Centre of Molecular Structure
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).
Preferred access modes:
Through the CIISB, with information available on the proposal submission page http://www.ciisb.org/open-access/proposal-submission
For Instruct country members ( https://www.structuralbiology.eu/content/countries–instruct ), access including partial cover of travel expenses can be obtained by means of the INSTRUCT European Research Infrastructure Consortium through its proposal submission web page https://www.structuralbiology.eu/submit-proposal?t=instruct
- BIOPHYSICAL TECHNIQUES
- 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
- CRYSTALLIZATION OF PROTEINS AND NUCLEIC ACIDS
- Crystallisation robot
- Crystallisation plate set-up
- Crystallisation hotel
- Remote crystallisation inspection
- DIFFRACTION TECHNIQUES
- Crystal structure
- X-ray Diffraction
- In-situ diffraction
- STRUCTURAL MASS SPECTROMETRY
- Coupled HPLC
- Hydrogen/Deuterium exchange
- Covalent labelling
The preferred way of booking CMS services / use of CMS equipment is through the CEITEC on-line booking system. On-line registration to access this booking system is available as indicated below:
- For the CMS-crystallisation CF (CMS-C), scientist in charge Dr Jiří Pavlíček https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-C
- For the CMS-diffraction CF (CMS-D), scientists in charge Drs Jiří Pavlíček and Karla Fejfarova https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-D
- For the CMS-Biophysical measurements CF (CMS-BF), scientist in charge Dr Tatsiana Charnavets https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-BF
- For the CMS-Structural Mass Spectrometry CF (CMS-MS), scientist in charge Dr Petr Pompach https://idm.ics.muni.cz/fed/registrar/?vo=ceitec&group=CMS-MS
In addition, the services/equipment may be accessed through the European Instruct (Integrating Biology) network (https://www.structuralbiology.eu/ ).
Finally, reservations may also be made by contacting directly the scientists in charge of the respective Core Facilities http://www.biocev.eu/corefacilit/centrum-molekularni-struktury/#head_of_program.
Team and contacts
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
Dr. Jan Dohnálek
- Guarantor of the CMS
- Biocev representative of Czech infrastructure for integrative structural biology - CIISB, Biocev representative of European infrastructure Instruct
- +420 325 873 758
Karla Fejfarová, Ph.D.
- Scientist in charge of the X-ray diffraction service
- +420 325 873 759
- 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
- Technician, half time at CMS
- +420 325 873 787
Where to find us in BIOCEV center
- 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.
- Size (diameter): from 0.3 nm to 5 microns;
- Molecular weight measurement down to 10 kDa;
- Temperature range 0-90°C.
- Particle size analysis (hydrodynamic radius);
- Temperature range 0-90°C.
- 25 µl of sample and the same volume of “empty“ buffer ;
- For protein solutions, concentrations of at least 0.2 mg/ml;
- For the measurements of zeta potential in folded capillary cells, 0.75 ml of sample is required.
- The label and immobilization free determination of protein binding to Ions, nucleic acids, small molecules and sugars (with an affinity of interaction in the range of 10 nM to mM).
- Concentration range of tryptophan-containing protein: 100 nM-10 µM;
- Final concentration of unlabeled molecule should be at least an order of magnitude or more above the expected Kd value ;
- Molecular weight range: 10-107 Da;
- Determination of thermal transition temperatures and stability of proteins.
- Protein must contain tryptophans in order to detect protein unfolding;
- Sample concentration range: from 5 µg/ml to 250 mg/ml;
- Prepare at least 20 µl of your samples;
- For thermal unfolding experiments no assay development or special sample preparation is needed
- 190-1100 nm;
- 50-1500 µl of sample;
- Scanning, dual beam
- Temperature control with Peltier element, scan-range 5-95ºC.
- Proteins and DNA thermostability measurements;
- 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;
- In-situ (in the crystallisation plates) testing of crystal diffraction using the ISX stage;
- Testing of diffraction using mounted crystals and / or measurement of X-ray diffraction data;
- Diffraction data processing, providing a data file (such as an MTZ file);
- Assistance / advice to solve a 3D structure (including a full 3D structure determination service on request);
- Protein molecular weight determination by ultra-high resolution FT-ICR mass spectrometer with sequence confirmation by Top-down approach using different fragmentation techniques (collision induced dissociation, electron transfer/capture dissociation);
- Peptide mass fingerprinting – identification of proteins from gel or solution including larger protein mixtures by using MALDI or ESI ;
- Characterization of posttranslational modification such as phosphorylation, glycosylation or disulphide bonds ;
- Structural mass spectrometry: limited proteolysis, hydrogen/deuterium exchange, chemical cross-linking, covalent labelling;
- HPLC separation of peptides, proteins and small molecules (metabolites) coupled with mass spectrometric detection by FT-ICR;
- Processing and interpretation of mass spectrometric data.
