3D-Strukturanalyse von Molluskenhämocyaninen aus elektronenmikroskopischen Bildern [Elektronische Ressource] / Gatsogiannis Christos

Meissner

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 26 Mo

Extrait

3D Strukturanalyse von Mollusken-
hämocyaninen aus
elektronenmikroskopischen Bildern



Diss ertation
zur Erlangung des Grades


„ D o k t o r
d e r N a t u r w i s s e n s c h a f t e n “


am Fachbereich Biologie
der Johannes Gutenberg Universität Mainz





Gatsoggiannis CChristos
Geb. am 13.05.1981 in Karditsa/Griechenland

Mainz, 2009


















Dekan:

1. Berichterstatter:

2. Berichterstatter:

Tag der mündlichen Prüfung:





Für r


















































"Nature does nothing without purpose or uselessly."

Aristotle of Stagira (384–322 BC)





















Table of contents I
I. Table of Contents
I. ............................................................................................................... I 
II.  List of Figures ............................................................................................................... V 
III.  List of Tables .............................................................................................................. VII 
A INTRODUCTION 1 
1.  Oxygen transporters .................................................................................................................... 1 
2.  Molluscan hemocyanins .............................................................................................................. 5 
2.1  Available X-ray structures of functional units .......................................................................... 12 
2.2  Medical relevance of molluscan hemocyanins 15 
3  CryoEM and Single particle analysis ........................................................................................ 17 
4  Objectives of the present work .................................................................................................. 18 
B MATERIALS AND METHODS ...................................................................................... 21 
1.  Materials and equipment ........................................................................................................... 21 
1.1  Animals ..................................................................................................................................... 21 
Megathura crenulata .................................................................................................................................... 21 
Nautilus pompilius.......... 22 
1.2  Electron microscopes ................................................................................................................ 24 
1.3  Computer-hardware ................................................................................................................... 25 
1.4  Further hardware ....................................................................................................................... 25 
1.5  Software .................................................................................................................................... 26 
2.  Biochemical Methods ................................................................................................................ 29 
2.1   KLH1-purification ..................................................................................................................... 29 
2.2   NpH-purification ....................................................................................................................... 29 
2.3   Determination of hemocyanin concentration and absorption spectrum .................................... 29 
2.4  Negative staining ....................................................................................................................... 30 
3.  Cryo-electron microscopy ......................................................................................................... 30 
3.1  Preparation of cryo-EM grids .................................................................................................... 30 
3.2  Data collection in the Cryo-Electron microscope...................................................................... 30 II Table of contents

KLH1 ........................................................................................................................................................... 31 
NpH .............................................................................................................................................................. 31 
4.  Bioinformatical Methods ........................................................................................................... 31 
4.1  Scanning of micrographs .... 31 
4.2  Particle selection ........................................................................................................................ 32 
4.3  CTF-correction .......................................................................................................................... 33 
4.4  Bandpass filter ........................................................................................................................... 35 
4.5  Multiple Reference Alignment (MRA) ..................................................................................... 37 
Translational alignment ................................................................................................................................ 38 
Rotational alignment .................................................................................................................................... 38 
4.6  MSA- Classification .................................................................................................................. 40 
MSA- multivariate statistical analysis .......................................................................................................... 40 
HAC-Classification and averaging ............................................................................................................... 41 
4.7  Orientation determination .......................................................................................................... 43 
Euler Angles ................................................................................................................................................. 43 
Angular reconstitution .................................................................................................................................. 45 
Projection m atching ...................................................................................................................................... 48 
4.8  3D Reconstruction ..................................................................................................................... 49 
4.9 Iterative refinements ................................................................................................................. 50 
4.10  Visualization .............................................................................................................................. 53 
4.11 Resolution assessment ............................................................................................................... 53 
4.11  Sharpening ................................................................................................................................. 54 
4.12 3D-masking ............................................................................................................................... 55 
4.13 Modelling the NpH polypeptide chain ...................................................................................... 57 
4.14 Molecular modelling of the KLH1-polypeptide chains ............................................................. 60 
4.15 Further methods ......................................................................................................................... 62 
Alignment of molecular models ................................................................................................................... 62 
Generation of 3D-electron density maps from PDB files ............................................................................. 62 
Fitting of one density map into another ........................................................................................................ 62 
C RESULTS AND DISCUSSION ............................