Small Angle Scattering Part A: Methods for Structural Investigation

Front Cover
Small Angle Scattering Part A: Methods for Structural Investigation
Copyright
Contents
Contributors
Preface
Chapter One: Advanced sample environments and sample requirements for biological SAXS
	1. Introduction
	2. Correlation between the physical process of the scattering event and sample requirements
		2.1. The scattering process and increasing the intensity of the signal
		2.2. Accounting for background subtraction with a second measurement
		2.3. Addressing the influence of structure factor with a concentration series
		2.4. Summary of general sample requirements
	3. Automated sample loading robotics
		3.1. Examples
		3.2. Designing/performing and assessing a SAXS experiment in batch mode: Automated sample loading robot
	4. Coupling of online purification systems
		4.1. Examples
		4.2. Designing/performing and assessing a SAXS experiment in SEC-SAXS mode
	5. Specialized sample environments
		5.1. General thoughts on designing ``non-standard´´ experiments
	References
Chapter Two: Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis
	1. Introduction
	2. Background: SAXS and CV-SAXS
		2.1. The origin of any SAXS signal: Electron density differences (or contrast) between molecule and background solvent
		2.2. Contrast variation SAXS
	3. What information can be extracted from CV-SAXS data?
		3.1. Special case: Analysis at the match point
		3.2. Measurements at different contrast values: Zero-angle scattering and radius of gyration
		3.3. Structural information can be obtained from the entire SAXS profile: Pair distance distribution function P(R), recon ...
	4. Experimental considerations
		4.1. General beamline details
		4.2. Buffer subtraction
		4.3. Contrast agent
		4.4. Biomolecule considerations: Molecular weight, purity, concentration
		4.5. Preparing the samples: Biomolecules in buffer and its buffer background
		4.6. Loading the sample at the beamline
	5. Doing the experiment
		5.1. Finding and exploiting the match point
		5.2. Performing a contrast series to reveal the structures of all components of a complex
		5.3. Benchmarks to assess data quality and effectiveness of the method
	6. Examples of information extracted
		6.1. At the match point: First example shows primary microRNA binding to a microprocessor protein
			6.1.1. Complex stoichiometry can be inferred from I(0) and validated with CV-SAXS
			6.1.2. Radius of gyration can indicate nucleic acid conformational changes upon protein binding
			6.1.3. Interpreting structural changes through distance distribution analysis and shape reconstructions
		6.2. At the match point with changing conformations, equilibrium and time resolved studies: Second example reports studie ...
			6.2.1. P(R) analysis provides in depth information about the changing DNA structures
			6.2.2. Structural ensemble modeling provides essential information about the conformations present in equilibrium salt ti ...
			6.2.3. CV-SAXS experiments can be performed in a time-resolved mode, to watch real time sequence of events
			6.2.4. Additional controls: Compare with other methods (validate)
		6.3. Analysis of contrast series: Third example to extract protein as well as RNA structure
			6.3.1. Structure of a non-enveloped virus (or virus like particle)
			6.3.2. Analysis via P(R)—Gateway for modeling
	7. Conclusions and outlook
	Acknowledgments
	References
Chapter Three: Deuteration for biological SANS: Case studies, success and challenges in chemistry and biology
	1. Deuteration for small angle neutron scattering
		1.1. Manipulating contrast
	2. Protein SANS
		2.1. Protein deuteration for SANS with contrast variation
		2.2. Matching out all but the protein: Invisible detergents and nanodiscs
	3. Membranes: Sterols and physiologically relevant lipids
	4. Deuteration by chemical synthesis
		4.1. Carboxylic acids, alcohols, amines, alkyl halides, thiols, alkanes
		4.2. Unsaturated fatty acids
		4.3. DDM/OG surfactants
		4.4. Ionic liquids
		4.5. Phospholipids—many examples
		4.6. Glycerides—Monoolein and Triolein
		4.7. Oleic acid and its derivatives
		4.8. Questions of deuteration on the physical characteristics of the molecules
	5. Examples of uses of deuteration for neutron scattering
	6. Deuteration facilities
	7. Deuteration facility use: Costs, feasibility, and quality of the experiment
	8. The naming of Isotopologues: A problem for structural biologists, industry, and databases
		8.1. The identification of isotopologues
		8.2. A biosynthetically-derived material: Deuterated cholesterol
		8.3. The naming of variously deuterated oleic acid
		8.4. Important lipids that are understood to be mixtures
	9. Proposals for the registration of deuterated compounds
	10. Concluding remarks
	References
Chapter Four: Planning, executing and assessing the validity of SANS contrast variation experiments
	1. Introduction
	2. Experiment planning
		2.1. Contrast and I(0) calculations
		2.2. Starting model structures
		2.3. Optimal sample concentrations
		2.4. Model SANS intensity curves
		2.5. Rg and Dmax for the complex and components
		2.6. Optimal instrument configuration(s)
		2.7. Rg and decomposition analyses
	3. Data collection and assessment of validity
		3.1. Data collection
		3.2. Assessment of validity
	4. Further data analysis and model refinement
	5. Concluding remarks
	Acknowledgments
	Disclaimer
	References
Chapter Five: Technical considerations for small-angle neutron scattering from biological macromolecules in solution: Cro ...
