M ETHODS IN M OLECULAR B IOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed. Structural Proteomics

High-Throughput Methods

Third Edition

Edited by Raymond J. Owens

The Rosalind Franklin Institute, Harwell Science Campus, Didcot, UK Editor Raymond J. Owens The Rosalind Franklin Institute Harwell Science Campus Didcot, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1405-1 ISBN 978-1-0716-1406-8 (eBook) https://doi.org/10.1007/978-1-0716-1406-8

© Springer Science+Business Media, LLC, part of Springer Nature 2008, 2015, 2021 Chapter 9 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/). For further details see license information in the chapters. This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover Illustration Caption: EM of SARS-CoV-2 spike protein.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Preface

2021 marks the 50th Anniversary of the Protein Data Bank (PDB), the global initiative for the curation and dissemination of macromolecular structures to the international research community. It is therefore appropriate that the opening chapter in this third edition of Structural Proteomics in the Methods in Molecular Biology series, by Velankar and colleagues from the PDB, summarizes the current status of the PDB and its role in the future of structural biology. The PDB contains over 160,000 protein structures, but this is still a fraction of the total number of protein sequences available from genomic data. Therefore, in silico methods for predicting protein structure from sequence play an important part in structural proteomics. In the following two chapters, Edmunds and McGuffin and Madhusudhan et al. provide comprehensive and complementary user guides to the bioin- formatics tools and resources for ab initio modeling of proteins and complexes, including ligand-docking algorithms. Structural proteomics has been one of the key drivers for the development of stream- lined workflows for sample preparation. The production of high-quality samples for struc- tural studies, particularly mammalian membrane proteins and protein complexes, remains challenging. New protocols for tackling these difficult-to-express targets in higher eukaryote cells (insect and mammalian cells) are described in the chapters by Krasnoselska and van den Heuvel for the transient expression of membrane proteins in mammalian and insect cells, respectively. Novel approaches to protein production include the genome engineering of either the expression host or vector. Poterszman et al. describe a gene editing protocol to introduce purification tags into endogenous proteins for purification of macromolecular complexes. In the next two chapters, the crossover into synthetic biology is covered by Berger and Kubick and colleagues. Protocols for modifying the genome of the baculovirus, a widely used expression vector, to produce a novel synthetic virus are reported by the Berger group. In the next chapter, Kubick et al. describe incorporating non-natural amino acids using mammalian cell-free expression to produce fluorescent labeling of antibodies. Isotopic labeling of endogenous proteins for NMR has now been extended to mamma- lian cells, and Baldus et al. describe the production of isotopically labelled microtubules and analysis of their interaction with MT-associated protein by solid-state NMR. X-ray crystal- lography remains a key technique for structural analysis; Orville and Aller present the state- of-the-art in the use of electron-free lasers for time-resolved crystallography. Since the publication of the last edition of Methods in Molecular Biology focused on Structural Proteomics, advances in detector technology and software algorithms have brought micros- copy (cryo-EM) to the forefront of structural biology. Therefore, five chapters in this third edition are devoted to aspects of the use of electrons in structural biology. The so-called resolution revolution now means that the structures of large proteins and complexes can now be routinely determined at near-atomic resolution. Experimental and data analysis workflows are described in the chapters by Renault and Sorzano, respectively. The introduc- tion of phase contrast methods has contributed to increasing resolution, and a guide to setting up and troubleshooting the Volta phase plate in cryo-EM data collection is detailed in the chapter by von Loeffelholz and Klaholz. In addition to single particle techniques, modern methods in cryo-EM include cryoelectron tomography and Microcrystal Electron Diffraction (MicroED). The combination of using focused ion beam milling to prepare v vi Preface lamella thin enough for electrons to penetrate with cryo-EM imaging is providing protein structural information within cells. The cryoelectron tomography workflow for sample preparation and analysis is described in the chapter by Nova´cˇek et al. MicroED described by Danelius and Gonen is the newest cryo-EM technique enabling the rapid determination of peptide and organic molecule structures from microcrystalline powders. The technique has important applications in the structural analysis of pharmaceutical compounds and natural products. I am grateful to all the contributors to this book for sharing their experience and expertise. I would also like to thank the Methods in Molecular Biology series editor, John Walker, for his guidance in preparing this volume and Springer for the opportunity to edit the third edition.

