INSTRUCT - Core Centre G: Max Planck Institute of Biophysics
Within ESFRI and INSTRUCT (“Integrated Structural Biology Infrastructure for Europe”) we run the core Centre G, which is devoted to membrane proteins.
As outlined before we try to understand membrane proteins based on an accurately known structure. One emphasis of our work therefore is the structure determination of proteins and protein complexes integrated into the cellular membranes of living organisms. The importance for life of membrane proteins is due to the fact that integral membrane proteins perform a huge variety of duties, for the communication of the cell with its environment, like signal exchange, as well as energy transduction and specific uptake and/or release of substrates of all kind.
Four different systems will be open to external users of the core centre:
1.) Mass spectrometry facility : A mass spectrometer (MS) equipped with a quadrupol and collision cell for MS/MS experiments and a time-of-flight chamber for highest available mass resolution. Two liquid chromatography systems will be coupled to the MS: For protein identification of transmembrane proteins using tryptic digests, a nano liquid chromatography system will be directly connected to the MS by an online ESI source. For lipid analysis, another liquid chromatography system will be linked to both the mass spectrometer and an evaporative light scattering detector for simultaneous qualitative and quantitative analysis of the sample.
2.) A crystallization robot consisting of two pipetting stations for screen development and crystallization plate setup, respectively, and six incubators sharing three imaging stations. The system can be enlarged by an additional crystallization plate pipetting station, to double the throughput.
3.) Two very powerful microfocus rotating anode X-ray generators equipped at one port with a CCD detector on a very high flux optics, to be used in first preference for crystal screening, and at the second port equipped with a high resolution optic and an image plate for precise data collection. Both screening stations will be equipped with automatic crystal mounting and centering robots.
4.) A MicroCal Capillary Differential Scanning Calorimeter (DSC) is available for determining the stability of investigated proteins. This is of particular relevance for membrane proteins, because their stability depends on the detergent used. The instrument will be equipped with an autosampler for high throughput.
Liquid chromatography - electrospray ionization quadrupol TOF mass spectrometry
The mass spectrometer system has a very small foot-print, since the TOF chamber is mounted on top of the machine. This leads to an overall height of the spectrometer of more than 3 meters. The system itself is mounted on a vibration damping stand, sitting directly on the lab floor in a room specifically designed to accommodate this mass spectrometer. The new damping is one of the reasons why the maXis is able to reach the high accuracy of below 1ppm, otherwise only possible with cyclic flight chambers. The nanoLC and the mass spectrometer are linked by an online ESI source. For standard protein identification, the sample is typically cleaved using a tryptic digest, the fragments separated on a LC reverse phase column and directly submitted to tandem-MS analysis using electrospray ionization of the eluate.
Lipid molecules bind to pockets of the hydrophobic surface of membrane proteins forming protein-lipid complexes. Specific lipids are often essential for membrane protein stability and function. If these essential lipid molecules are lost during the purification process, addition of the correct lipid molecules to the crystallization buffer could improve the crystal quality or allow crystallization at all. For lipid analysis different solvent systems and stationary phases are required. Thus a second LC system will be coupled to the MS, with a simultaneous link to an evaporative light scattering detector for quantification of the lipids bound to the target transmembrane protein.
Mass spectrometry is often used to determine prior to crystallization the accurate mass of the protein. However, this mass may not correspond to the mass of the protein which subsequently crystallizes. Protein-degradation or incorporation of more than one form of the same protein in the crystal could influence its quality. Thus, the knowledge of the accurate mass present in the protein can be used to aid structure solution or to improve crystal quality.
Automated protein crystallization platform
The crystallization robot automates several operations, which involved manual interaction up to now:
The heart of the system is the IntegrationModule containing a robot arm mounted to rails running all the way from the left to the right end of the module. On one side are the pipetting stations located and on the other side are the incubator pairs with shared imaging system attached to the IntegrationModule. The robot arm is responsible for moving the plates from one station to the next. Controlled is the system by the CrystalTrak software.
The control software will be installed on a redundant server system with a fiber-channel attached RAID system. The CrystalMation system itself may be stopped by a computer or hardware failure, but the images remain accessible due to this redundant system.
The ease of detecting crystals in the crystallization wells can be improved by upgrading the MinstrelHT with UV units. To improve the crystal quality by increasing or decreasing the speed of the crystallization process, we provide with each of the six incubators a different crystallization temperature. We offer crystallization screens optimized for membrane proteins.
Two X-Ray generators each equipped with CCD Detector and VHF Optics on one port and Image Plate with HR Optics on the other
The lab is equipped with an extremely powerful micro-focus rotating anode generator. The FR-E+ generator is by a factor of 2-3 more powerful than the MicroMax-007HF generator. Together with the Saturn 944+ CCD detector which is by a factor of 3 to 4 more efficient than an image plate, the new crystal screening station gains an enormous increase in X-ray power in our lab. This increase is often necessary to avoid synchrotron trips only for screening crystals. For efficient high throughput it is necessary to obtain a real advice, whether new crystallization conditions improved the crystal quality, using our in-house equipment. To improve the throughput even further, the crystal screening stations will be equipped with Actor crystal mounting robots. Automatic alignment of the mounted crystal is achieved by centering the center of the cryo-loop. To secure correct centering of tiny crystals of the loop center, an edge detecting system will be necessary.
Capillary differential scanning calorimeter
Differential scanning calorimetry is unsurpassed as a method to determine the stability of biological materials. It measures directly heat quantity changes that occur during a controlled increase or decrease in temperature. An unfolding transition shows up prominently in a plot vs. temperature. The heat or enthalpy of unfolding (ΔHº) and the heat capacity of unfolding (ΔCp) at the transition midpoint (T0) can be readily determined from these data. The method can be used to study interactions of proteins with lipids, small ligands or other proteins. Addition of a ligand to the protein solution can either stabilize the folded or the denatured state and thus evoke a new transition midpoint (TM). The binding constant between ligand and protein KB can be determined from T0, TM, ΔHº, ΔCp, and the concentration of the added ligand. Although it is applicable to interactions of any strength, it is often the method of choice of ultra-tight interactions, when other methods fail.
BMBF: INSTRUCT start up grant
EU-INSTRUCT preparatory phase grant
Dr. Juergen Koepke
Phone: +49 (0) 69 6303 1055
Fax: +49 (0) 69 6303 1002