Centrum molekulární struktury
The Centre of Molecular Structure offers services focused on the characterization of biological molecules and of their complexes with ligands, protein and nucleic acids. The services offered include structure analysis with the use of X-ray diffraction, mass spectrometry and specialized analyses of biomolecular targets (target identification, modifications, ligand binding, folding state etc).
- BIOFYZIKÁLNÍ TECHNIKY
- Circular dichroism
- UV/Vis spectrophotometry
- Surface plasmon resonance
- Microscale thermophoresis
- Label-free thermophoresis
- Tyr and Trp fluorescence
- Isothermal titration calorimetry
- Differential scanning calorimetry
- Dynamic light scattering
- Static light scattering
- KRYSTALIZACE PROTEINŮ A NUKLEOVÝCH KYSELIN
- Crystallisation robot
- Crystallisation plate set-up
- Crystallisation hotel
- Remote crystallisation inspection
- DIFRAKČNÍ TECHNIKY
- Crystal structure
- X-ray Diffraction
- In-situ diffraction
- STRUKTURÁLNÍ HMOTNOSTNÍ SPEKTROMETRIE
- Coupled HPLC
- Hydrogen/Deuterium exchange
- Covalent labelling
Upřednostněný způsob rezervace CMS (Centrum molekulární struktury) podpory nebo použití zařízení v CMS je pomocí rezervačního systému CEITEC.
Registrace pro přístup do tohoto rezervačního systému je možná přes následující odkazy:
- Pro CMS-krystalizace (CMS-C), odpovědná osoba Dr. Jiří Pavlíček https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-C
- Pro CMS-dikrakce (CMS-D), odpovědná osoba Dr. Jiří Pavlíček a Dr. Karla Fejfarová https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-D
- Pro CMS-biofyzikální měření (CMS-BF), odpovědná osoba Dr. Tatsiana Charnavets https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-BF
- Pro CMS-strukturní hmotnostní spektrometrie (CMS-MS), odpovědná osoba Dr. Petr Pompach https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-MS
Do CMS je možný také přístup přes „European Instruct (Integrating Biology) network“ na stránkách: https://www.structuralbiology.eu/.
Jednotlivé rezervace mohou být také provedeny po dohodě s odpovědnou osobou za každou část CMS. http://www.biocev.eu/corefacilit/centrum-molekularni-struktury/#head_of_program.
Tým a kontakt
Frédéric Vellieux, Ph.D., HDR
- Managing scientist of the Centre of Molecular Structure
- +420 325 873 786
Tatsiana Charnavets, Ph.D.
- Scientist in charge of the biophysical measurements service
- +420 325 873 789
Ing. Jan Dohnálek, Ph.D.
- Guarantor of the CMS
- Biocev representative of Czech infrastructure for integrative structural biology - CIISB, Biocev representative of European infrastructure Instruct
- +420 325 873 758
Mgr. Karla Fejfarová, Ph.D.
- Scientist in charge of the X-ray diffraction service
- +420 325 873 759
Ing. Hana Hejnalová
- Administration, half time at CMS
- +420 325 873 703
RNDr. Jiří Pavlíček, Ph.D.
- Scientist in charge of the crystallization and X-ray diffraction service
- +420 325 873 787
RNDr. Petr Pompach, Ph.D.
- Scientist in charge of the mass spectroscopy service
- +420 325 873 785
Ing. Karel Pufler
- Technician, half time at CMS
- +420 325 873 787
Kde nás najdete v BIOCEV centru
- Quantification of binding affinity and kinetics;
- Determination of binding specificity and the number of binding sites;
- Characterization of membranes, lipids, nucleic acids and micellar systems.
- Concentration of ligand depends on the level of immobilization desired, generally 10–200 μg/ml. For kinetic analysis the best results are obtained by using a 100-fold range of analyte concentrations, 0.1–10xKd;
- Immobilization of one interacting partner is essential. The service can provid with a sensor chip, or with the user bringing own chip;
- The ProteOn acetate buffer (at pH 4.0, 4.5, 5.0, or 5.5) is recommended as immobilization buffer;
- Detection range: 170-1150 nm
- Peltier temperature control.
- Determination of protein folding;
- Characterization of protein secondary structure and DNA conformation;
- Detection of the changes in protein structure upon mutagenesis;
- Studying of conformational stability of proteins and DNA (pH stability, denaturant stability, temperature, buffers addition of stabilizers).
- The CDNN software package is available for detailed model-based analysis and predicting secondary structure using CD data;
- Software Global Analysis of multiwavelength kinetic data is available to fit multi-dimensional experimental data to one of a number of specified models.
- Measurement of CD spectrum for the determination of secondary structure of protein requires 160 µl of 0.1 – 0.2 mg/ml protein solution;
- Measurement of CD spectrum for the determination of DNA conformation requires 160 µl of 20 µM of solution or 1400 µl of 2 µM solution;
- Determination using a fluorescent dye or fluorescent protein of the affinity of interaction from 1nM to mM.
- Concentration of fluorescent labeled molecule: 10 nM – 10 mM;
- Final concentration of unlabeled molecule should be at least two orders of magnitude above the expected Kd value. To perform simulations of binding events and to help choose the appropriate concentration, the “Concentration Finder” software is available on the device control panel;
- At least 20 µl samples per capillary is needed.
- Direct measurement of submilimolar to nanomolar binding constants (10 3 – 10 9 M -1);
- Thermodynamic characterization of the molecular interaction in a single experiment (stoichiometry, Kd, ∆H and ∆S values);
- Calorimetric measurement over a range of biologically relevant conditions (temperature, salt, pH, etc.).