Didcot, UK Raymond J. Owens Contents

Preface ...... v Contributors...... ix

PART ISTRUCTURAL BIOINFORMATICS

1 The Protein Data Bank Archive ...... 3 Sameer Velankar, Stephen K. Burley, Genji Kurisu, Jeffrey C. Hoch, and John L. Markley 2 Computational Methods for the Elucidation of Protein Structure and Interactions ...... 23 Nicholas S. Edmunds and Liam J. McGuffin 3 Methods for Molecular Modelling of Protein Complexes ...... 53 Tejashree Rajaram Kanitkar, Neeladri Sen, Sanjana Nair, Neelesh Soni, Kaustubh Amritkar, Yogendra Ramtirtha, and M. S. Madhusudhan

PART II PROTEIN PRODUCTION

4 High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus ...... 83 Ester Behiels and Jonathan Elegheert 5 Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies ...... 105 Ganna O. Krasnoselska, Maud Dumoux, Nadisha Gamage, Harish Cheruvara, James Birch, Andrew Quigley, and Raymond J. Owens 6 Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells ...... 129 Maren Schubert, Manfred Nimtz, Federico Bertoglio, Stefan Schmelz, Peer Lukat, and Joop van den Heuvel 7 SynBac: Enhanced Baculovirus Genomes by Iterative Recombineering ...... 141 Hannah Crocker, Barbara Gorda, Martin Pelosse, Deepak Balaji Thimiri Govinda Raj, and Imre Berger 8 Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification...... 153 Sylvain Geny, Simon Pichard, Arnaud Poterszman, and Jean-Paul Concordet

vii viii Contents

9 Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems ...... 175 Marlitt Stech, Nathanae¨l Rakotoarinoro, Tamara Teichmann, Anne Zemella, Lena Thoring, and Stefan Kubick

PART III STRUCTURE DETERMINATION

10 Solid-State NMR Spectroscopy for Studying Microtubules and Microtubule-Associated Proteins...... 193 Yanzhang Luo, Shengqi Xiang, Alessandra Lucini Paioni, Agnes Adler, Peter Jan Hooikaas, A. S. Jijumon, Carsten Janke, Anna Akhmanova, and Marc Baldus 11 Dynamic Structural Biology Experiments at XFEL or Synchrotron Sources...... 203 Pierre Aller and Allen M. Orville 12 From Tube to Structure: SPA Cryo-EM Workflow Using Apoferritin as an Example ...... 229 Christoph A. Diebolder, Rebecca S. Dillard, and Ludovic Renault 13 Image Processing in Cryo-Electron Microscopy of Single Particles: The Power of Combining Methods ...... 257 Carlos Oscar S. Sorzano, Amaya Jime´nez-Moreno, David Maluenda, Erney Ramı´rez-Aportela, Marta Martı´nez, Ana Cuervo, Robert Melero, Jose Javier Conesa, Ruben Sa´nchez-Garcı´a, David Strelak, Jiri Filipovic, Estrella Ferna´ndez-Gime´nez, Federico de Isidro-Gomez, David Herreros, Pablo Conesa, Laura del Can˜o, Yunior Fonseca, Jorge Jime´nez de la Morena, Jose Ramon Macı´as, Patricia Losana, Roberto Marabini, and Jose-Maria Carazo 14 Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection...... 291 Ottilie von Loeffelholz and Bruno P. Klaholz 15 Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology ...... 301 Jana Moravcova´, Radka Dopitova´, Matya´ˇs Pinkas, and Jirˇı´ Nova´cˇek 16 Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED ...... 323 Emma Danelius and Tamir Gonen

