Scattering Methods in Structural Biology, Part B, Volume 676 in the Methods in Enzymology serial, highlights advances in the field, presenting chapters on Quality controls, Refining biomolecular structures and ensembles by SAXS-driven molecular dynamics simulations, Data analysis and modelling of small-angle scattering data with contrast variation, Observing protein degradation in solution by the PAN-20S proteasome complex: state-of-the-art and future perspectives of TR-SANS as a complementary tool to NMR, crystallography and Cryo-EM, Extracting structural insights from chemically-specific soft X-ray scattering, Reconstruction of 3D density of biological macromolecules from solution scattering, ATSAS- present state and new developments in computational methods, and much more.
Additional chapters cover Modeling Structure and Dynamics of Protein Complexes with SAXS Profiles (FoXSDock and MultiFoXS), Validation of macromolecular flexibility in solution by SAXS, Combining NMR, SAXS and SANS to characterize the structure and dynamics of protein complexes, Application of Molecular Simulation Methods to Analyze SAS Data, and more.
Provides the authority and expertise of leading contributors from an international board of authors
Presents the latest release in the Methods in Enzymology serial
Updated release includes the latest information on Small Angle Scattering Methods for Structural Interpretation

"And yet it moves"--This phrase, attributed to Galileo Galilei, emphasizes the critical importance of defining matter with its movement over time for understanding a system.
Advances in experimental and computational structural methods are increasingly making transformative improvements in defining and predicting biological mechanisms. At the heart of these improvements is the critical ability to visualize both structural detail at the level of their molecular building blocks along with their changing conformation and assemblies as a function of time. Prior Methods in Enzymology volumes on diffraction methods and cryo-electron microscopy have presented detailed techniques for accurate and often high-resolution structures for crystallized and cryo-cooled biological macromolecules and assemblies. Here the contributing authors for Scattering Methods for Biological Macromolecules focus on procedures employing the scattering of X-rays (SAXS) and neutrons (SANS) by biological macromolecules, as divided into Volume 678, Part A, Methods for Structural Investigation and Volume 679, Part B, Methods for Structural Interpretation. These scattering methods can comprehensively measure and model molecular structures in solution, including their flexibility and dynamic functional conformations and assemblies. The ana-lyses not only can approach near physiological conditions but also can broadly evaluate solution states and conditions so as to better understand the relationships between macromolecular structures, their solution environments, and their functionally important flexibility and dynamics. In essence, defining the time-dependent conformation and assembly state of macromolecules in their biological environment is foundational and predictive knowledge for their activities and regulation. In this context, these volumes encompass the capabilities that make scattering methods a premier approach for measuring molecular conformations and assemblies in both space and time, as well as for efficiently leveraging information on atomic models from all other techniques.
Each volume is organized into sections and topics pertaining to the spectrum of scattering methods and the logical sequence of steps involved in first investigating (Part A) and then interpreting structure (Part B). Part A is primarily devoted to the experimental aspects of scattering exper-iments and data, starting from sample preparation and handling, followed by methods of data collection and data assessment. Part B primarily discusses data and structural interpretation, covering various aspects of scattering measurements and methods for their analysis as well as the resulting struc-tural insights. Armed by the information in these articles, the reader can pro-ceed knowledgeably and productively toward their structural goals with an appreciation of the practical and fundamental aspects of each technique, including its potential to substantially augment combined and integrated structural approaches.
In providing these two volumes on scattering methods, the authors and I collectively strived to attain a threefold overall goal: (1) to give biochemists and biologists an introduction to the field of macromolecular structure anal-ysis by scattering, offering them practical guidance to available techniques and pathways to gain functionally important insights into macromolecular structures; (2) to give scattering practitioners a comprehensive spectrum of current techniques available to them from robust to state-of-the-art and into pushing the technology's frontiers; and (3) to provide sufficient knowledge and guidance for interested researchers to effectively apply scattering approaches to combined methods and integrated structural biology. The authors of these chapters include both those upon whose shoulders we stand in developing new methods as well as established leaders and new pioneers in extending the reach and impact of scattering methods for structural biology.
In Volume 677 Graewert and Svergun provide the strong foundation for Part A, Methods for Structural Investigation in Chapter 1, by comprehensively describing sample environments and requirements for biological SAXS. In Chapters 2-5, Pollack, Duff, Krueger, and Jeffries and colleagues provide expert information on equipment and procedures to harness the power of neutrons for contrast and other experiments. In Chapters 6-7, Rosenberg and Vachette and their colleagues detail considerations and methods for coupling size-exclusion chromatography with scattering (SEC-SAXS). Tidow and Gabel and their colleagues then describe powerful approaches to time-resolved SAXS (Chapter 8) and SANS (Chapter 9). Vestergaard and Langkilde (Chapter 10) provide an essential guide for the application of SAXS to the biologically and medically important process of protein fibrillation. Pioneering methods for high-pressure SAXS experiments and for soft X-ray scattering are described by Gillilan (Chapter 11) and Gomez and colleagues (Chapter12),respectively. In Chapter 13,Yang discusses SAXS on lipid membranes and in Chapter 14 Todow and colleagues present SANS on membrane protein by stealth constitution systems. Predicting scattering with explicit solvent is detailed by Hub and Chatzimagas, who describe the WAXSiS web server for calculation of SAXS/WAXS curves based on explicit-solvent molecular dynamics, as a useful tool to extract maximum information from the SAXS profile(Chapter 15).In Chapter 16, Skepö and colleagues describe sample prepa-ration and data collection considerations for applying SAXS to highly flexible protein samples. Wang and colleagues (Chapter17) consider SAXS analyses of RNA, including an approach to simplifying exploration of conformational space and tying this in with SAXS constraints. In Chapter 18, Bernadó and Sagar provide unique information for enhanced biological insights from SAS data, including conformational rearrangements and transient biomolecular complexes, to anchor this volume.
