Three-dimensional electron microscopy of structurally heterogeneous biological macromolecules [Elektronische Ressource] / submitted by Florian Hauer

Schraeder

Sujets

• Biologie

Informations

• Publié par: georg-august-universitat_gottingen
• Publié le: 01 janvier 2009
• Nombre de lectures: 43
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Three-dimensional electron microscopy of
structurally heterogeneous biological
macromolecules

PhD Thesis

in partial fulfilment of the requirements
for the degree “Doctor of Philosophy (PhD)”
in the Molecular Biology Graduate Program
at the Georg August University Göttingen
Faculty of Biology

submitted by

Florian Hauer

born in

Karlsruhe, Germany



2009




























Members of Thesis Committee

First Referee: Prof. Dr. Holger Stark
Second Referee: Prof. Dr. Ralf Ficner
Third Referee: Prof. Dr. Reinhard Jahn


















Affidavit
I hereby declare in lieu of oath that this thesis has been written independently and with no
other sources and aids than quoted.

Göttingen, 31.05.2009


Florian Hauer

List of Publications

1. Sirajuddin, M., Farkasovsky, M.. Hauer, F.. Kuhlmann, D., Macara, I. G.,
Weyand, M.. Stark, H., Wittinghofer, A.., Structural insight into filament formation
by mammalian septins. Nature, 2007. 449(7160): p. 311-5.
2. Kastner, B., Fischer, N., Golas, M. M., Sander, B., Dube, P., Boehringer, D.,
Hartmuth, K., Deckert, J., Hauer, F., Wolf, E., Uchtenhagen, H., Urlaub, H.,
Herzog, F., Peters, J. M., Poerschke, D., Luhrmann, R., Stark, H., GraFix: sample
preparation for single-particle electron cryomicroscopy. Nat Methods, 2008. 5(1):
p. 53-5.
3. Schmeisser, M., Heisen, B.C., Luettich, M., Busche, B., Hauer, F., Koske, T.,
Knauber, K.-H., Stark, H, Parallel, distributed and GPU computing technologies in
single-particle electron microscopy. Acta Crystallogr D Biol Crystallogr, 2009.
65(Pt 7): p. 659-71. i
Table of Contents
List of Figures ....................................................................................................................... iv
List of Tables ........................ vi
List of Abbreviations ........................................................................................................... vii
Acknowledgements ............... 1
Abstract .................................................................................................................................. 2
1 Introduction ................................................................................................................... 3
1.1 Transmission electron microscopy ........ 4
1.1.1 Image formation in transmission electron microscopy...................................... 4
1.1.2 Phase contrast transfer function ......................................... 7
1.2 Image processing in single-particle electron microscopy...... 9
1.2.1 General procedures in 3D reconstruction .......................................................... 9
1.2.2 Alignment of single-particle electron microscopy images .............................. 10
1.2.3 Multivariate statistical analysis of particle images .......................................... 11
1.2.4 Angular reconstitution ..................................................... 13
1.2.5 Three-dimensional reconstruction ................................... 14
1.2.6 Resolution assessment ..................... 15
1.3 Analysis of flexibility and structural heterogeneity in single particle electron
microscopy ...................................................................................................................... 16
1.3.1 Structural heterogeneity of biomacromolecular complexes ............................ 16
1.3.2 Image processing techniques ........................................................................... 17
1.4 The GraFix protocol ................................................................ 28
1.5 Macromolecular complexes studied in this work ................ 31
1.6 The 50S Ribosomal subunit of Thermotoga maritima ........................................ 32
1.7 The Eukaryotic Initiation Factor 3 (eIF3) complex ............. 35
1.8 The Vacuolar ATPase (V-ATPase) of Thermus thermophilus ............................ 38
2 Material and Methods .................................................................................................. 43
2.1 Materials .............. 43
2.1.1 Software ........................................................................................................... 43
2.1.2 Chemicals ........ 43
2.1.3 Laboratory materials ........................................................................................ 44
2.1.4 Special equipment ........................... 44
2.1.5 Buffers ............................................................................................................. 45
2.2 Biochemical methods .......................... 46 ii

2.2.1 Isolation and purification of biomacromolecular complexes .......................... 46
2.2.2 GraFix preparation of biomacromolecular complexes .................................... 46
2.3 Preparation of samples for single-particle electron microscopy ......................... 47
2.3.1 Preparation of grids for cryopreparation of samples on carbon foil ............... 47
2.3.2 Preparation of negatively stained samples ...................................................... 48
2.3.3 Preparation of unstained cryo samples ............................ 49
2.4 Electron microscopy analysis .............................................. 49
2.4.1 Transmission electron microscopy .................................. 49
2.4.2 Processing of raw images ................ 50
2.4.3 Image processing ............................................................................................. 50
2.5 The MaverickTilt Software ................. 51
2.5.1 Basic algorithms .............................. 53
2.5.2 Determination of the initial reference point set ............................................... 55
2.5.3 Iterative detection of tilt pairs ......................................... 58
3 Results ......................................................................................................................... 61
3.1 The MaverickTilt software .................. 61
3.1.1 Benchmarking ................................. 61
3.2 The 50S Ribosomal subunit of Thermotoga maritima ........ 68
3.2.1 The L7/L12 stalk of T. maritima ..................................... 68
3.2.2 Expansion segments of the T. maritima large ribosomal subunit ................... 70
3.2.3 Flexibility of the L1 stalk in T. maritima ........................................................ 72
3.3 Translation initiation factor 3 (eIF3) from Saccharomyces cerevisiae ............... 74
3.3.1 GraFix preparation of eIF3 complexes ............................ 74
3.3.2 RCT analysis of negatively stained eIF3 complexes ...................................... 74
3.3.3 Refinement of three-dimensional reconstructions ........... 78
3.3.4 Cross-validation of refined three-dimensional reconstructions ...................... 79
3.4 The V-ATPase of Thermus thermophilus ........................................................... 81
3.4.1 Central cavities in the reconstruction of V-ATPases ...................................... 83
3.4.2 Conformational heterogeneity of stalk connections ........ 84
3.4.3 Flexibility of the cytosolic V domain ............................................................ 86 1
4 Discussion ................................................................................... 88
4.1 The MaverickTilt software .................................................. 88
4.1.1 Applicability of the software ........... 88
4.1.2 Implementation notes ...................................................... 88 iii
4.1.3 Performance of the MaverickTilt program on noisy data................................ 88
4.1.4 De-noising of particle coordinate datasets....................................................... 89
4.1.5 Application to experimental data ..... 90
4.2 The 50S Ribosomal subunit of Thermotoga maritima ........................................ 91
4.2.1 The extended ribosomal stalk of T. maritima .................. 91
4.2.2 Expansion segments of the large ribosomal subunit ....... 93
4.2.3 Conformational flexibility of L1 in the large ribosomal subunit..................... 94
4.3 Translation initiation factor 3 (eIF3) from Saccharomyces cerevisiae ............... 94
4.3.1 Checkpoints for the evaluation of initial models obtained from averaged RCT
reconstructions ............................................................................................................. 95
4.4 The V-ATPase of Thermus thermophilus ............................................................ 96
4.4.1 GraFix preparation of the the V-ATPase of Thermus thermophilus ............... 96
4.4.2 Validation of refined reconstructions .............................. 97
4.4.3 Flexibility of the cytosolic V domain ........................................................... 101 1
4.4.4 Functional implications of structural heterogeneity ...................................... 101
5 Conclusions .....................................................