Index ...... 343 Contributors

AGNES ADLER • NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands ANNA AKHMANOVA • Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands PIERRE ALLER • Diamond Light Source Ltd, Didcot, UK KAUSTUBH AMRITKAR • Indian Institute of Science Education and Research Pune, Pashan, Pune, India MARC BALDUS • NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands ESTER BEHIELS • Univ. Bordeaux, CNRS, Interdisciplinary Institute for NeuroScience (IINS), UMR5297 CNRS/UB, Bordeaux, France IMRE BERGER • Bristol Synthetic Biology Centre BrisSynBio, Biomedical Sciences, School of Biochemistry, University of Bristol, Bristol, UK; School of Chemistry, Max Planck Bristol Centre for Minimal Biology, Bristol, UK FREDERICO BERTOGLIO • Department of Biotechnology, Institut fuer Biochemie, Biotechnologie und Bioinformatik, Technische Universitaet Braunschweig, Braunschweig, Germany JAMES BIRCH • Diamond Light Source Ltd, Didcot, UK STEPHEN K. BURLEY • Research Collaboratory for Structural Bioinformatics Protein Data Bank, Center for Integrative Proteomics Research, Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, NJ, USA; Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ, USA; Rutgers Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA; Skaggs School of Pharmacy and Pharmaceutical Sciences and San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA, USA LAURA DEL CAN˜ O • National Centre for Biotechnology (CSIC), Madrid, Spain JOSE-MARIA CARAZO • National Centre for Biotechnology (CSIC), Madrid, Spain HARISH CHERUVARA • Diamond Light Source Ltd, Didcot, UK JEAN-PAUL CONCORDET • Laboratoire Structure et Instabilite´ des Ge´nomes, Inserm U1154, CNRS UMR 7196, Museum National d’Histoire Naturelle, Paris, France JOSE JAVIER CONESA • National Centre for Biotechnology (CSIC), Madrid, Spain PABLO CONESA • National Centre for Biotechnology (CSIC), Madrid, Spain HANNAH CROCKER • Bristol Synthetic Biology Centre BrisSynBio, Biomedical Sciences, School of Biochemistry, University of Bristol, Bristol, UK ANA CUERVO • National Centre for Biotechnology (CSIC), Madrid, Spain EMMA DANELIUS • Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA; Department of Physiology, University of California Los Angeles, Los Angeles, CA, USA; Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, CA, USA CHRISTOPH A. DIEBOLDER • The Netherlands Centre for Electron Nanoscopy (NeCEN), Leiden University, Leiden, The Netherlands REBECCA S. DILLARD • The Netherlands Centre for Electron Nanoscopy (NeCEN), Leiden University, Leiden, The Netherlands ix x Contributors