	1. Introduction
	2. Nuclear cross sections
		2.1. The coherent scattering length–bc
		2.2. The incoherent scattering length—bi
		2.3. Neutron scattering contrast
		2.4. Estimating neutron scattering contrast
		2.5. Procedure: Calculation of neutron scattering contrast
	Notes
	3. Small angle neutron scattering instruments
		3.1. Neutron wavelengths and guides
		3.2. Choosing instrument configurations, qmin
		3.3. Procedure: Optimization of configurations using SASCalc
		3.4. Choosing instrument configurations, qmax
		3.5. q-Resolution
		3.6. q-Calibration
		3.7. Procedure: q-Calibration
	4. The measurement approach
		4.1. Transmission measurements
		4.2. Procedure: Transmission measurement
		4.3. Scattering measurements
		4.4. Procedure: Scattering measurements
		4.5. Procedure: Measurement approach
		4.6. Procedure: Data reduction (brief overview)
	5. Summary
	Acknowledgments
	References
Chapter Six: Size exclusion chromatography coupled small angle X-ray scattering with tandem multiangle light scattering a ...
	1. Introduction
	2. Endstation design
		2.1. SEC-SAXS-MALS layout
		2.2. SEC-SAXS flow cell design
	3. Sample preparation for SEC-SAXS-MALS
		3.1. Equipment
		3.2. Reagents
		3.3. Procedure
		3.4. Notes
	4. Data acquisition
		4.1. Equipment
		4.2. Reagents
		4.3. SEC-SAXS-MALS sequence setup
		4.4. Setting up the SIBYLS SAXS process GUI
		4.5. Notes
	5. Data processing
		5.1. Processing MALS, QELS, and UV data
		5.2. Processing SEC-SAXS data
		5.3. Notes
	6. Data validation and analysis
		6.1. Self-validation and analysis of SEC-SAXS data
		6.2. Sample validation through SEC-MALS
		6.3. Notes
	7. Summary and conclusion
	Acknowledgments
	References
Chapter Seven: SEC-SAXS: Experimental set-up and software developments build up a powerful tool
	1. Introduction
	2. The instrument
		2.1. Columns
		2.2. Experimental protocol
	3. Data processing
		3.1. The capillary fouling issue
		3.2. Simple case: Well-resolved peaks
		3.3. More complex cases
	4. Comparative analysis of SEC-SAXS datasets using the various available programs
		4.1. BSA
			4.1.1. US-SOMO HPLC module
			4.1.2. EFA analysis
			4.1.3. REGALS analysis
			4.1.4. Results
		4.2. Aldolase
	5. Conclusion
	Acknowledgments
	References
Chapter Eight: Stopped-flow-time-resolved SAXS for studies of ligand-driven protein dimerization
	1. Introduction
	2. Sample preparation
		2.1. Additional sample characterisation
		2.2. Protein concentration considerations
	3. Stopped-flow experiment: The measurement
	4. Data analysis and interpretation
	5. Singular value decomposition and OLIGOMER analysis of time-resolved SAXS data
	6. Fitting a kinetic model to extract rate constants from SF-SAXS data
	7. Summary and conclusions
	Acknowledgments
	References
Chapter Nine: Time-resolved small-angle neutron scattering (TR-SANS) for structural biology of dynamic systems: Principle ...
