Technische Universität München Max-Planck-Institut für Biochemie Abteilung für Molekulare Strukturbiologie Strategies for cryo-electron tomography of the mycobacterial cell envelope and its pore proteins and functional studies of porin MspA from Mycobacterium smegmatis Christian Werner Hoffmann Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ. – Prof. Dr. J. Buchner Prüfer der Dissertation: 1. Hon. – Prof. Dr. W. Baumeister 2. Univ. – Prof. Dr. S. Weinkauf 3. Univ. – Prof. Dr. W. Liebl Die Dissertation wurde am 28.04.2010 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 14.07.2010 angenommen. TABLE OF CONTENTS A Table of contents A Table of contents .................................................................................................................... I B Abbreviations ........................................................................................................................ V C Zusammenfassung............................................................................................................ VIII D Summary ................................................................................................................................ X 1 Introduction ........................................................................................................................... 1 1.1 The genus Mycobacterium .............................................................................................. 1 1.1.1 Taxonomy .................................................................................................................. 1 1.1.2 Medical relevance of mycobacteria ........................................................................... 2 1.2 The global architecture of the mycobacterial cell envelope ........................................... 4 1.3 The porin MspA of Mycobacterium smegmatis.............................................................. 7 1.4 Aim of the thesis ........................................................................................................... 11 2 Cryo-electron microscopy and tomography ..................................................................... 12 2.1 Contrast formation in cryo-electron microscopy .......................................................... 15 2.2 Phase contrast transfer function .................................................................................... 16 2.3 CTF correction in cryo-electron tomography ............................................................... 17 3 Cryopreparation of biological samples ............................................................................. 20 3.1 Cryosectioning of vitrified biological material ............................................................. 21 3.2 Cryopreparation by focused ion beam (FIB) micromachining ..................................... 22 4 Materials and Methods ....................................................................................................... 24 4.1 Materials and Instruments ............................................................................................. 24 4.2 Bacterial strains, plasmids, oligonucleotides and enzymes .......................................... 25 4.3 Media, buffers and solutions ......................................................................................... 26 4.3.1 Media ....................................................................................................................... 26 4.3.2 Buffers and solutions ............................................................................................... 27 4.4 General Methods ........................................................................................................... 28 4.5 Bacterial growth conditions .......................................................................................... 28 I TABLE OF CONTENTS 4.6 Detachment of the outer membrane .............................................................................. 29 4.7 Extraction and purification of MspA ............................................................................ 29 4.7.1 Growing and harvesting of the cells ........................................................................ 29 4.7.2 Extraction of MspA ................................................................................................. 29 4.7.3 Acetone precipitation ............................................................................................... 29 4.7.4 Chromatographic purification .................................................................................. 29 4.8 Preparation of proteovesicles for CET .......................................................................... 30 4.8.1 Preparation of lipid vesicles by extrusion ................................................................ 30 4.8.2 Reconstitution of MspA into lipid vesicles .............................................................. 30 4.9 Constructing mutants of MspA ..................................................................................... 30 4.9.1 Site-directed mutagenesis by combined polymerase chain reaction (CCR) ............ 30 4.9.2 Construction of the MspA loop 6 (L6) deletion mutant .......................................... 31 4.9.3 Electroporation of M. smegmatis ML10 cells ......................................................... 32 4.10 Conductivity assays ...................................................................................................... 32 4.10.1 Preparation of liposomes ........................................................................................ 32 4.10.2 Reconstitution of wild-type MspA and the mutants in GUVs ................................ 33 4.10.3 Planar lipid bilayer formation ............................................................................... 33 4.10.4 Data recording and analysis .................................................................................. 34 4.11 Electron microscopy ..................................................................................................... 34 4.11.1 Sample preparation ............................................................................................... 34 4.11.2 Data acquisition .................................................................................................... 35 4.11.3 Extended data acquisition scheme for CTF correction of tilt series ........................ 36 4.12 Image processing .......................................................................................................... 36 4.12.1 CTF determination and correction of tilted projections ........................................ 36 4.12.2 Correction of the modulation transfer function (MTF)......................................... 37 4.12.3 Reconstruction of tilt series .................................................................................. 38 4.12.4 Image analysis of the cell wall structures ............................................................. 38 4.12.5 Averaging of subtomograms ................................................................................. 39 4.12.6 Visualization of electron density maps ................................................................. 39 5 Results .................................................................................................................................. 40 5.1 Cryo-electron tomography reveals the native architecture of bacterial cell envelopes 40 II TABLE OF CONTENTS 5.1.2 The outer layer is revealed as a lipid bilayer in cryo-electron tomograms ............ 43 5.1.3 Vitreous cryosections confirm the bilayer structure of mycobacterial outer membranes ............................................................................................................. 44 5.1.4 The asymmetric structure of the outer membrane of Escherichia coli is visualized in cryo-electron tomograms ................................................................................... 47 5.1.5 Mycolic acids are an essential part of the outer membrane in Corynebacterium glutamicum ............................................................................................................. 48 5.2 CET with FIB-micromachined M. smegmatis cells ...................................................... 50 5.2.1 Specimen thickness and milling approaches .......................................................... 50 5.2.2 Wedge-shaped milling results in areas thin enough for CET ................................ 52 5.3 CET of MspA reconstituted into lipid vesicles ............................................................. 57 5.3.1 Reconstitution of MspA .......................................................................................... 57 5.3.2 Extended exposure-scheme for CTF-correction of tilted projections ..................... 58 5.3.3 CTF determination and correction in tomograms of reconstituted MspA ............... 60 5.3.4 Sub-tomogram averaging reveals the effects of CTF- and MTF-correction and shows the situation of MspA in a lipid membrane .................................................. 64 5.4 Biophysical analyses of the ion conductivity of MspA ................................................ 68 5.4.1 Construction of the MspA mutants – Strategy .......................................................