RADKA DOPITOVA´ • CEITEC, Masaryk University, Brno, Czech Republic MAUD DUMOUX • The Rosalind Franklin Institute, Didcot, UK NICHOLAS S. EDMUNDS • School of Biological Sciences, University of Reading, Reading, UK JONATHAN ELEGHEERT • Univ. Bordeaux, CNRS, Interdisciplinary Institute for NeuroScience, IINS, Bordeaux, France ESTRELLA FERNA´ NDEZ-GIME´ NEZ • National Centre for Biotechnology (CSIC), Madrid, Spain JIRI FILIPOVIC • Masaryk University, Brno, Czech Republic YUNIOR FONSECA • National Centre for Biotechnology (CSIC), Madrid, Spain NADISHA GAMAGE • Diamond Light Source Ltd, Didcot, UK SYLVAIN GENY • Laboratoire Structure et Instabilite´ des Ge´nomes, Inserm U1154, CNRS UMR 7196, Museum National d’Histoire Naturelle, Paris, France TAMIR GONEN • Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA; Department of Physiology, University of California Los Angeles, Los Angeles, CA, USA; Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, CA, USA BARBARA GORDA • School of Biochemistry, Bristol Synthetic Biology Centre BrisSynBio, Biomedical Sciences, University of Bristol, Bristol, UK DAVID HERREROS • National Centre for Biotechnology (CSIC), Madrid, Spain JOOP VAN DEN HEUVEL • Department of Structure and Function of Proteins, Helmholtz Zentrum fu¨r Infektionsforschung GmbH, Braunschweig, Germany JEFFREY C. HOCH • BioMagResBank, Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT, USA PETER JAN HOOIKAAS • Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands FEDERICO DE ISIDRO-GO´ MEZ • National Centre for Biotechnology (CSIC), Madrid, Spain CARSTEN JANKE • Institut Curie, PSL Research University, CNRS UMR3348, Orsay, France; Universite´ Paris Sud, Universite´ Paris-Saclay, CNRS UMR3348, Orsay, France A. S. JIJUMON • Institut Curie, PSL Research University, CNRS UMR3348, Orsay, France; Universite´ Paris Sud, Universite´ Paris-Saclay, CNRS UMR3348, Orsay, France AMAYA JIME´ NEZ-MORENO • National Centre for Biotechnology (CSIC), Madrid, Spain TEJASHREE RAJARAM KANITKAR • Indian Institute of Science Education and Research Pune, Pashan, Pune, India BRUNO P. K LAHOLZ • Centre for Integrative Biology (CBI), Department of Integrated Structural Biology, IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Illkirch, France; Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France; Institut National de la Sante´ et de la Recherche Me´dicale (Inserm) U964, Illkirch, France; Universite´ de Strasbourg, Strasbourg, France GANNA O. KRASNOSELSKA • Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, UK STEFAN KUBICK • Branch Bioanalytics and Bioprocesses (IZI-BB), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam, Germany; Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus-Senftenberg, The Brandenburg Medical School Theodor Fontane and the University of Potsdam, Senftenberg, Germany GENJI KURISU • Protein Data Bank Japan, Institute for Protein Research, Osaka University, Suita, Osaka, Japan Contributors xi

OTTILIE VON LOEFFELHOLZ • Department of Integrated Structural Biology, Centre for Integrative Biology (CBI), IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Illkirch, France; Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France; Institut National de la Sante´ et de la Recherche Me´dicale (Inserm) U964, Illkirch, France; Universite´ de Strasbourg, Strasbourg, France PATRICIA LOSANA • National Centre for Biotechnology (CSIC), Madrid, Spain PEER LUKAT • Department of Structure and Function of Proteins, Helmholtz Zentrum fu¨r Infektionsforschung GmbH, Braunschweig, Germany YANZHANG LUO • NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands JOSE RAMON MACI´AS • National Centre for Biotechnology (CSIC), Madrid, Spain M. S. MADHUSUDHAN • Indian Institute of Science Education and Research Pune, Pashan, Pune, India DAVID MALUENDA • National Centre for Biotechnology (CSIC), Madrid, Spain ROBERTO MARABINI • Universidad Autonoma de Madrid, Madrid, Spain JOHN L. MARKLEY • BioMagResBank, Biochemistry Department, University of Wisconsin- Madison, Madison, WI, USA MARTA MARTI´NEZ • National Centre for Biotechnology (CSIC), Madrid, Spain LIAM J. MCGUFFIN • School of Biological Sciences, University of Reading, Reading, UK ROBERT MELERO • National Centre for Biotechnology (CSIC), Madrid, Spain JANA MORAVCOVA´ • CEITEC, Masaryk University, Brno, Czech Republic JORGE JIME´ NEZ DE LA MORENA • National Centre for Biotechnology (CSIC), Madrid, Spain SANJANA NAIR • Indian Institute of Science Education and Research Pune, Pashan, Pune, India MANFRED NIMTZ • RG Cellular Proteomics, Department of Structure and Function of Proteins, Helmholtz Zentrum fu¨r Infektionsforschung GmbH, Braunschweig, Germany JIRˇ I´ NOVA´ Cˇ EK • CEITEC, Masaryk University, Brno, Czech Republic ALLEN M. ORVILLE • Diamond Light Source Ltd, Didcot, UK RAYMOND J. OWENS • Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, UK; The Rosalind Franklin Institute, Didcot, UK ALEASSANDRA LUCINI PAIONI • NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands MARTIN PELOSSE • Bristol Synthetic Biology Centre BrisSynBio, Biomedical Sciences, School of Biochemistry, University of Bristol, Bristol, UK SIMON PICHARD • Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Integrated Structural Biology, Equipe labellise´e Ligue Contre le Cancer, Illkirch, France; Centre National de la Recherche Scientifique, UMR7104, Illkirch, France; Institut National de la Sante´ et de la Recherche Me´dicale, U1258, Illkirch, France; Universite´ de Strasbourg, Illkirch, France MATYA´ Sˇ PINKAS • CEITEC, Masaryk University, Brno, Czech Republic ARNAUD POTERSZMAN • Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Integrated Structural Biology, Equipe labellise´e Ligue Contre le Cancer, Illkirch, France; Centre National de la Recherche Scientifique, UMR7104, Illkirch, France; Institut National de la Sante´ et de la Recherche Me´dicale, U1258, Illkirch, France; Universite´de Strasbourg, Illkirch, France ANDREW QUIGLEY • Diamond Light Source Ltd, Didcot, UK xii Contributors

