Jingjing Zhao Push the limitations of crystal structure determination by 3D Push the limitations of crystal structure determination by 3D electron diffraction 3D electron by of crystal structure determination the limitations Push electron diffraction From inorganic porous materials to biomolecules Jingjing Zhao Jingjing Zhao was born in China. She received Master degree in Material Science and Engineering from University of Science and Technology of China. She started her PhD study at Stockholm University in 2017. She has expertise in electron crystallography. ISBN 978-91-7911-448-0 Department of Materials and Environmental Chemistry Doctoral Thesis in Physical Chemistry at Stockholm University, Sweden 2021 Push the limitations of crystal structure determination by 3D electron diffraction From inorganic porous materials to biomolecules Jingjing Zhao Academic dissertation for the Degree of Doctor of Philosophy in Physical Chemistry at Stockholm University to be publicly defended on Friday 11 June 2021 at 09.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B. Abstract Structure elucidation is fundamental to understanding the chemical and physical properties of a material. Three-dimensional electron diffraction (3D ED) has shown great power for structure determination of nanometer- or submicrometer-sized crystals that are either too small or too complex for X-ray diffraction. 3D ED can be applied to a wide range of crystalline materials from inorganic materials, small organic molecules, to macromolecules. In this thesis, continuous rotation electron diffraction (cRED), also known as micro-crystal electron diffraction (MicroED) in macromolecular crystallography, has been applied for the determination of interesting novel crystal structures. New methods and protocols have been developed to push the current limitations of crystal structure determination by 3D ED. The structure of silicate zeolite PST-24 is highly disordered. A combination of cRED with high-resolution transmission electron microscopy (HRTEM) revealed its unique channel system with varying dimensionality from 2D to 3D. The aluminum metal-organic framework CAU-23 nanocrystals form aggregates and are very beam sensitive. Its structure, as determined by cRED, is built by twisted helical Al-O chains connected by TDC2- linkers, forming a chiral structure with square channels. The unique structure of CAU-23 provides high stability and high water adsorption capacity, making it an ideal material for ultra-low temperature adsorption driven chillers. A simple pressure-assisted specimen preparation method, denoted Preassis, has been developed to overcome the challenges in the application of MicroED on biological samples with high viscosity and low crystal concentration. It has been successfully applied for the specimen preparation of several bio-molecular crystals including a novel R2lox metalloenzyme, which was crucial for its structure determination. Furthermore, an investigation of the influence of radiation damage on lysozyme crystals was performed to improve the data quality and final structural model. Finally, the crystal structure of acetylated amyloid-β fragment Ac-Aβ16-20, related to Alzheimer’s disease, has been studied. The crystal has an active optical wave-guiding property with an excitation wavenumber of 488 nm due to its unique packing of Ac-KLVFF β–sheets. Keywords: electron crystallography, 3D electron diffraction, cryo-EM specimen preparation, structure determination, porous materials, biomolecules. Stockholm 2021 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-192517 ISBN 978-91-7911-448-0 ISBN 978-91-7911-449-7 Department of Materials and Environmental Chemistry (MMK) Stockholm University, 106 91 Stockholm PUSH THE LIMITATIONS OF CRYSTAL STRUCTURE DETERMINATION BY 3D ELECTRON DIFFRACTION Jingjing Zhao Push the limitations of crystal structure determination by 3D electron diffraction From inorganic porous materials to biomolecules Jingjing Zhao ©Jingjing Zhao, Stockholm University 2021 ISBN print 978-91-7911-448-0 ISBN PDF 978-91-7911-449-7 Printed in Sweden by Universitetsservice US-AB, Stockholm 2021 To my family. Doctoral Thesis 2021 Department of Materials and Environmental Chemistry Stockholm University, Sweden Faculty opponent: Dr. Tim Grüne Centre for X-ray Structure Analysis, Faculty of Chemistry University of Vienna, Austria Evaluation committee: Prof. Eva Olsson Department of Physics Chalmers University of Technology, Sweden Assoc. Prof. Alexey Amunts Department of Biochemistry and Biophysics Stockholm University, Sweden Dr. Philippe Boullay Laboratoire de Cristallographie et Sciences des Materiaux Normandie Universite, France Substitute: Prof. Gunnar Svensson Department of Materials and Environmental Chemistry Stockholm University, Sweden Abstract Structure elucidation is fundamental to understanding the chemical and physical properties of a material. Three-dimensional electron diffraction (3D ED) has shown great power for structure determination of nanometer- or submicrometer-sized crystals that are either too small or too complex for X- ray diffraction. 