Sticky triangles [Elektronische Ressource] : new tools for experimental phasing of biological macromolecules / vorgelegt von Tobias Beck

Tobias Beck

Informations

• Publié par: georg-august-universitat_gottingen
• Publié le: 01 janvier 2010
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Sticky triangles: New tools for
experimental phasing of biological
macromolecules
Dissertation
zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades
“Doctor rerum naturalium”
der Georg-August-Universita¨tGo¨ttingen
vorgelegt von
Diplom-Chemiker
Tobias Beck
aus Hannover
Gottingen 2010¨D7
Referent: Prof.George M. Sheldrick, Ph.D.
Korreferent: Prof.Dr.Oliver Einsle
Tag der mu¨ndlichen Pru¨fung: 16. September 2010Table of contents
Scope of this thesis 1
I Crystallographic background 3
1 From experiment to structure 5
1.1 X-ray diffraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2 The phase problem in crystallography . . . . . . . . . . . . . . . . . . . . . 7
2 Experimental phasing 11
2.1 X-ray absorption and radiation damage . . . . . . . . . . . . . . . . . . . . 12
2.2 Isomorphous replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 Anomalous dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4 Substructure determination . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5 Density modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3 Heavy-atom derivatisation 25
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2 Soaking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.3 Co-crystallisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
II Materials and methods 29
4 Synthesis of sticky triangles 31
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.2 Iodinated compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.3 Brominated compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5 Crystallisation and heavy-atom derivatisation 39
5.1 Crystallisation of small molecules . . . . . . . . . . . . . . . . . . . . . . . 40
5.2 General remarks on protein crystallisation and heavy-atom derivatisation . 40
5.3 Soaking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.4 Co-crystallisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
vvi Table of contents
6 Data collection, processing and refinement 45
6.1 Small-molecule crystallography . . . . . . . . . . . . . . . . . . . . . . . . 46
6.2 In-house data collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
6.3 Data collection at the synchrotron . . . . . . . . . . . . . . . . . . . . . . . 46
6.4 Integration and scaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6.5 Experimental phasing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6.6 Model building, refinement and validation . . . . . . . . . . . . . . . . . . 47
6.7 Radiation damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
III Results and discussion 49
7 I3C - The magic triangle 53
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
7.2 I3C crystal structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
7.3 Experimental phasing with I3C . . . . . . . . . . . . . . . . . . . . . . . . 56
7.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
8 B3C - The MAD triangle 63
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
8.2 B3C crystal structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
8.3 MAD phasing with B3C . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
8.4 SAD phasing with B3C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
8.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
9 More sticky triangles 75
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
9.2 Crystal structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
9.3 Experimental phasing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
9.4 Binding sites for B3M, B3O and B3C . . . . . . . . . . . . . . . . . . . . . 82
9.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
10 B4C - The MAD tetragon 87
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
10.2 B4C crystal structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
10.3 Experimental phasing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
10.4 B4C binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
10.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
11 Exploiting the triangles for phasing 97
11.1 Searching for triangles: TRIFIND . . . . . . . . . . . . . . . . . . . . . . . 98
11.2 Difficult structures with I3C . . . . . . . . . . . . . . . . . . . . . . . . . . 100
11.3 Tests with SHELXD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
11.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Table of contents vii
12 What makes the triangles sticky? 107
12.1 Interaction modes of the phasing tools . . . . . . . . . . . . . . . . . . . . 108
12.2 Triangles as additives in crystallisation screens . . . . . . . . . . . . . . . . 116
12.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
13 Summary and perspective 119
IV Appendix 121
A Novel protein structures solved with the magic triangle I3C 123
B Crystallographic parameters 129
C Crystallographic data for proteins 135
D Crystallographic data for small molecules 139
E Experimental data for small molecules 145
F Sticky triangles in the news 149
Bibliography 151
List of abbreviations 165
List of figures 169
List of tables 173
Scientific contributions 175
Acknowledgments 179
Curriculum Vitae 181viiiScope of this thesis
X-ray crystallography is the prime method for the elucidation of three-dimensional molec-
ular structures. It enables structure determination of biological macromolecules such as
proteins and nucleic acids. These detailed structural models form the basis of molecular
biology. The determination of novel macromolecular structures usually requires the in-
corporation of heavy atoms, which are exploited for experimental phasing. Conventional
heavy-atom derivatisation with heavy-metal salts often suffers from non-specific binding,
resulting in low occupancy of the heavy-atom sites or derivatisation failing completely. In
addition many such soaks require the use of toxic chemicals and therefore stringent safety
precautions.
Wehavedevelopedanewclassofcompoundsthatcombinesheavyatomsforexperimen-
talphasingwithfunctionalgroupsforinteractionwithbiologicalmacromolecules. Thelead
structure is based on a benzene ring that provides a rigid scaffold. The ring is substituted
with three functional groups and three heavy atoms, iodine or bromine, respectively.
The three functional groups, e.g. carboxyl or amino groups, but also hydroxyl or
methoxy moieties, mayinteract withproteinresidues andthereforeshow enhanced binding
properties compared with traditional heavy-atom compounds. The three halogen atoms
provide a strong anomalous signal and may be used for experimental phasing via the tech-
niques SAD and SIRAS (iodine) or SAD, SIRAS and MAD (bromine). The three heavy
atoms form an equilateral triangle, which is easily recognised in the heavy-atom substruc-
ture.
Since compounds similar to the ones shown above have been used as contrast agents in
medical imaging (Yu and Watson, 1999), a low toxicity is expected for phasing tools based
on halogen atoms.
The present work is positioned at the interface of chemistry and structural biology
and spans the design, synthesis, characterisation and utilisation of the sticky compounds;
the compounds were used for experimental phasing of biological macromolecules, includ-
ing test proteins and novel structures. This work combines methods from small-molecule
crystallography and macromolecular crystallography.
12