NATHANAE¨L RAKOTOARINORO • Branch Bioanalytics and Bioprocesses (IZI-BB), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam, Germany ERNEY RAMI´REZ-APORTELA • National Centre for Biotechnology (CSIC), Madrid, Spain YOGENDRA RAMTIRTHA • Indian Institute of Science Education and Research Pune, Pashan, Pune, India LUDOVIC RENAULT • The Netherlands Centre for Electron Nanoscopy (NeCEN), Leiden University, Leiden, The Netherlands RUBEN SA´ NCHEZ-GARCI´A • National Centre for Biotechnology (CSIC), Madrid, Spain STEFAN SCHMELZ • Department of Structure and Function of Proteins, Helmholtz Zentrum fu¨r Infektionsforschung GmbH, Braunschweig, Germany MAREN SCHUBERT • Department of Biotechnology, Institut fuer Biochemie, Biotechnologie und Bioinformatik, Technische Universitaet Braunschweig, Braunschweig, Germany NEELADRI SEN • Indian Institute of Science Education and Research Pune, Pashan, Pune, India NEELESH SONI • Indian Institute of Science Education and Research Pune, Pashan, Pune, India CARLOS OSCAR S. SORZANO • National Centre for Biotechnology (CSIC), Madrid, Spain MARLITT STECH • Branch Bioanalytics and Bioprocesses (IZI-BB), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam, Germany DAVID STRELAK • National Centre for Biotechnology (CSIC), Madrid, Spain TAMARA TEICHMANN • Branch Bioanalytics and Bioprocesses (IZI-BB), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam, Germany DEEPAK BALAJI THIMIRI GOVINDA RAJ • Council for Scientific and Industrial Research CSIR, Pretoria, South Africa LENA THORING • Branch Bioanalytics and Bioprocesses (IZI-BB), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam, Germany SAMEER VELANKAR • Protein Data Bank in Europe, European Molecular Biology Laboratory–European Bioinformatics Institute, Wellcome Genome Campus, Cambridge, UK SHENGQI XIANG • NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands; MOE Key Lab for Membrane-less Organelles & Cellular Dynamics, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China ANNE ZEMELLA • Branch Bioanalytics and Bioprocesses (IZI-BB), Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam, Germany