Certifying Central Facility Beamlines for Biological and Chemical Crystallography and Allied Methods

For the new user of a beamline, details such as its beam focus sizes and fluxes onto the sample will be of keen interest. Likewise, instrument scientists are keen to document their evidence for their beamlines’ performances.

Neutrons used in crystallography research are generated in two types of sources: nuclear reactors and spallation neutron sources. While reactors produce neutrons continuously, spallation sources produce pulses of neutrons due to the pulsed nature of the particle accelerators used. The generated neutrons have energies of mega electron volt (MeV) and thus have wavelengths that would be extremely short for our diffraction purpose. The emitted neutrons are therefore passed through materials known as “moderators” to reduce the neutron energies to the milli electron volt (meV) range. According to the type of moderator the neutron beams are called “cold” having wavelengths of ~5Å or “thermal” or “hot” neutrons having wavelengths of ~1Å.

At the beamline it is generally assumed that the electronic area detector’s calibration has been undertaken by the supplying manufacturer and/or beamline scientist. These calibrationsCalibrationlinearity of detector response include correcting for its non-uniformity of intensity response across its area and its non-linearity of intensity measurements as well as spatial distortions.

Beam line front ends are vital for protecting the machine vacuum. The lifetime of the beam in the ring depends on the vacuum level. Hence, the beam lines emanating from the ring are separated from the main ring vacuum chamber.

First and foremost, of course, is crystallisation for the crystallographic study (see e.g. Chayen et al. in Macromolecular Crystallization and Crystal Perfection, 2010) and single particle grids for cryoelectron microscopy (cryoEM) work (see e.g. Weissenberger et al. in Nat Methods 18:463–471, 2021). Secondly, an evaluation of whether your crystal diffracts, and whether it cools in an acceptable manner for cryo work should be done. Likewise, for cryoEM it can be the case that the access to a ‘high end microscope’ at a central facility requires demonstration of the quality of the grids.

Aragao and Cowieson (Acta Cryst A 78, 2022) describe the checks which the beamline scientists do, including daily, for their beamlines. They nicely describe quality control systems which give a maintenance record of checks.

The user predominantly is focussed on their sample. They will be as certain as they can be from their several biophysical and biochemical characterization methods that they know their sample. In two ways this has been called into question for metal containing macromolecules.

Fixed wavelength beamlines have been established as they are somewhat easier and less costly to build and operate, as well as provide a higher intensity, than tuneable beamlines. They have increasingly come into fashion as protein structure determination using molecular replacement programs in macromolecular crystallography have been easier to use and, especially, the number of unique protein folds in the PDB have grown substantially.

TheseTuneable wavelength usage beamlines seek to exploit the changes in the resonant X-ray scattering of specific atom or atoms in a crystal structure.

ThereLaue diffraction are quite a variety of configurations for this type of beamline. Principally these are categorised on whether they provide a wide wavelength bandpassBandpass of wavelengths (e.g. 0.5–2 Å) or a narrow wavelength bandpassBandpass of wavelengths (e.g. 0.9–1.1 Å). The latter can also be called ‘pink beams’ or in neutron Laue DiffractionLaue diffraction beamlines, ‘quasi-Laue’.

The impetus for this type of beamline arose out of planning the European Synchrotron RadiationSynchrotron radiation Project (ESRP) which of course became the ESRF, the F being for Facility. In the Foundation Phase Report (ESRF 1987) the chapters on macromolecular crystallography (Helliwell (1987)) included one for microfocus. Microfocus beamlines provision has grown substantially at the 3rd generation SR sources.

Stimulated by the XFELsXFEL introducing serial delivery of samples in liquid jets or extruded gels (see Chap. 13 below) synchrotron crystallography beamlines introduced similar approaches. [This section is placed here, ahead of the XFELS, to complete the synchrotron crystallography beamlines as a group of chapters.]

XFELsXFEL deliver a beam intensity in tens of femtoseconds that a typical synchrotron beamline delivers in a millisecond. The short intense pulse of the X-ray laser allows radiation damage mechanisms that occur on time scales longer than tens of femtoseconds to be avoided.

Crystallography has expanded into the time domain, which follows its success in yielding up very large numbers of ‘static’ crystal structures in both structural chemistry and then structural biology.

The stronger scattering strength of electrons than X-rays by approximately a million times has led to a revolution in single particle cryoEM (see Chap. 20 ). But also, electron nanocrystallography has seen a steady evolution for some 50 years.

NeutronsNeutron crystallography have a unique role to play in determining the structure and dynamics of biological macromolecules and their complexes. The similar neutron scattering magnitude from deuterium (2H), carbon (C), nitrogen (N), and oxygen (O) nuclei, means that the effect of atomic vibration in lowering the visibility of these atoms in Fourier maps is no worse for the deuterium atoms, unlike the X-ray case. Moreover, the negative scattering length of the common protium isotope (1H) and the positive scattering length of deuterium allows the well-known neutron 1H/2H contrast variation method to be applied. Furthermore, as there is no problem with radiation damage using neutrons as the diffraction probe, unlike X-rays, room temperature neutron data collection studies are completely viable. Unfortunately, even with these advantages, the low flux of existing neutron facilities means that neutron biological crystallography is not going to be a high throughput technique due to the long measuring runs at present e.g. typically between one to two weeks or more.