Trewhella provides the foundation for Volume 676, Part B, Methods for Structural Interpretation in Chapter 1, with insights into the method and its development into a mainstream technique at the frontier of integrative structural biology, including key standards for data quality, model validation, publication, and data sharing. In Chapter 2,Hub and Chatzimagas describe a rigorous protocol for Bayesian interpretation of SAXS-driven MD simu-lations, models, and methods that will aid understanding of biological function by enabling understanding of conformational dynamics. Whitten and Jeffries describe SANS data analysis and modeling (Chapter 3), including a clear theoretical overview plus a step-by-step procedure for available software. Gabel describestime-resolved small-angle neutron scattering (Chapter 4) with an exemplary macromolecular system including the practical use of temperature and thermophilic systems to control kinetics. In Chapter 5, Gomez and colleagues describe informative procedures for predicting scattering contrast, identifying energies plus methods for inter-pretation suitable for measurements using resonant soft X-ray scattering (RSoXS) and near-edge X-ray absorption fine structure(NEXAFS)spec-troscopy to gain structural insights. Grant (Chapter 6) describes his ab initio modeling algorithm DENSS, which allows examination of low-resolution density fluctuations inside the particle envelope, which would seem to have many biological applications. In Chapter 7, Mertens considers the components and developments in the ATSAS package for its user-driven applications informing data reduction, analysis, and modeling strategies. Schneidman and colleagues describe a protocol for accurate inte-grative modeling of antibody-antigen complexes in Chapter 8, including SAXS profiles, protein-protein docking, deep-learning models, and statisti-cal potentials. In Chapter 9, Sattler and colleagues present the integration of SAS and NMR as complementary solution structural techniques to better describe structure with conformational dynamics with application guidelines depending upon the nature of the system. Skepö and coauthors present SAXS spectra analyses for highly flexible protein sample solutions in Chapter 10 with the definable parameters, model-free analysis methods, plus advanced methods to examine ensembles and crowded solutions. In Chapter 11, Brosey and colleagues present an efficient protocol for applying high-throughput SAXS to screening the binding of chemical ligands. In Chapter 12, Tsutakawa and colleagues consider SAXS applications to improved structure prediction. Vestergaard and Langkilde (Chapter 13) describe analysis of protein fiber structures by SAXS. In Chapter 14, Hura and Murray reveal elegant web-accessible approaches to analyses of similarity and correlated SAXS data sets to complete the volume.
In practice it is nearly impossible to navigate through scattering exper-iments, controls, data validation and analysis, and data interpretation, which can be conceptually simple but are usually technically sophisticated, without more methods detail than is available in published papers, which typically are space limited and focused upon new results. In their sharing of techniques and methods that they have painstakingly developed, validated, and opti-mized by years of tests and efforts, the authors herein made a remarkable contribution and investment toward the reproducibility and advanced impact of scattering for structural biology. In this regard, I acknowledge the generosity of all the contributing authors who strived to reveal their insights and explicit instructions on how to utilize their valuable methods to our readers. We furthermore thank our many reviewers, whose time and attention contributed valuable suggestions to improve the chapters. I am grateful to Anna Pyle and David Christianson (Co-Editors-in-Chief of Methods in Enzymology) for the invitation and strong support. Most credit for the quality of the two volumes should go to the authors and reviewers, along with David Christianson, who was a force of nature in implementing reviewing and editing. I thank Paulo Mendoza and the Elsevier staff for their patient and expert efforts at all stages to strengthen the volumes for readers. I acknowledge my National Cancer Institute R35CA220430 support for my efforts on the project "Mesocale and Nanoscale Technologies Integrated by Structures for DNA Repair Complexes (MANTIS-DRC)." I admit that I am responsible for any mistakes and omissions. Yet, due to the combined efforts of authors, reviewers, and editors, the chapters in these two volumes provide a depth of coverage and clarity of presentation suitable for readers spanning from students to experts.
Furthermore, the combined chapters show that scattering methods have advanced to the point that these articles are likely to become classic "go to" references that stand the test of time. So we expect that many laboratories will find these collected methods helpful as a starting point to adapt for their own systems and to extend these methods for in-depth understanding of mechanism, regulation, and functions. Reflecting on these chapters, it is an honor to be the editor of both volumes, where leaders and innovators I have admired, along with early investigators who are pushing methods forward into new frontiers, provide inspiration and insights to me and others working on hybrid methods to unveil biology at the nanoscale. In my view, the collected articles in these two volumes provide a critical enabling path to defining macromolecular structures and mechanisms in solution, including biologically relevant flexibility in both functionally important complexes and conformations.
JOHN A. TAINER
University of Texas MD Anderson Cancer Center