- The buffer solution, containing both the macromolecule and the ligand of interest, should be the same.
- The volume of the sample placed in the cell must be at least 300 µl. Preferably, the solutions of macromolecules should be dialysed against the buffer solution used for the ITC measurement;
- The ligand solution (the sample placed in the injection syringe) must have a volume at least 70.0 µl. Normally the ligand concentration should be 10 times as high as the concentration of macromolecule;
- In the case of high affinity interactions, the minimum concentration of macromolecule (that causes measurable heat effects) is 10 µM. For low affinity interactions the macromolecule sample concentration should be at least 5 times the Kd value;
- The buffers used should have low ionization enthalpies (e.g. phosphate, citrate, acetate);
- If the presence of reducing agent is required for a protein stability, then ß‑mercaptoethanol (at a concentration lower than 5 mM) or TCEP (lower than 2 mM) should be used rather than DTT.
Three-dimensional structure determination by macromolecular crystallography (protein/NA crystallization, X-ray diffraction structure determination)
Core Facility Services
The available state-of-the-art equipment for the biophysical characterization of biomacromolecules enables to perform studies on proteins, nucleic acids or complexes thereof for a wide range of molecular biology or structural biology research projects. Individual techniques are provided either as full service including analysis, or as the supply of dedicated machine time to trained users.
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.
The protein and nucleic acid crystallization facility provides the technologies for successful macromolecular crystallization, and subsequent steps (monitoring of the crystallization plates, crystal cryo-cooling (vitrification) in liquid nitrogen and long-term storage. The facility welcomes guests to use either complex approach to target crystallization with the use of all options available at CMS, or individual access to the facility equipment: “normal” (room) temperature, plus high and low temperature crystallisation (with dedicated high quality stereomicroscopes available for these temperatures), pre-crystallization characterization of samples by sub-microliter dynamic light scattering, time-line observation of macromolecular crystallization in combined UV/VIS and DLS monitoring for difficult projects, automated crystallization monitoring with remote access (in a crystallization hotel), crystal cryo-cooling (vitrification) equipment.
The X-ray diffraction facility enables in situ (i.e. in crystallisation trays) automated screening of crystals for X-ray diffraction without disturbing the crystallization drop and its precise condition, single crystal diffraction quality screening and X-ray diffraction data collection at room temperature and at cryo-temperatures (80-300 K). Diffraction data processing and structure solution and refinement can be provided on request. The facility can also provide regular synchrotron data collection and data analysis. Services are provided either as access to dedicated machine time for trained users, as partial services (for example up to data processing) and, to a limited extent, also as a full service.
The Structural Mass Spectrometry facility provides access to novel biomolecular mass spectrometry (MS) methods in order to make the characterization of protein structure and dynamics more rapid and routine. Methods include non-denaturing mass spectrometric approaches in combination with hydrogen-deuterium exchange, chemical crosslinking and other labeling techniques together with computational approaches. This toolbox will be made available to the broader scientific community, and will greatly enhance our ability to design new drugs and ensure the quality and efficacy of biopharmaceuticals, thereby benefiting human health.
Reference a publikace
SPOLUPRÁCE S MEZINÁRODNÍMI VÝZKUMNÝMI INFRASTRUKTURAMI A KONSORCII
Centrum molekulární struktury je součástí České infrastruktury pro integrativní strukturní biologii (CIISB; Czech Infrastructure for Integrative Structural Biology) – přidruženého národního centra Evropského strategického fóra pro výzkumné infrastruktury Instruct (ESFRI; European infrastructure for structural biology Instruct). Členové CIISB reprezentují českou strukturní biologii a Českou republiku jakožto jednu z ustavujících zemí konsorcia INSTRUCT.
BioRad ProteOn XPR36
Label-free quantitative analysis of biomolecular interactions by the technique of surface plasmon resonance (SPR).
The ProteOnTM XPR36 protein interaction array system enables label-free quantitative analysis of biomolecular interactions in real time using SPR technology. The ProteOn system allows to screen analytes simultaneously against 36 different targets of interest, enabling rapid comparison among large numbers of interactions.
SPR can be used for:
The recommended running buffer for most applications is the ProteOn phosphate buffered saline, pH 7.4 (10 mM sodium phosphate and 150 mM sodium chloride with 0.005% Tween 20).
Applied Photophysics Chirascan Plus ™ spectrometer
Measurement of circular dichroism spectra and absorbance as function of temperature, pH and concentration to determine the secondary structure of proteins and peptides, conformation of RNA and DNA, as well as to detect conformational changes.
The Chirascan Plus CD spectropolarimeter with avalanche photodiode detector – provides fast scanning and high sensitivity. This instrument can simultaneously measure accurate CD, absorbance and fluorescence data.
Circular dichroism can be used for:
NanoTemper Monolith NT.150
Used to study biomolecular interactions. The device allows to characterize protein-protein and protein-ligand (small molecule, DNA, RNA, peptides, sugars, lipids) interactions that can be measured under close to native conditions based on thermophoretic effect. Protein labeling is required with this device.
The Monolith NT.115 MST device allows to detect changes in hydration shell, charge or size of molecules and thus to detect biomolecular interactions.
MST can be used for:
Malvern Microcal iTC200
Label-free solution studies of biomolecular interactions.
The Malvern iTC200 instrument is used for the characterization of biomolecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids etc.
The iTC200 device can be used for: