PROXIMA-1 beamline for macromolecular crystallography measurements at Synchrotron SOLEIL

The undulator beamline PROXIMA-1 at Synchrotron SOLEIL scheduled its first users in March 2008. The endstation is dedicated to biomolecular crystallography experiments, with a layout designed to favour anomalous data recording and studies of crystals with large cell dimensions. In 12 years, the beamline has accommodated 4267 shifts of 8 h and more than 6300 visitors. By the end of 2020, it saw 1039 identified published scientific papers referring to 1415 coordinates deposited in the Protein Data Bank. The current paper describes the PROXIMA-1 beamline, including the recent specific implementations developed for the sample environment. The setup installed in the experimental station contains numerous beam-shaping equipment, a chi-geometry three-axis goniometer, a single-photon-counting pixel-array X-ray detector, combined with a medium-throughput sample exchange robot. As part of a standard experimental scheme, PROXIMA-1 can also be accessed via `mail-in' services or remotely.

Protein-crystal detection with a compact multimodal multiphoton microscope

There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.

Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization

Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.

Twilightreloaded: the peptide experience

Thede factocommoditization of biomolecular crystallography as a result of almost disruptive instrumentation automation and continuing improvement of software allows any sensibly trained structural biologist to conduct crystallographic studies of biomolecules with reasonably valid outcomes: that is, models based on properly interpreted electron density. Robust validation has led to major mistakes in the protein part of structure models becoming rare, but some depositions of protein–peptide complex structure models, which generally carry significant interest to the scientific community, still contain erroneous models of the bound peptide ligand. Here, the protein small-molecule ligand validation toolTwilightis updated to include peptide ligands. (i) The primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) a new method used to score peptide-ligand models is presented; (iii) a few instructive and specific examples, including an electron-density-based analysis of peptide-ligand structures that do not contain any ligands, are discussed in detail; (iv) means to avoid such mistakes and the implications for database integrity are discussed and (v) some suggestions as to how journal editors could help to expunge errors from the Protein Data Bank are provided.

Only seeing is believing – the power of evidence and reason

Biomolecular crystallography is a mature science that provides an instructive example for modern inductive reasoning as a model for Bayesian epistemology in empirical science. Fundamental scientific epistemology requires that a strong claim is supported by strong and convincing proof. Biomolecular crystallography, based on solid foundations of rich experimental data and extensive prior knowledge provides a prime example for modern, evidence based reasoning that strongly relies on assessments of plausibility based on prior knowledge while at the same time constantly delivering some of the most novel and exciting results based on new experimental evidence. As a consequence of the solid underlying physical principles and its mathematical rigor, crystallography as a mature science could be almost fool proof – were it not for the human element.

Quasi-Groups and the Configuration Space of Symmetry-Constrained Motions

In this work, the set of all possible positions and orientations of a large molecule within the crystallographic asymmetric unit is equated to the coset space of the continuous group of proper rigid-body motions modulo the chiral space group of the macromolecular crystal. Since every chiral space group is a co-compact subgroup in the full group of rigid-body motions (which is a six-dimensional Lie group), the resulting coset space is a compact 6D manifold. However, since none of the crystallographic groups are normal in the full group of rigid-body motions, this coset space is not a group. However, it can be endowed with an operation that satisfies all of the group axioms except for associativity, thereby giving it the structure of a quasi-group. The quasi-group properties of such spaces are explored in this work as an example of generalized symmetry. The mathematical formulation presented here, which builds on the author's prior work cited below, is relevant to both the Molecular Replacement (MR) method in biomolecular crystallography, and in the design of new engineered crystals.

Crystallography and New Medicines: Examples from Influenza and Cell Death

Biomolecular crystallography underpins contemporary drug discovery. The author’s experiences in early (influenza) and recent (cancer) examples mark progress in the sophistication of approaches that have enabled a shift from simpler problems, as in enzyme inhibition, to complex problems, as in blocking protein–protein interactions.