Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1847 Structural Studies of Large dsDNA Viruses using Single Particle Methods HEMANTH KUMAR NARAYANA REDDY ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0732-9 UPPSALA urn:nbn:se:uu:diva-391671 2019 Dissertation presented at Uppsala University to be publicly examined in Room C2:301, BMC, Husargatan 3, Uppsala, Friday, 11 October 2019 at 13:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Sarah Butcher (University of Helsinki). Abstract Narayana Reddy, H. K. 2019. Structural Studies of Large dsDNA Viruses using Single Particle Methods. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1847. 72 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0732-9. Structural studies of large biological assemblies pose a unique problem due to their size, complexity and heterogeneity. Conventional methods like x-ray crystallography, NMR, etc. are limited in their ability to address these issues. To overcome some of these limitations, single particle methods were used. In these methods, each particle image is manipulated individually to find the best possible set of images to reconstruct the 3D structure. The structural studies in this thesis, exploit the advantages of single particle methods. The large data set generated by the SPI study of PR772 provides better statistics about the sample quality due to the use of GDVN, a container-free sample delivery method. By analyzing the diffusion map, we see that the use of GDVNs as a sample delivery method produces wide range of particle sizes owing to the large droplet that are created. The high-resolution structure of bacteriophage PR772 confirmed the speculation about the heteropentameric nature of the penton and revealed the new architecture of the vertex complex consisting of a hetero-pentameric penton formed with three copies of P5 and two copies of P31. The beta propeller region of P2, formed by domains I and II is bound to the N-terminal domain of P5. The structure also reveals new conformations of N-terminal and C-terminal region of P3 which play an important role in particle assembly and structural stability. The study of Melbournevirus revealed the protein composition in a packed particle. The CryoEM structure of Melbournevirus reveals a T=309 capsid with an inner lipid membrane. A dense body was found in the viral particle, a feature not observed in other viruses of the Marseilleviridae family. The density of this body is similar to a nucleic acid-protein complex. This observation, along with the histone-like protein identified during study, suggest genome organization in the viral particle, similar to higher organisms. The soft X-ray microscope operated in the water-window shows the progression of the Cedratvirus lurbo infection in the host cell without the use of chemical fixation, staining, sample dehydration or polymer embedding. The study revealed a significant bioconversion from the host cell to the viral particle at later stages of infection. Keywords: PR772, phage, PRD1, Bacteriophage, coliphage, Melbournevirus, Cedratvirus, lurbo, Pithovirus, CryoEM, Single particle imaging, Coherent, Diffractive, Imaging, Soft X- ray, Microscopy, Microscope, GDVN, High resolution, XFEL, aerosol, Injection, electrospray, gas dynamic virtual nozzle, CDI, CXI, FEL Hemanth Kumar Narayana Reddy, Department of Cell and Molecular Biology, Molecular biophysics, Box 596, Uppsala University, SE-75124 Uppsala, Sweden. © Hemanth Kumar Narayana Reddy 2019 ISSN 1651-6214 ISBN 978-91-513-0732-9 urn:nbn:se:uu:diva-391671 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-391671) Dedicated to Vanajakshi, Narayana Reddy, Shruthi List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Reddy HKN et al. Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source. Scientific Data 4 170079 (2017). II Reddy HKN et al. Electron cryo-microscopy of Bacteriophage PR772 reveals the elusive vertex complex and the capsid architecture. elife (submitted) III Okamoto K*, Miyazaki N*, Reddy HKN et al. Cryo-EM structure of a Marseilleviridae virus particle reveals a large internal microassembly. Virology 516 239-245 (2018). IV Kördel M, Svenda M, Reddy HKN et al. Giant DNA virus infection dynamics investigated by quantitative soft x-ray microscopy. Manuscript in preparation Reprints were made with permission from the respective publishers. * Authors have contributed equally. List of Additional Papers V Kurta RP et al. Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses. Physics Re- view Letters. 119 (15):158102 (2017). VI Okamoto K et al. Structural variability and complexity of the giant Pithovirus sibericum particle revealed by high-voltage electron cryo-tomography and energy-filtered electron cryo-mi- croscopy. Scientific Reports. 7(1):13291 (2017). VII Lundholm I V, Sellberg JA et al. Considerations for three-di- mensional image reconstruction from experimental data in co- herent diffractive imaging. IUCrJ. 5(5):531-541 (2018). VIII Munke A et al. Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source. Scientific Data. 3(1):160064 (2016). IX Hantke MF, Bielecki J et al. Rayleigh-scattering microscopy for tracking and sizing nanoparticles in focused aerosol beams. IU- CrJ. 5(6):673–680 (2018). X Bielecki J, Hantke MF et al. Electrospray sample injection for single-particle imaging with x-ray lasers. Science Advanced. 5(5):8801 (2019). Contents 1 Introduction ............................................................................................11 2 Viruses ...................................................................................................12 2.1 Classification of Viruses ..................................................................12 2.2 Bacteriophages ................................................................................13 2.2.1 Tectiviridae ..............................................................................13 2.3 Giant Viruses ..................................................................................15 2.3.1 Marseilleviridae .......................................................................16 2.3.2 Pithoviridae .............................................................................17 3 Structure Determination of Biomolecules ................................................19 3.1 Conventional Methods.....................................................................23 3.1.1 X-ray Crystallography ..............................................................23 3.2 Single Particle Methods ...................................................................25 3.2.1 Electron Cryo-Microscopy .......................................................25 3.2.2 Single Particle Coherent X-ray Diffractive Imaging ..................26 3.2.3 X-ray Cryo-Microscopy ...........................................................28 4 Sample Preparation and Characterization ................................................30 4.1 Bacteriophage PR772 ......................................................................30 4.2 Melbournevirus ...............................................................................32 4.3 Cedratvirus lurbo ............................................................................35 4.4 Sample Optimization .......................................................................36 4.4.1 Electron cryo-microscopy ........................................................36 4.4.2 Single Particle - Coherent X-ray Diffractive Imaging ...............37 4.4.3 X-ray Cryo-Microscopy ...........................................................39 5 Structural Studies of Bacteriophage PR772 from Tectiviridae .................40 5.1 Introduction .....................................................................................40 5.2 SP-CDI of PR772 ............................................................................42 5.2.1 Experimental setup ...................................................................42 5.2.2 Data Collection and Analysis ...................................................42 5.2.3 Results .....................................................................................43 5.2.4 Data Deposition .......................................................................44 5.3 Electron Cryo-Microscopy of PR772 ...............................................45 5.3.1 Data Analysis ...........................................................................45 5.3.2 Model Building ........................................................................45 5.3.3 Results and Discussion .............................................................46 6 Structural Studies of Giant Viruses .........................................................50 6.1 Structural studies of Melbournevirus ...............................................50 6.1.1 Identification of Viral Proteins in the Melbournevirus ..............50 6.1.2 Results .....................................................................................50 6.1.3 Electron Cryo-Microscopy .......................................................51 6.1.4 Data Analysis ...........................................................................51 6.1.5 Results