	1. Introduction, principles and limitations
	2. Examples from diverse classes of biological systems
	3. Considerations on sample state and practical guidelines for the design of TR-SANS experiments
	4. Conclusions and future perspectives
	Acknowledgment
	References
Chapter Ten: Protein fibrillation from another small angle: Sample preparation and SAXS data collection
	1. Introduction
	2. General method and safety notes
		2.1. Safety when handling amyloid and amyloid-like material
		2.2. Fibrillation ``in batch´´ or in plate reader?
		2.3. Synchrotron radiation or laboratory instrument SAXS data: In situ or ex situ experiments?
	3. Feasibility, design and optimization
		3.1. Equipment
		3.2. Reagents
		3.3. Procedure
		3.4. Notes
	4. The main experiment: SAXS data collection
		4.1. Equipment
		4.2. Reagents
		4.3. Procedure
		4.4. Notes
	5. Summary and conclusions
	Acknowledgments
	References
Chapter Eleven: High-pressure SAXS, deep life, and extreme biophysics
	1. Introduction
	2. Structural effects of pressure on biomolecular systems
	3. Pressure dependence of SAXS
		3.1. Prefactor and contrast
		3.2. Form factor
			3.2.1. Radius of gyration
			3.2.2. Forward scattering and molecular mass
			3.2.3. Unfolding and disorder
	4. Methods
		4.1. Design
		4.2. Protocol
		4.3. Sample preparation
		4.4. Baroresistant buffers
		4.5. Recommended steps
			4.5.1. Pre-HP screening protocol
			4.5.2. HP-SAXS protocol
	5. Summary and conclusions
	Acknowledgments
	References
Chapter Twelve: Characterization of biological materials with soft X-ray scattering
	1. Introduction
	2. Sample preparation for dry or dehydrated materials
		2.1. Materials
		2.2. Procedure
			2.2.1. Preparation of onion outer epidermal peel
			2.2.2. Mounting sample onto silicon nitride RSoXS substrate
		2.3. Notes
	3. Sample preparation for liquids and hydrated solids
		3.1. Materials
		3.2. Procedure
		3.3. Notes
	4. Storing and transporting samples
		4.1. Materials
		4.2. Procedure
		4.3. Notes
	5. Mounting samples onto a sample bar
		5.1. Materials
		5.2. Procedure
		5.3. Notes
	6. Performing NEXAFS experiments
		6.1. Materials
		6.2. Procedure
		6.3. Notes
	7. Performing RSoXS experiments
		7.1. Materials
		7.2. Procedure
			7.2.1. Selecting sample spot
			7.2.2. Acquiring RSoXS data
		7.3. Notes
	8. Opportunities in instrumentation for resonant soft X-ray scattering of biological systems
	9. Conclusion and outlook
	Acknowledgments
	References
Chapter Thirteen: Scattering measurements on lipid membrane structures
	1. Introduction
	2. Information contents in the scattering data
	3. General experimental planning considerations
	4. Biological membranes
	5. Model membrane structures
		5.1. Simple lipid-water dispersions
		5.2. Solution-based model systems
			5.2.1. Sample preparation and data collection
			5.2.2. Data interpretation
		5.3. Substrate-supported membrane structures
			5.3.1. Sample preparation and data collection
			5.3.2. Data interpretation
	6. Concluding remarks
	References
Chapter Fourteen: Studying integral membrane protein by SANS using stealth reconstitution systems
	1. Introduction
	2. Contrast variation in small-angle neutron scattering (SANS) by selective deuteration of lipids and proteins for use as ...
	3. Reconstitution of integral membrane proteins (IMP) into stealth carrier systems/example: Reconstitution of MsbA into s ...