The evolution of photon and neutron central facilities’ data policies in the past two decades or so has seen substantial changes. These changes have reflected the practicality of being able to archive large i.e. ‘Big Data’ level of quantities. However, there has also been an evolution of policy thinking especially by the funding agencies as they increasingly realised that commercial publishers were making large profits (even around 50%) out of taxpayers’ funded research.

As a modern example of facility publication policy let’s refer to the ESRF, which as a pan European project involves at least 10 participating countries with national and EU laws to navigate and whose policy will be an amalgamation of those countries’ national laws.

The most commonly available sample condition used is that of a cryo-temperature. Cooling a crystal in smaller molecule crystallography for diffraction data collection at around 100 K is the norm. In biological crystallography where X-ray radiation damage at ambient temperature is a problem this challenge is routinely ‘solved’ by cooling in an acceptable manner the crystal using a cryogen such as liquid nitrogen.

As particle preservation techniques at cryo-temperatures improved, as well as the sensitivity of detectors, and their time-resolution, to the electrons transmitted through the single particles improved, the spatial resolution achievable with electron microscopy took a revolutionary leap to the atomic level. This was about ten years ago. There is an interesting restriction that the macromolecules or their complexes need to be a certain size such as above 100 kDa, depending on the spatial resolution achieved.

A major treatise by Harris et al. (2009) describes the whole field, which is very broad. NMRNMR responds to the short-range environment of relevant atoms and is not directly influenced by long-range order. It can therefore be applied to amorphous materials as well as crystalline ones and the solution state.

Such SAXS/WAXS (X stands for X-ray) beamlines are usually available at all synchrotrons and XFEL facilities and are too numerous to summarise here. Likewise, SANS (N stands for neutrons) beamlines are usually available at all neutron facilities and are too numerous to summarise. The IUCr Commission on Small Angle ScatteringSmall angle scattering (X-ray and neutron) is described at: https://www.iucr.org/resources/commissions/small-angle-scattering and the IUCr Commission on Neutron Scattering is described at: https://www.iucr.org/iucr/commissions/neutron-scattering .

X-raysX-ray absorption spectroscopy (XAS) are absorbed by matter primarily through the photoelectric effect. Photoelectric absorption occurs when a bound electron (e.g. K shell) is excited to a continuum state by an incident photon of energy. It is thereby possible to discriminate between different elements in a given sample. By using not only K but also L edges and possibly M edges all elements in the periodic table are accessible using synchrotron radiationSynchrotron radiation. Since the effect is dependent only on the presence of an excitable atom and an ordered local environment of that atom, the sample can be disordered (e.g. amorphous, solution) or ordered (e.g. crystalline); the only constraint being that sufficient material be present and the concentration of the excitable atom be enough for a reasonable signal-to-noise ratio. Information concerning the electronic structure and/or immediate environment about the primary absorbing atom can be obtained by the accurate measurement of: the position (point of maximum inflection) of the absorption edge, the details of the edge structure, and the intervals and amplitudes of theExtended X-ray Absorption Fine Structure (EXAFS) Extended X-ray Absorption Fine Structure (EXAFS) ripples.

Mass spectroscopy (also known asMass spectrometry mass spectrometry) is an analytical technique that is used to measure the mass to charge ratio of ions with the measurements presented as a mass spectrum. It is used in many different fields and is applied to pure samples as well as complex mixtures. These instruments are provided commercially such as described here https://www.agilent.com/ .

Fourier transformUV/Vis spectroscopy UV–Vis, (FT)–IR, RamanInfra-red spectroscopy spectroscopies, in addition to NMRNMR spectroscopy, are some of the most routinely used techniques in chemistry laboratories. These methods provide complementary information to crystallography but in solution.

A highlySoftware for data analysis effective way to judge the quality of a beamline’s performance, whether by a user or by a beamline scientist, is the data analysis. Across the domains of crystallography, diffraction, scattering, and spectroscopy, as well as cryoEMCryoEM and NMRNMR, there are software analysis packages available. Within crystallography itself there are distinct sub-domains for small molecule and for macromolecule data analysis and structure determination and analysis.

In readiness for an experiment, it is vital to plan the safety of everything to do with it. This includes pre-beamtime trainingSafety of chemicals and preparation proceduresbeamline and facility training videos which will be provided by the facility. These are in my experience online and so can be viewed at one’s convenience but must be completed before arriving at the facility’s site. Sometimes the conclusion of viewing a video will be a quiz. If you fail one of the questions you may well have to repeat your viewing and the quiz.

All experiments are preceded with some level of theory, even if it is a basic question like “what happens if we do this?”. This is referred to as “science pull”. The wider context of an individual scientist is the degree of sophistication of the technology available. Advances in technology create a push to what scientists can think of doing, referred to as “technology push”. Theory can help by defining ideal cases as benchmarks.

There are two perspectives that are special to an experimental run at a synchrotron, XFELXFEL or neutron facility: that of the user and that of the facility staff. The user looks to the website for their beamline of interest as a first step to their certification of it i.e. that it will do what they envisage. The facility needs to provide clear information and policies.

The Facility has scheduled your experiment. Its staff will have worked hard to ensure the smooth running of its beamline and instruments for your experiment. So, onto the briefing. An onsite experiment tends to be more complicated than a remote experiment. The briefing will likely involve the Principal Investigator (PI) leading a discussion with the measuring team leader (a senior PostDoc let’s say) and PhD students. If I may use a military analogy the PI as Commander will describe the overall strategy and the measuring team leader is the General on the ground, with the troops, and who will decide tactics commensurate with achieving the overall strategy.