Applied Photophysics Chirascan Plus ™ spectrometer
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:
Malvern Zetasizer Nano ZS90
Measurement of molecular size using Dynamic Light Scattering (DLS), zeta potential and molecular weight using Static Light Scattering.
The Zetasizer Nano ZS90 instrument is used for the measurement of particle and molecular size using Dynamic Light Scattering, with the option of measuring zeta potential and electrophoretic mobility, and molecular weight using Static Light Scattering.
Established methodologies and provided services
Characterization of protein-ligand interactions based on thermophoretic effect, using the intrinsic tryptophan fluorescence. No sample modification is required with this device.
The NT.LabelFree MST instrument uses intrinsic tryptophan fluorescence for microscale thermophoresis detection, thereby allowing label-free and immobilization-free experiments.
MST can be used for:
Minimum sample volume used: 10 µl per sample
NanoTemper Prometheus NT.48
Measurement of protein stability using tyrosine and tryptophan fluorescence.
The Prometheus NT.48 instrument measure native DFS to determine protein thermal transition temperatures and stability of 48 up to samples at a time.
No dye is required, tryptophan fluorescence at 330 nm and 350 nm is detected;
Temperature range: from 15 °C to 95 °C.
DFS can be used for:
AnalyticJena SPECORD® 50 PLUS i
Molecular absorption spectroscopy with ultraviolet and visible radiation in the spectral range 190 to 1100 nm.
The AnalyticJena SPECORD 50 PLUS device is a UV/Vis double-beam spectrophotometer with split-beam technology that combines high energy throughput with good stability.
The spectrometer can be used for:
With or without stirrer can be used for enzyme kinetics.
Malvern Microcal VP-DSC
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).
Crystallization of proteins and nucleic acids
Formulatrix RI 1000 crystallisation hotel
Crystallisation plate storage and automated crystallization monitoring enclosure allowing remote access to crystallization images. Crystallisation drop images can be taken using visible light, polarized light and UV.
A service offered is the remote (web-based) access to crystallisation droplet images, and automated preliminary evaluation of crystallisation experiments.
ArtRobbins Gryphon dropsetter
A multi-channel (96 channels) pipetting robot for the easy set-up of nanodrop crystallisation plates.
This crystallisation robot equipment allows the robotic setup of 96-well crystallisation plates, for screening of crystallisation conditions (and also for “routine crystal production”) - for proteins, nucleic acids, complexes of biological macromolecules;
In drop dynamic light scattering measurements in Terasaki 72-well plates. This equipment allows to check the monodispersity of the macromolecule in drops, thereby allowing to check the suitability of the sample for crystallisation.
Bruker D8 Venture diffractometer
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.
This equipment allows to offer the following services:
Upon request: Measurement of X-ray diffraction data sets at synchrotron radiation sources.
Structural mass spectrometry
Bruker Daltonics 15T-Solarix XR FT-ICR mass spectrometer (associated with Agilent Technologies 1200 HPLC system)
Bruker Daltonics 15T-Solarix XR FT-ICR mass spectrometer, with electrospray and MALDI ion sources. This ultra-high resolution mass spectrometer is used mostly for the determination of the precise mass of biological macromolecules, and the characterization of their posttranslational modifications. Further possibilities include peptide mass fingerprinting, detection of small molecule/metabolites, monitoring of protein structural changes and protein-protein interactions under physiological conditions by hydrogen-deuterium exchange, chemical cross-linking and covalent labelling.
Agilent Technologies 1200 HPLC system (usually coupled to the 15T-SolariX XR mass spectrometer) for the separation of complex peptide mixtures, proteins and metabolites.
This equipment allows to provide the following services:
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.
References and Publications
CONNECTION TO INTERNATIONAL RESEARCH INFRASTRUCTURES OR CONSORTIA
The Centre of Molecular Structure belongs to the Czech Infrastructure for Integrative Structural Biology (CIISB) – an affiliate center of the European infrastructure for structural biology Instruct (ESFRI). Members of CIISB represented the Czech structural biology community and the Czech Republic as one of the countries at the establishment of the Instruct consortium.