Contributors

Preface xv

1. Data quality assurance, model validation, and data sharing for biomolecular structures from small-angle scattering 1

Jill Trewhella

1. Becoming a mainstream structural biology technique 2

2. The path to standards and data sharing 4

3. Draft publication guidelines and plans for data archiving 8

4. A data archive for SAS as part of a federated system for integrative structural biology 11

5. The current publication guidelines and data archiving requirements 11

6. Quantifying data reproducibility and establishing a consensus experimental benchmark data set 15

7. Future opportunities 16

8. Conclusions 17

Acknowledgments 17

References 17

2. Structure and ensemble refinement against SAXS data: Combining MD simulations with Bayesian inference or with the maximum entropy principle 23

Leonie Chatzimagas and Jochen S. Hub

1. Introduction 24

2. SAXS-driven molecular dynamics simulations 27

3. SAXS-driven MD as a tool for Bayesian inference of molecular structures 37

4. Maximum-entropy ensemble refinement against SAXS data 41

5. Discussion: Conceptual considerations and recommendations 48

6. Applications 49

7. Summary 50

Acknowledgments 51

References 51

3. Data analysis and modeling of small-angle neutron scattering data with contrast variation from bio-macromolecular complexes 55

Andrew E. Whitten and Cy M. Jeffries

1. Introduction 56

2. Analysis of the forward scattering intensity, /(0), and calculation of contrast 59

3. Analysis of the radius of gyration 67

4. Composite scattering functions 74

5. Dummy-atom (bead) modeling against contrast variation data 78

6. Rigid body modeling against contrast variation data 85

7. Summary 91

Acknowledgments 92

References 92

4. Observing protein degradation in solution by the PAN-20S proteasome complex: Astate-of-the-art example of bio-macromolecular TR-SANS 97

