Mission To support the research and educational goals of members of the University in all areas of structural biology. In particular, to provide

Transcription

1

2 Mission To support the research and educational goals of members of the University in all areas of structural biology. In particular, to provide access to state-of-the-art instrumentation and expert advice in all major areas of contemporary structural biology research, including X-ray crystallography, electron microscopy, computational studies, and modeling.

3 Structural biology is a branch of molecular biology, biochemistry, and biophysics concerned with the molecular structure of biological macromolecules, especially proteins and nucleic acids, how they acquire the structures they have, and how alterations in their structures affect their function. This subject is of great interest to biologists because macromolecules carry out most of the functions of cells, and because it is only by coiling into specific three-dimensional shapes that they are able to perform these functions. This architecture, the "tertiary structure" of molecules, depends in a complicated way on the molecules' basic composition, or "primary structures." Biomolecules are too small to see in detail even with the most advanced light microscopes. Wikipedia

4

5 Macromolecular structures can range in molecular weight from a few thousands of Daltons to millions of Daltons. They can be individual proteins or nucleic acids or complicated complexes of them Range in size from tens of Ångstroms to hundreds of Ångstroms.

6 Structural information is crucial to understand the function of biological macromolecules. The structure does not only provide information on the general architecture and fold of a macromolecule, but also on the way it interacts with other molecules. From : Sci Rep Sep 8;6: doi: /srep Structural insights into Parkin substrate lysine targeting from minimal Miro substrates. Klosowiak JL, Park S, Smith KP, French ME, Focia PJ, Freymann DM, Rice SE.

7 Structural studies have become integral to drug design studies, from the design stage to validation From : PLoS One Jun 26;8(6):e doi: /journal.pone Print Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction. Watterson DM, Grum-Tokars VL, Roy SM, Schavocky JP, Bradaric BD, Bachstetter AD, Xing B, Dimayuga E, Saeed F, Zhang H, Staniszewski A, Pelletier JC, Minasov G, Anderson WF, Arancio O, Van Eldik LJ.

8 Structural studies of large multicomponent assemblies have become possible in the last few years thanks to major technological advances From : Nature May 11;533(7603): doi: /nature Near-atomic resolution visualization of human transcription promoter opening. He Y, Yan C, Fang J, Inouye C, Tjian R, Ivanov I, Nogales E.

9 Brief history Started as Computational Facility supported by the Cancer Center ~25 years ago. No personnel Expanded to ~20 years ago adding 3 people and providing support for X-ray crystallography both in-house and at the Advanced Photon Source Constant renovation of the Facility, including stopping support for in-house X-ray crystallography Merged with CryoEM Facility in 2016

10 Organization

11 Major services Support and advice on all aspects of macromolecular crystallography experiments, including sample preparation, crystallization, data collection, and structure determination Support and advice on macromolecular EM, including sample preparation, data collection, and data analysis Support and advice on many different types of computational projects Crystallographic and EM data collection services Crystallographic and EM data analysis services Complete crystallographic and EM analysis services

12 Major resources Organize and facilitate access to the Life Sciences Collaborative Access team (LS CAT) at the Advanced Photon Source, Argonne National Laboratory Access to crystallization robots for macromolecular crystallography Access to large computational servers for data storage and processing Access to cryogenic Electron Microscope (cryoem) and smaller EM Access and support to large group of data processing and analysis programs from crystallography, NMR, electron microscopy, molecular modeling, etc.

13 Human resources Pamela Focia, Ph.D. Expert in macromolecular crystallography with extensive experience in all aspects of the work Jason Pattie. Senior computer systems administrator Jonathan Remis, M.Sc. Expert in electron microscopy, including single particle analysis and tomography. Valerie Tokars, Ph.D. Expert in macromolecular crystallography, including drug design.

14

15 The Advanced Photon Source

16

17 LS CAT Current membership Institution Northwestern University (Managing Partner) University of Michigan Van Andel Research Institute Michigan State University Wayne State University Vanderbilt University Medical Center University of Wisconsin-Madison University of Illinois Grand Valley State University Cayman Chemical Glaxo Smith Kline Beryllium Biostructures Genentech Corporation Medical College of Wisconsin Rice University Type Member Member Member Member Member Member Member Member Associate Member Associate Member Associate Member Associate Member Associate Member Associate Member Associate Member

18 DND-CAT Management Board NU VP Research Jay Walsh LS-CAT Management Board LS CAT Management NU Synchrotron Research Center Michael Bedzyk, Co-Chair Alfonso Mondragón, Co-Chair DND-CAT Denis Keane, Director LS-CAT Alfonso Mondragón, Scientific Director Keith Brister, Manager

19 LS CAT Design 21-ID-D MD-2 Diffractometer with mini-kappa CATS Robot system with SPINE and ACTOR support Dectris 6M Eiger detector 21-ID-F and 21-ID-G MD-2 Diffractometer with mini-kappa CATS Robot system with SPINE and ACTOR support Rayonix MX225 (21-ID-F) Rayonix MX300 (21-ID-G)

20 What can you do with bad crystals and a good beamline? Bacterial RNase P holoenzyme in complex with trna

21 Experimental electron density map Refinement: 3.8 Å resolution R work / R free: 25 % / 27% protein trna 5 P RNA Reiter, N.J., Osterman, A., Torres-Larios, A., Swinger K. K., Pan T., Mondragón A. Structure of a bacterial ribonuclease P holoenzyme in complex with trna. Nature, 468, , 2010.

22 What if something does not crystallize or if it is too big or has many different conformation? Electron microscopy is emerging as the technique of choice for many structural biology problems

23 CryoEM of P22 procapsid images Field Emission Gun, 400 kv Top: Close to focus. Defocus value ~ 0.95 µm Bottom: Defocus value ~ 2.7 µm 500 Å

24

25 JEOL 1400 JEOL 3200FS with Gatan K2 Summit Direct Electron Detector

26 Tomography

27 Negative stain structure of GyrA- CTD

28 From : Nature May 11;533(7603): doi: /nature Near-atomic resolution visualization of human transcription promoter opening. He Y, Yan C, Fang J, Inouye C, Tjian R, Ivanov I, Nogales E.

29 Both EM and crystallography generate enormous amounts of raw data that needs to be processed. Moving the data from the data collection instruments to the cluster used for analysis is a challenge in itself. The Eiger detector generates tens of gigabytes per data set. The CryoEM can generate terabytes per data set. The computer infrastructure needed is very similar for many of our applications: large amount of storage space, fast access to the data, fast processors, sophisticated workstations for visualization.

30 Where do go from here? Increase our involvement with outside groups, both in academic institutions and companies Increase the number and type of services we provide More involvement in routine crystallographic investigations, for example in drug discovery studies More support for neophyte research groups More data collection services for laboratories that do not want to collect their own data EM data collection services and analysis for outside groups New areas that we want to explore and develop: Cryo-tomography Micro electron diffraction (microed)

31 Challenges Constant upgrading of the equipment Incorporate new instrumentation advances, for example, phase plate Maintain and upgrade our computer infrastructure, improve speed and reliability New instruments Expand our user base User satisfaction

32 Explore opportunities to form partnership with other Institutions to be able to afford state-of-the-art instrumentation that is difficult to afford by one Institution and in this way also increase the user base. CryoEM may be the obvious target for these efforts as new EM microscopes cost between $5-10M and are expensive to maintain. NIH is considering a new initiative on CryoEM that may suit the needs of the structural biology community.

33

34

35

36

37 Acknowledgments Pamela Focia Jason Pattie Jonathan Remis Valerie Tokars