3D ED can be applied to a wide range of crystalline materials from inorganic materials, small organic molecules, to macromolecules. In this thesis, continuous rotation electron diffraction (cRED), also known as micro- crystal electron diffraction (MicroED) in macromolecular crystallography, has been applied for the determination of interesting novel crystal structures. New methods and protocols have been developed to push the current limitations of crystal structure determination by 3D ED. The structure of silicate zeolite PST-24 is highly disordered. A combination of cRED with high-resolution transmission electron microscopy (HRTEM) revealed its unique channel system with varying dimensionality from 2D to 3D. The aluminum metal-organic framework CAU-23 nanocrystals form aggregates and are very beam sensitive. Its structure, as determined by cRED, 2- is built by twisted helical Al-O chains connected by TDC linkers, forming a chiral structure with square channels. The unique structure of CAU-23 provides high stability and high water adsorption capacity, making it an ideal material for ultra-low temperature adsorption driven chillers. A simple pressure-assisted specimen preparation method, denoted Preassis, has been developed to overcome the challenges in the application of MicroED on biological samples with high viscosity and low crystal concentration. It has been successfully applied for the specimen preparation of several bio- molecular crystals including a novel R2lox metalloenzyme, which was crucial for its structure determination. Furthermore, an investigation of the influence of radiation damage on lysozyme crystals was performed to improve the data quality and final structural model. Finally, the crystal structure of acetylated amyloid- fragment Ac-A16-20, related to Alzheimer’s disease, has been studied. The crystal has an active optical wave-guiding property with an excitation wavenumber of 488 nm due to its unique packing of Ac-KLVFF β– sheets. Keywords: electron crystallography, 3D electron diffraction, cryo-EM specimen preparation, structure determination, porous materials, biomolecules. i List of publications This thesis is based on the following papers: Paper I: PST-24: A zeolite with varying intracrystalline channel dimensionality D. Jo,† J. Zhao,† J. Cho,† J.H. Lee, Y. Liu, C.J. Liu, X. Zou, and S.B. Hong Angew. Chew. Int. Ed. 2020, 59, 17691 – 17696. DOI: 10.1002/anie.202007804 Scientific contributions: I collected the cRED data and HRTEM images. I did the data processing, structure determination, image processing, and structure analysis of the disorder. I contributed to the ED pattern simulation, proposed three polytypes. I contributed to the main figure making and the manuscript writing, especially the structure-related part. Paper II: A metal-organic framework for efficient water-based ultra-low- temperature-driven cooling D. Lenzen, J. Zhao, S.J. Ernst, M. Wahiduzzaman, A.K. Inge, D. Fröhlich, H. Xu, H.J. Bart, C. Janiak, S. Henninger, G. Maurin, X. Zou, and N. Stock Nat. Commun. 2019, 10:3025. DOI: 10.1038/s41467-019-10960-0 Scientific contributions: I contributed to the cRED data collection, processing, and structure determination. I also contributed to the topology analysis. I made figures and wrote the part of manuscript related to cRED experiments and structure discussions. Paper III: A simple pressure-assisted method for MicroED specimen preparation J. Zhao, H. Xu, H. Lebrette, M. Carroni, H. Taberman, M. Högbom, and X. Zou Nat. Commun. 2021, under review Scientifc contributions: I contributed to the setup building, modifications, and also the design of experiments. I performed major part of the experiments, including specimen preparation, diffraction and image collection, and also analysis. I wrote the initial manuscript of this work. ii Paper IV: Solving a new R2lox protein structure by microcrystal electron diffraction H. Xu, H. Lebrette, M.T.B. Clabbers, J. Zhao, J.J. Griese, X. Zou, and M. Högbom Sci. Adv. 2019, 5:eaax4621. DOI: 10.1126/sciadv.aax4621 Scientific contributions: I contributed to the MicroED specimen preparation and data collection. Paper V: Limiting the effects of radiation damage in MicroED through dose selection during data processing H. C. Bwanika, J. Zhao, G. Hofer, U. Sauer, X. Zou, H. Xu J. Synchrotron Radia. 2021, under review Scientific contributions: I was responsible for the study of the radiation- induced displacement