization trays.
Basic characterization of solution of biological macromolecules by SAXS Determination of a low resolution 3-D shape of biological macromolecules by SAXS SAXS characterization of non-biological nanostructures Test of a diffraction quality of protein crystals, derivatives, cryoprotectants etc prior data collection Collection of diffraction data with crystals of biological macromolecules Data collection and solving of the crystal structures with non-biological single crystals Collection of high angle diffraction data with non-biological single crystals Collection of diffraction data with small and/or weekly diffracting non-biological single crystals
Besides providing the access to the spectrometers, the core facility offers consultations on the choice of suitable NMR measurements, support in setup and running the experiments, processing the data and evaluation of the results. The facility can run the latest multidimensional measurements using non-uniform sampling methods and direct carbon detection. In collaboration with the research groups of Protein Structure and Dynamics and Structural Biology of Gene Regulation, the users can in collaborative projects utilize the expertise of the groups in studies of the structures, dynamics and interactions of proteins and nucleic acids, including the use of available hardware and software.
The cryo-EM core facility provides access and support to collect cryo-electron microscopy images for both single particle as well as for electron tomography applications. Furthermore, the facility provides support for sample preparation, namely plunge freezing of purified protein complexes as well as cryo-FIB lamella milling for thick biological objects such as cells. Additional support is given in experiment design, setting up data collection and data analysis (i.e. image processing). Interested non-specialists could receive training in using the electron microscopes or develop a collaboration with the CryoEM research group, particularly in these areas: high-resolution structure determination of macromolecular complexes and assemblies structural studies of intracellular compartments and host-pathogen interactions time-resolved electron microscopy of transient macromolecular complexes
The utilization of high-resolution mass spectrometry (15T FT-ICR MS) to determine the composition of molecules (metabolites, nucleic acid, proteins, and carbohydrates) based on accurate mass measurements and fragment pattern. FT-ICR MS is equipped with atmospheric pressure ionization technique (electrospray) and various types of vacuum ionization techniques (laser desorption, matrix-assisted laser desorption). FT-ICR MS is able to perform sustained off-resonance irradiation, collision-induced and electron transfer/capture dissociations. Mass spectrometric cutting-edge analysis of post-translational modifications, and of structural states of proteins and complexes in solution. Combination of covalent surface labeling, isotope (H/D) exchange, chemical cross-linking.
motorized stage for in-situ X-ray diffraction experiments, enabling screening of diffraction properties in crystallization trays
Agilent Technologies 1200 HPLC system (usually coupled to the 15T-SolariX XR mass spectrometer) for the separation of complex peptide mixtures, proteins and metabolites
small dual beam microscope (SEM/FIB) equipped with a Quorum Technologies PP3010T cryo-preparation system for delivery of vitrified samples into the microscope. System is optimal for preparation of thin lamellas from vitrified cells for the purposes of in situ structural biology studies 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
Spectrolight 600 - in drop dynamic light scattering measurements in Terasaki 72-well plates
Transmission electron microscope operating at 20–200 kV equipped with Schottky field emission gun (SFEG), and a FEI Eagle 4k x 4k CCD camera. Sample is inserted using either side-entry cryo-holders (Gatan 626 and Gatan 914) or room-temperature side-entry holders. The microscope is well suited for optimization of cryoEM samples and automated data collection by single particle analysis or (cryo-)electron tomography
Robotized macromolecular diffraction system with ACTOR sample changer optimized for work at Cu-Kα wavelength
Rigaku BioSAXS-1000 SAXS camera for small angle X-ray scattering from solutions of biological macromolecules
ArtRobbins Gryphon dropsetter - a multi-channel (96 channels) pipetting robot for the easy set-up of nanodrop crystallisation plates
Formulatrix RI1000 crystallisation hotel - a crystallisation plate storage and automated crystallization monitoring enclosure allowing remote access to crystallization images
FEI Titan Krios – high-end transmission electron microscope operating at 80 - 300 kV equipped with Schottky field emission gun (XFEG) providing highly coherent electron beam with ultimate stability. The microscope is equipped with hole free Volta phase plate for increase of contrast, Thermo Fisher Scientific Falcon3EC direct electron detector, and Gatan K2 direct electron detector positioned at the end of post-column imaging energy filter (GIF Quantum LS imaging filter). Automated collection of single particle analysis data can be performed with EPU software, cryo-electron tomography data can be acquired using Tomography4 or SerialEM software