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Crystallography Unit

Coordinator

Sebastiano Pasqualato

"Almost all aspect of life are engineered at the molecular level, and without understanding molecules we can only have a sketchy understanding of life itself" Francis Crick (1988), What Mad Pursuit: A Personal View of Scientific Discovery, p. 61

Mission and services of the Crystallography Unit
X-ray crystallography is the foremost technique used in structural biology to determine the three-dimensional structure of macromolecules. Over the years, it has allowed paramount achievements, such as the elucidation of the DNA structure, the mechanisms of action of enzymes, what ribosomes, molecular motors or viruses look like and how they work, how proteins interact with each other and with DNA.

During the last decade, X-ray crystallography have greatly benefit from important technological developments, that have widely broaden its accessibility. High-throughput robotics allows the quick set up of crystallization trials using minimal amounts of sample; use of brilliant synchrotron light sources and cryo-temperatures permit very fast data collections; improvement of programs and computing power greatly smoothened the steps of structure determination and model refinement.

Still, a great deal of attention has to be paid to sample preparation and characterization, crucial parameters to increase the chances of success of the crystallization step.

The Crystallography Unit supports scientific groups which are interested in developing structural biology aspects of their projects. It provides both its experienced assistance and specific services for protein expression, purification, characterization, crystallization and structure determination.

We manage a platform for protein expression in insect cells, purification and characterization of proteins and macromolecular complexes and state-of-the-art infrastructures for X-ray Crystallography.

  • Research activities

    Crystallography


    The Unit offers the following services:

    • assistance with target design and cloning strategies, protein production and purification;
    • protein expression in insect cells via the baculovirus expression system;
    • biochemical characterization of macromolecular samples;
    • measurements of binding affinity of macromolecular complexes;
    • set up of crystallization trials and crystal optimization;
    • crystal harvesting, data collection, structure determination and analysis.


    Assistance with target design, protein expression and purification
    To increase the chances of crystallization, macromolecular samples should ideally be highly pure, concentrated and monodisperse. To match these criteria, expression of the best protein constructs in terms of stability and solubility has to be achieved.
    The Unit provides assistance in analysis of secondary structure predictions, as well as in strategies of protein expression and purification. Our experience in proteins co-expression and complex purifications might be particularly helpful to those interested in studying molecular mechanisms of complex biological systems.

    Protein expression in insect cells via the baculovirus expression system
    The baculovirus expression system permits high levels of expression of recombinant proteins in insect cells. With respect to bacteria, it has the advantages of allowing production of larger targets, which will get properly post-translationally modified by the eukaryotic cell. A recent development of this technology is the MultiBac system, which was design to express multi-protein complexes.
    At the Crystallography Unit, we routinely express proteins and multi-protein complexes in insect cells with the MultiBac system, and offer a complete service that goes from gene cloning to protein production.

    Biochemical characterization of macromolecular samples
    To determine oligomeric states of macromolecular samples in solution, a Static Light Scattering instrument is available. The sample is run on a size exclusion chromatography and the eluted material is analyzed through a triple detector array that allows the determination of the absolute molecular weight of the species in solution.
    Protein stability in different buffer conditions can be assessed by Differential Scanning Fluorimetry. This technique is also successfully used to identify low molecular weight ligands that bind and stabilize purified proteins.
    The Unit offers these analyses as service to users.

    Measurements of binding affinity of macromolecular complexes
    Protein-protein, protein-DNA or protein-ligand binding affinities and interaction stoichiometries can be measured using an Isothermal Titration Calorimetry instrument. If the complex involves binding of a protein to small polypeptides or oligonucleotides, its binding affinity can also be determined using lower amounts of samples through Fluorescence Polarization.
    The Unit provides assistance in designing and performing these measurements.

    Set up of crystallization trials and crystal optimization
    We have established an automated platform for the high-throughput crystallization of macromolecular samples, in order to maximize the success rate of initial trials with minimal amounts of sample.
    Initial crystallization trials, chosen among a panel of a dozen commercial screens available at the Unit, are automatically set up by a nano-dispenser robot. This allows screening of 288 conditions with 50 μl of sample, using the sitting-drop vapor diffusion method with 200 nl drops in 96 well plates. These screening can be stored at 20 °C or at 4 °C. Plates at 20 °C are automatically imaged on a standard schedule, and drop images are available online to users, who can follow remotely crystal appearance and growth.
    Optimization of initial hits is typically performed manually in 24 well plates with the hanging drop method in 2 μl drops. In this cases, tailored screens are prepared based on initial hits conditions.
    The Crystallography Unit offers a complete service, from trials set up to crystal optimization, or simple access to reagents and instrumentation for expert users.

    Data collection and structure determination
    In order to determine the atomic resolution structure of macromolecules, crystals need to be irradiated with a monochromatic and highly collimated X-ray beam and the diffraction pattern images have to be collected and processed.
    The Crystallography Unit offers the service of data collection at synchrotron light sources and data processing, which leads to structure determination. Thanks to the close collaboration with the scientific group of Marina Mapelli, the Unit routinely gets peer-reviewed allocation of beamtime at the Europena Synchrotron Radiation Facility (ESRF -Grenoble -F) and Swiss Light Source (SLS - Villigen - CH) beamlines. Data are processed in-house with crystallographic software suites installed and maintained by the Unit with the help of the IEO ICT service.

A SMALL GESTURE IS A GREAT HELP FOR US

PARTNERSHIP

Università degli Studi di Milano Ecancer Medical Science IFOM-IEO Campus

CREDITS

Ministero della Salute Joint Commission International Breastcertification bollinirosa

© 2013 Istituto Europeo di Oncologia - via Ripamonti 435 Milano - P.I. 08691440153

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