		3.1. Equipment
		3.2. Chemicals
		3.3. Protocol
		3.4. Notes
	4. SANS data acquisition and analysis of IMP/stealth nanodisc samples
	5. Summary and conclusions
	Acknowledgments
	References
Chapter Fifteen: Predicting solution scattering patterns with explicit-solvent molecular simulations
	1. Introduction
	2. Implicit-solvent methods
		2.1. Displaced solvent
		2.2. Hydration layer (HL)
		2.3. Risk of overfitting solvent-related parameters
	3. Explicit-solvent SAS predictions with the WAXSiS method
	4. Theory
		4.1. Comparing to experimental data
	5. Workflow
		5.1. A: With any GROMACS
		5.2. B: With GROMACS-SWAXS
			5.2.1. Specify the atomic form factors/neutron scattering lengths
			5.2.2. Generating the envelope
			5.2.3. Generating the solute and solvent run-input files
			5.2.4. Compute the SAS curve
	6. Practical considerations
		6.1. Convergence
		6.2. Orientational average
		6.3. Envelope size
		6.4. Solvent density correction
		6.5. Atomic fluctuations
		6.6. Water models
		6.7. Computational costs
	7. Summary
	Acknowledgments
	References
Chapter Sixteen: From dilute to concentrated solutions of intrinsically disordered proteins: Sample preparation and data  ...
	1. Introduction
	2. What is measured during an experiment?
	3. Before a measurement
		3.1. Dilute conditions
		3.2. Concentrated conditions
	4. Performing a measurement
		4.1. Size exclusion chromatography in combination with SAXS
		4.2. Automated BioSAXS robot: Batch measurements
		4.3. Static capillary measurements
	5. After a measurement
	6. Summary and conclusions
	Acknowledgments
	References
Chapter Seventeen: Deriving RNA topological structure from SAXS
	1. Introduction
	2. Outline of methods and experimental strategies
	3. Detailed descriptions of methods and experimental strategies
		3.1. RNA sample preparation for SAXS experiments
			3.1.1. In vitro transcription
			3.1.2. RNA purification
			3.1.3. Sample conditioning and buffer matching
				3.1.3.1. Buffer composition
				3.1.3.2. Sample transfer to matching buffer
				3.1.3.3. Sample concentration and amount
			3.1.4. Pre-examination before X-ray scattering
		3.2. Computing three-dimensional topological structures of RNA molecules based on SAXS and secondary structure
			3.2.1. Basic principle and algorithm
			3.2.2. CPU and system requirements
			3.2.3. Procedure
			3.2.4. Input file format
				3.2.4.1. Section 1
				3.2.4.2. Section 2
				3.2.4.3. Section 3
			3.2.5. Analysis of results
		3.3. Ab initio calculation of molecular envelopes, generation of atomic models and divide-and-conquer strategy for large  ...
	4. Case studies
		4.1. Computing 3D topological structures from SAXS data and RNA secondary structures
		4.2. The three-dimensional topological structure of the HIV Rev response element (RRE) RNA
			4.2.1. Background
			4.2.2. Sample preparation and conditioning
			4.2.3. Deriving the 3D topological structure of the HIV-1 RRE RNA
		4.3. Long non-coding subgenomic flavivirus RNAs from dengue virus 2
			4.3.1. Background
			4.3.2. Sample preparation and conditioning
			4.3.3. Deriving the 3D topological structure
		4.4. The 3X and 5BSL3.2 domains of hepatitis C virus and their distal complexes
			4.4.1. Background
			4.4.2. Experimental strategy and ionic conditions
			4.4.3. Sequence design
			4.4.4. Model building and evaluation
			4.4.5. Topological structure of the 3X domain in monomeric and dimeric states
			4.4.6. Topological structure of the 5BSL3.2 subdomain and its distal complex with domain 3X
	5. Summary and conclusions
	Acknowledgments
	References
Chapter Eighteen: Disentangling polydisperse biomolecular systems by Chemometrics decomposition of SAS data
	1. Introduction
	2. General description of COSMiCS
		2.1. Mathematical considerations of MCR-ALS
		2.2. Constraints used in COSMiCS
		2.3. Practical aspects of COSMiCS
	3. Considerations for SAXS measurements
	4. Procedure for COSMiCS decomposition
		4.1. Decomposition steps
		4.2. Notes
	5. Indications of unreliable/incorrect decomposition and possible solutions
	6. Summary and conclusions
	Acknowledgments
	References
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