draft requirements for presenting biological macromolecule small-angle scattering data
It is not the intention of this document to define a quality requirement for SAS experiments that would be acceptable for publication. Rather, the purpose is to outline the way in which SAS experiments should be presented in order to enable the reader to independently assess the quality of any interpretations made by the authors.
High sample quality is required for SAS experiments. As such the authors must demonstrate as clearly as possible that any sample is of sufficient quality to yield meaningful results in a SAS experiment.
A complete description of the sample must be made available; including sequences for proteins and polynucleotides (including purification tags), modifications and cofactors.
Sample purification procedure must be reported, along with an estimate of the final purity and how this was estimated.
Solvent blank composition (including pH for aqueous systems) must be reported.
For SANS contrast variation experiments, a statement describing the level of deuteration in biomolecules and their solvents and how it was determined is required.
Sample concentration(s) and method(s) of determination must be reported, including extinction coefficients when 280 nm absorbance measurements are used.
A SAS-independent assessment of monodispersity (DLS and/or aggregate-free gel filtration and/or MALLS) gives the reader confidence in the results and should be reported for globular proteins where available.
A statement describing how the solvent blank was obtained (e.g. dialysis, column flow through) should be made.
Details on the execution of the SAS experiment must be provided.
Instrument type (e.g. model or beamline) and configuration (point or line source, collimation details, detector details) must be reported. In the case of SANS there may be several configurations (e.g. detector positions, number of guides, apertures etc.) for a single experiment.
Wavelength (including Δλ/λ for neutrons) and measured q-range must be reported.
Sample environment (including cell pathlengths, temperature) and exposure times.
Standards measured and controls [secondary scattering standards for assessing I(0) data (e.g. lysozyme, water)].
For synchrotron X-ray studies, radiation damage must be monitored and the ways of ensuring the absence of the damage must be reported (addition of scavengers, sample flow, analysis of time frames).
Data reduction protocol and software should be reported.
Where a line source is used, beam geometry must be provided [either in terms of dimensions of a defined shape (e.g. parameters of a trapezoidal profile), or as a plot of the beam profile file].
In order for a reader to be able to assess the quality of a SAS experiment, it is necessary that the data be presented in a clear, well described manner.
Where possible, scattering profiles [I(q) versus q] and P(r) profiles [P(r) versus r] should be reported in the bulk manuscript. If space is restricted, plots may be reported in supplementary materials. I(q) plots should be presented either as linear X–log Y or log X–log Y axes. The linear X–linear Y representation should be avoided. Where possible, data should be placed on an absolute scale; however, multiple curves may be offset on the same plot for clarity, provided that this is explained in the figure caption.
For structural characterisation of isotropic samples, Guinier plots must be shown. The Guinier range should be given explicitly and the linear fitting should be displayed in the range not exceeding qRg = 1.3. Gunier plots may be included as stand-alone figures, insets to I(q) versus q plots, or reported in the supplementary materials.
The plots containing experimental data must show propagated errors (usually based on counting statistics).
If the presented data are desmeared (to correct for beam geometry or polychromaticity), this fact must be reported as well as the method for desmearing.
Molecular weight estimates using either I(0) or the molecular volume determined by Porod invariant must be reported, including uncertainties in the parameters used in the determination.
The data must be recorded at multiple solute concentrations to eliminate the possibility of concentration-dependent oligomerisation or interparticle interference.
For contrast variation experiments (both SAXS and SANS) the nature and number of contrast points should be reported. The plot of normalized ±√I(0) versus solvent density particle matching point must be presented.
Theoretical contrasts and molecular weights for globular samples should be reported along with the method of calculation.
Extrapolation to infinite dilution is desirable to avoid interference effects. Where no change in Rg or I(0)/C is observed with increasing concentration (C), this fact should be reported.
For the contrast variation experiments, Stuhrmann plots of Rg2 versus the reciprocal of contrast are desirable. Extracted component scattering functions (including cross-term) are desirable.
Where the experimenter is looking to support a three-dimensional model, any modelling must be justified and described thoroughly.
All software used for modelling [including generating P(r) profiles] must be reported.
χ2 values and a plot of the model fit to the experimental I(q) versus q must be shown for at least the best model.
Analysis of the ambiguity of the reconstruction (averaging or clustering) must be done.
For rigid-body modelling, a description of how the starting models were obtained (e.g. crystal structure of a domain, homology model etc.) as well as any connectivity or distance constraints and how they were chosen.
Any modelling assumptions (e.g. symmetry) must be stated.
If multiple modelling protocols were utilized, all χ2’s and fits to the data should be shown (in the supplementary materials if necessary).
Any additional experimental evidence supporting modelling assumptions and therefore enabling modelling restraints should be reported.