Frank Gabel

1. The interest of TR-SANS for dynamic bio-macromolecular systems 98

2. Specific protein degradation in biological cells 99

3. A concrete example of bio-macromolecular TR-SANS: Insight into structural dynamics of substrate processing by the archaeal PAN-proteasome system 103

4. Sample conditions, instrumental setup and data reduction 104

5. Experimental TR-SANS results and mechanistic model of protein degradation 106

6. Conclusions and outlook 115

Acknowledgments 117

References 117

5. Extracting structural insights from soft X-ray scattering of biological assemblies 121

Sintu Rongpipi, Joshua T. Del Mundo, Enrique D. Gomez, and Esther W. Gomez

1. Introduction 122

2. Predicting RSoXS scattering contrast from NEXAFS spectra 124

3. Reduction of RSoXS 2D data into 1D 129

4. Interpretation of scattering data 132

5. Identification of the real-space structure that leads to an observed scattering profile 136

6. Opportunities for application of new RSoXS analysis approaches to biological assemblies 138

7. Conclusion and outlook 139

Acknowledgments 140

References 140

6. Reconstruction of 3D density from solution scattering 145

Thomas D. Grant

1. Introduction 146

2. Theory 148

3. DENSS software 158

4. Preparing the data with denss.fit_data.py 159

5. Running a single reconstruction with denss.py 162

6. Alignment and averaging 173

7. Analysis and interpretation of results 178

8. SANS 182

9. Materials science applications 184

10. Publication guidelines and SASBDB deposition 184

11. Summary 186

Acknowledgments 187

Funding 187

References 187

7. Computational methods for the analysis of solution small-angle X-ray scattering of biomolecules: ATSAS 193

Haydyn D.T. Mertens

1. Introduction 194

2. Calculation and simulation of scattering data 196

3. Primary data processing 205

4. Structural modeling from SAXS data 213

5. ATSAS summary 231

References 231

8.Multi-state modeling of antibody-antigen complexes with SAXS profiles and deep-learning models 237

Tomer Cohen, Matan Halfon, Lester Carter, Beth Sharkey, Tushar Jain, Arvind Sivasubramanian, and Dina Schneidman-Duhovny

1. Introduction 238

2. Materials and methods 241

3. Results 248

4. Protocol 255

5. Discussion 256

Acknowledgments 256

References 257

9. Combining NMR, SAXS and SANS to characterize the structure and dynamics of protein complexes 263

Florent Delhommel, Santiago Martinez-Lumbreras, and Michael Sattler

1. Introduction 264

2. Sample preparation 266

3. NMR spectroscopy 269

4. Small angle X-ray and neutron scattering (SAXS/SANS) 275

5. Integration of NMR and SAS 279

6. Conclusions and future perspectives 288

Acknowledgments 290

References 290

10.From dilute to concentrated solutions of intrinsically disordered proteins: Interpretation and analysis of collected data 299

Samuel Lenton, Eric Fagerberg, Mark Tully, and Marie Skepo

1. Introduction 300

2. How to tell if a protein is an IDP? 303

3. The conformational ensemble 305

4. Ensemble optimization methods 311

5. Special considerations for crowded IDP solutions 314

6. Beyond analytical models 316

7. General notes on the treatment of hydration layers of IDPs 321

8. Summary and conclusions 323

Acknowledgments 324

References 324

11. Applying HT-SAXS to chemical ligand screening 331

Chris A. Brosey, Runze Shen, Davide Moiani, Darin E. Jones, Kathryn Burnett, Greg L. Hura, and John A. Tainer

1. Introduction 332

2. Considerations for SAXS target and library selection 333

3. HT-SAXS sample preparation 337

4. Benchmarking a pilot HT-SAXS screen 337

5. Ligand screen design and assembly 338

6. Analysis of HT-SAXS screening datasets 342

7. Summary and future perspectives 346

Simple Scattering deposition 347

Acknowledgments 347

References 348

12. Combining small angle X-ray scattering (SAXS) with protein structure predictions to characterize conformations in solution 351

Naga Babu Chinnam, Aleem Syed, Greg L. Hura, Michal Hammel, John A. Tainer, and Susan E. Tsutakawa

