Native SAD Research Articles

Facing the phase problem.

The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we - myself, my laboratory and the diffraction community - have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today's structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.

A crystal-processing machine using a deep-ultraviolet laser: application to long-wavelength native SAD experiments.

While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality. A crystal-processing machine that uses a deep-UV laser has been developed. The machine utilizes the pulsed UV laser soft ablation (PULSA) technique, which generates less heat than methods using infrared or visible lasers. Since protein crystals are sensitive to heat damage, PULSA is an appropriate method to process them. Integration of a high-speed Galvano scanner and a high-precision goniometer enables protein crystals to be shaped precisely and efficiently. Application of this crystal-processing machine to a long-wavelength X-ray diffraction experiment significantly improved the diffraction data quality and thereby increased the success rate in experimental phasing using anomalous diffraction from atoms.

A solution-free crystal-mounting platform for native SAD

A solution-free crystal-mounting platform for native SAD

Automated crystal shaping to facilitate native SAD phasing

Automated crystal shaping to facilitate native SAD phasing

Crystallographic analysis and phasing of iron-assimilating protein 1 (FEA1) from Chlamydomonas reinhardtii.

As an essential component of protein cofactors, iron is important for all photosynthetic organisms. In Chlamydomonas reinhardtii, iron levels are strictly controlled by proteins such as iron-assimilating protein 1 (FEA1). This periplasmic protein is expressed under conditions of iron deficiency, but its mechanisms of function remain unknown. Because FEA1 has no amino-acid similarity to protein structures in the Protein Data Bank, its crystal structure cannot be solved by molecular replacement. Here, recombinant FEA1 protein lacking the N-terminal signal sequence was successfully purified and crystals of apo FEA1 were obtained by hanging-drop vapor diffusion. Neither selenomethionine substitution nor heavy-atom derivatization was successful; therefore, the phase problem of FEA1 crystals was solved by the native sulfur SAD method using long-wavelength X-rays (2.7 Å). Laser-cutting technology was used to increase the signal-to-noise ratio and derive an initial structure. This study will lead to further structural studies of FEA1 to understand its function and its links to the iron-assimilation pathway.

The copper(II)-binding tripeptide GHK, a valuable crystallization and phasing tag for macromolecular crystallography.

The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.

A solution-free crystal-mounting platform for native SAD.

The native SAD phasing method uses the anomalous scattering signals from the S atoms contained in most proteins, the P atoms in nucleic acids or other light atoms derived from the solution used for crystallization. These signals are very weak and careful data collection is required, which makes this method very difficult. One way to enhance the anomalous signal is to use long-wavelength X-rays; however, these wavelengths are more strongly absorbed by the materials in the pathway. Therefore, a crystal-mounting platform for native SAD data collection that removes solution around the crystals has been developed. This platform includes a novel solution-free mounting tool and an automatic robot, which extracts the surrounding solution, flash-cools the crystal and inserts the loop into a UniPuck cassette for use in the synchrotron. Eight protein structures (including two new structures) have been successfully solved by the native SAD method from crystals prepared using this platform.

Long-wavelength native SAD phasing at BL13-XALOC enabled by the presence of a helium cone

Long-wavelength native SAD phasing at BL13-XALOC enabled by the presence of a helium cone

Solving structures by native SAD at room temperature

Solving structures by native SAD at room temperature

Optimized diffraction data collection for native SAD phasing

Optimized diffraction data collection for native SAD phasing

Solving structures with native SAD on laboratory X-ray sources

Solving structures with native SAD on laboratory X-ray sources

Making routine native SAD a reality

Making routine native SAD a reality

Exploiting wavelengths longer than 3 Å for native SAD phasing

Exploiting wavelengths longer than 3 Å for native SAD phasing

Laser processing of protein crystals for native SAD data collection

Laser processing of protein crystals for native SAD data collection

Long-wavelength macromolecular crystallography - First successful native SAD experiment close to the sulfur edge.

Phasing of novel macromolecular crystal structures has been challenging since the start of structural biology. Making use of anomalous diffraction of natively present elements, such as sulfur and phosphorus, for phasing has been possible for some systems, but hindered by the necessity to access longer X-ray wavelengths in order to make most use of the anomalous scattering contributions of these elements. Presented here are the results from a first successful experimental phasing study of a macromolecular crystal structure at a wavelength close to the sulfur K edge. This has been made possible by the in-vacuum setup and the long-wavelength optimised experimental setup at the I23 beamline at Diamond Light Source. In these early commissioning experiments only standard data collection and processing procedures have been applied, in particular no dedicated absorption correction has been used. Nevertheless the success of the experiment demonstrates that the capability to extract phase information can be even further improved once data collection protocols and data processing have been optimised.

Native SAD data collection environment at the Photon Factory BL-1A

Native SAD data collection environment at the Photon Factory BL-1A

On the influence of crystal size and wavelength on native SAD phasing.

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g. air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.

Advances in experimental phasing.

Advances in experimental phasing.

Native SAD is maturing.

Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow 'highly accurate data' to be routinely collected. Today, native SAD sits on the verge of becoming a 'first-choice' method for both de novo and molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight both de novo native SAD structures and recent structures that were key to methods development.

In meso in situ serial X-ray crystallography of soluble and membrane proteins.

The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.