structural biology across length scales€¦ · structural biology §cutting edge instrumentation...
TRANSCRIPT
![Page 1: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/1.jpg)
Structural biology across length scales
Thomas R. Schneider, EMBL Hamburg
EIROforum School on InstrumentationNordwijk,13/05/2019
![Page 2: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/2.jpg)
Basic Research
Advanced Training
TechnologyDevelopment & TransferServices
Integration of life science research
EMBL’s Five Missions
Nuclear Pore Complex
Core Facilities
Synchrotron beamlinesEMBL-EM
5/9/192
§ How does life work?§ Architecture
-> Structural Biology?
![Page 3: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/3.jpg)
ESI, Geneva, May 201377
Structural Biology
Tries to determine the molecular structure of the component of cells and relate it to their function.
The Nuclear Pore Complex (‘NPC’)
https://en.wikipedia.org/?title=Nuclear_porehttp://book.bionumbers.org/how-big-is-a-human-cell/ 5/9/193
Linear dimensions: 3 – 100 µm
![Page 4: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/4.jpg)
The Nuclear Pore Complex (‘NPC’)
5/9/194
![Page 5: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/5.jpg)
The Nuclear Pore Complex (‘NPC’)
5/9/195
NPC by Cryo Electron Tomography: 3.2 nm
Bui et al. (2013). Integrated Structural Analysis of the Human Nuclear Pore
Complex Scaffold. Cell, 155:1233
~ 1000 protein molecules of 30 kinds, 120 MDa, 12 Mio atoms
assemble / disassemble
![Page 6: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/6.jpg)
Bui et al
5/9/196
Composition
Numerous proteins: e.g. hNup107, Nup358, Nup214, Nup88, Aladin,
hCG1, Nup153, Nup50
CNC by Single Particle Cryo Electron Microscopy: ~1 nm
Bui et al. (2013). Integrated Structural Analysis of the Human Nuclear Pore
Complex Scaffold. Cell, 155:1233
![Page 7: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/7.jpg)
Nuclear Pore Complex
5/9/197
Nup107 Composition
Nup107 consists of 10 proteinsSpatial arrangement by cross-
linking mass spectrometry
![Page 8: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/8.jpg)
Nuclear Pore Complex
5/9/198
Nup120 by X-ray Crystallography: 3.0 Å
Leksa et al. (2009). The Structure of the Scaffold Nucleoporin Nup120 Reveals a New and Unexpected
Domain Architecture. Structure, 17:1082pdbid: 3HXR
![Page 9: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/9.jpg)
5/9/199
Putting things back together
![Page 10: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/10.jpg)
Reconstructing the NPC bySuper Resolution Microscopy
Szymborska et al., 2013, Science 341:655GSDIM = ground state depletion microscopy followed by individual molecule return
confocal image raw GSDIM data superresolved image
5/9/1910
![Page 11: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/11.jpg)
Mapping the radial orientation of theY-shaped complex inside the NPC
Nup160-N Seh1 Nup85
52.5 �0.2 nm 39.9 �0.2 nm 38.2 �0.2 nm
Nup107 Nup160-C Nup37
48.2�0.2 nm 42.6�0.2 nm 45.5 �0.2 nm
Szymborska et al., 2013, Science 341:655
Nup133
50.1�0.2 nm
5/9/1911
![Page 12: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/12.jpg)
Putting the Y-shaped complex
5/9/1912
Szymborska et al., 2013, Science 341:655
![Page 13: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/13.jpg)
4Pi-STORM 3D reconstruction of the NPC
Nup160, U2OS
5/9/1913
STORM = Stochastic Optical Reconstruction Microscopy Imaging
![Page 14: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/14.jpg)
Crystallographic structure of the Y-shaped complex
5/9/1914
CNC (Nup107) by X-ray Crystallography: 7.4 Å
Stuwe et al. (2015) Architecture of the nuclear pore complex coat. Science 347:1148
![Page 15: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/15.jpg)
Putting things back together
5/9/1915
![Page 16: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/16.jpg)
Consistency?
5/9/1916
Stuwe et al. (2015)Science 347:1148
Szymborska et al., (2013)Science 341:655
![Page 17: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/17.jpg)
And what about the middle?
5/9/1917
Kosinski, J., Mosalaganti, S., Appen, von, A., Teimer, R., DiGuilio, A. L., Wan, W., Bui, K. H., Hagen, W. J. H., Briggs, J. A. G., Glavy, J. S., Hurt, E. & Beck, M. (2016). Science. 352, 363–365.
![Page 18: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/18.jpg)
5/9/1918
https://www.youtube.com/watch?v=7IqGiW2jZRg
![Page 19: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/19.jpg)
And what about the middle?
5/9/1919Lin, D. H., Stuwe, T., Schilbach, S., Rundlet, E. J., Perriches, T., Mobbs, G., Fan, Y., Thierbach, K., Huber, F. M., Collins, L. N., Davenport, A. M., Jeon, Y. E. & Hoelz, A. (2016). Science. 352, aaf1015.
![Page 20: Structural biology across length scales€¦ · Structural Biology §Cutting edge instrumentation is crucial for pushing the boundaries of Structural Biology (object size and imaging](https://reader034.vdocument.in/reader034/viewer/2022051915/6007476a738d5221e84b9bdc/html5/thumbnails/20.jpg)
Structural Biology
§ Cutting edge instrumentation is crucial for pushingthe boundaries of Structural Biology (object sizeand imaging resolution).
§ Whatever probe is useful will be used (light, e-, n, ...).§ Technology development, technology provision (as service),
technology dissemination (commercial) for research in the member states.
§ Biologists, biochemists, chemists, physicists, computer scientists, engineers, lawyers, and others working together makes the difference.
§ Transition from experiment to measurement needs to be made.§ Software becomes ever more important (download, web-
services).§ Centralized user facilities / ‘infrastructures’ are a (the only
possible?) way to deal with technical complexity and cost.
5/9/1920