1. Introduction 352

2. Obtaining a protein structure prediction 357

3. Prediction of SAXS curve from an atomic model 360

4. Comparison of experimental and predicted SAXS curves 362

5. Fitting of the protein structure prediction(s) to the experimental SAXS data 364

6. Example: XRCC1 solution state 367

7. Notes 369

8. Summary and conclusions 370

Acknowledgments 372

References 372

13. Protein fibrillation from another small angle--SAXS data analysis of developing systems 377

Annette Eva Langkilde and Bente Vestergaard

1. Introduction 378

2. General method notes 383

3. Essential check of SAXS data from fibrillating systems 383

4. Initial analysis of the background subtracted data 386

5. Decomposition of data-Manual approach 390

6. Decomposition of developing data using COSMiCS 395

7. Further interpretation 399

8. Alternative approaches 404

9. Summary and conclusions 405

Acknowledgments 406

References 406

14. Visualizing and accessing correlated SAXS data sets with Similarity Maps and Simple Scattering web resources 411

Daniel T. Murray, David S. Shin, Scott Classen, Chris A. Brosey, and Greg L. Hura

1. Introduction 412

2. Visualization of correlated SAXS data 416

3. Simple Scattering data set repository 430

4. Summary and outlook 437

Acknowledgments 438

References 438

Prof. John A. Tainer trained in X-ray crystallography, biochemistry, and computation. With this foundation, he contributed to structural biochemistry for the biology for DNA repair, reactive oxygen control, the immune response, and other stress responses for >20 years. His NCI-funded papers report robust structural and biophysical measurements to advance understanding of cellular stress responses that are evolutionarily conserved and important in preserving genome stability and preventing diseases in humans. His methods, results, and concepts have stood the test of time: they are often used and cited >30,000 total times. PA\At Scripps, Prof. Tainer created and ran the Scripps NSF Computational Center for Macromolecular Structure along with an NIH P01 on Metalloprotein Structure and Design. He also helped develop and utilize the Scripps share of the NSF San Diego Supercomputer Center. At LBL, he developed and directed the~$2.9 million/year DOE Program "Molecular Assemblies Genes and Genomics Integrated Efficiently” (MAGGIE) from 2004-2011. PA\At Berkeley, Prof. Tainer designed, developed, and directed the world's only dual endstation synchrotron beamline SIBYLS (Structurally Integrated BiologY for Life Sciences), used by >200 NIH labs. This unique technology integrates high flux small angle x-ray scattering (SAXS) and macromolecular X-ray crystallography (MX). At SIBYLS his lab develop, optimize, and apply technologies to determine accurate structures, conformations and assemblies both in solution and at high resolution. His lab defined an R-factor gap in MX revealing an untapped potential for insights on nanoscale structures by better modeling of bound solvent and flexible regions. PA\At the University of Texas MD Anderson Cancer Center, Prof. Tainer is joining biochemistry and biophysics to fluorescent imaging measures of protein and RNA interactions on DNA for mechanistic insights. He is integrating these data with cryo-EM, MX and SAXS structures by linking MD Anderson and SIBYLS facilities. PA\As an originator of applying proteins from thermophiles to defining dynamic structures and functional conformations, Prof. Tainer develop methods for measurements on structures including conformations, and assemblies in solution. Prof. Tainer has combined cryo-EM and X-ray structures with biochemistry to define functional assemblies. His lab introduced new equations for analyzing X-ray scattering for flexible macromolecules and complexes. His lab also defined a novel SAXS invariant: the first discovered since the Porod invariant ~60 years ago. The defined parameters quantitatively assess flexibility, measure intermolecular distances, determine data to model agreement, and reduce false positives. PA\Prof. Tainer has a track record of successful collaborations, completing projects, sharing innovating approaches and technologies, developing insights along with new structural data, and providing fundamentally important technologies that improve the ways others do their research. He has benefited from continuous peer-reviewed NCI funding since 1999. NCI support has allowed Prof. Tainer to develop expertise in the methods development and in the structural biology of DNA repair, immune responses, and other stress.

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