Structural Proteomics High-Throughput Methods

This updated and expanded volume reflects the current state of the structural protein field with improved and refined protocols that have been applied to particularly challenging proteins. Beginning with a section on structural bioinformatics, the book continues with sections covering the challenge of producing high quality samples for structural studies, particularly mammalian membrane proteins and protein complexes, as well as protocols for structure determination, including the use of electrons in structural biology and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Chapter 9 is available open access under a CC BY 4.0 license.

Part I: Structural Bioinformatics

 

1. The Protein Data Bank Archive

            Sameer Velankar, Stephen K. Burley, Genji Kurisu, Jeffery C. Hoch, and John L. Markley

 

2. Computational Methods for the Elucidation of Protein Structure and Interactions

            Nicholas S. Edmunds and Liam J. McGuffin

 

3. Methods for Molecular Modeling of Protein Complexes

            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

            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

            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

            Maren Schubert, Manfred Nimtz, Federico Bertoglio, Stefan Schmelz, Peer Lukat, and Joop van den Heuvel

 

7. SynBac: Enhanced Baculovirus Genomes by Iterative Recombineering

            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

            Sylvain Geny, Simon Pichard, Arnaud Poterszman, and Jean-Paul Concordet

 

9. Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems

            Marlitt Stech, Nathanaël 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

            Yanzhang Luo, Shengqi Xiang, Alessandra Lucini Paioni, Agnes Adler, Peter Jan Hooikaas, Jijumon A.S., Carsten Janke3, Anna Akhmanova, and Marc Baldus

 

11. Dynamic Structural Biology Experiments at XFEL or Synchrotron Sources

            Pierre Aller and Allen M. Orville

 

12. From Tube to Structure: SPA Cryo-EM Workflow Using Apoferritin as an Example

            Christoph A. Diebolder, Rebecca S. Dillard, and Ludovic Renault

 

13. Image Processing in Cryo-Electron Microscopy of Single Particles: The Power of Combining Methods

            Carlos Oscar S. Sorzano, Amaya Jiménez-Moreno, David Maluenda, Erney Ramírez-Aportela, Marta Martínez, Ana Cuervo, Robert Melero, Jose Javier Conesa, Ruben Sánchez-García, David Strelak, Jiri Filipovic, Estrella Fernández-Giménez, Federico de Isidro-Gómez, David Herreros, Pablo Conesa, Laura del Caño, Yunior Fonseca, Jorge Jiménez de la Morena, Jose Ramon Macías, Patricia Losana, Roberto Marabini, and Jose-Maria Carazo

 

14. Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection

            Ottilie von Loeffelholz and Bruno P. Klaholz

 

15. Cryo-Focused Ion Beam Lamella Preparation Protocol for In Situ Structural Biology

            Jana Moravcová, Radka Dopitová, Matyáš Pinkas, and Jirí Novácek

 

16. Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED

            Emma Danelius and Tamir Gonen