Experimental phasing opportunities for macromolecular crystallography at very long wavelengths

Abstract

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

DSAS: A new macromolecular substructure solution program based on the modified phase-retrieval algorithm

“…5 The best CC: the highest CC value in the whole iterations. 6 Phase error: the phase difference between the calculated substructure and the reference substructure. 7 Comparison: the nsites solved by SHELXD and Phenix.hyss.…”

“…Especially, SAD remains the dominant method for determining the structures of nucleic acids [4,5] and protein. [6,7] Based on the type of anomalous scatters, there are currently several commonly used variants of SAD: S-SAD, Se-SAD (labeling proteins with selenomethionine), and X-SAD (iodine, bromine, or metal ions).…”

Neutron Macromolecular Crystallography for Biological Samples—Current State and Future Perspectives

“…For a long time, the standard way to determine biological macromolecular structures at atomic resolution was to analyse X-ray diffraction patterns from a crystalline sample. A crystalline sample is required to overcome the weak diffraction provided by individual molecules (the ordered lattice of crystals allows scattered waves to add up in phase, amplifying the signal to observable intensities), while X-rays are required as the resolution able to be achieved is limited to approximately half the wavelength of the scattered particle (the wavelength of X-rays used on standard synchrotron beamlines typically ranges from 0.7 to 2 Å, with the most commonly used wavelength being ~1 Å [13]; for a comprehensive examination of the theory behind crystallography, please refer to [14]).…”

“…Many have access to laboratory-scale devices-typically utilizing rotating anode sources and charged-coupled device (CCD) detectors-which are capable of analysing crystals a few hundred µm in size over several hours. However, current-generation synchrotron facilities, which offer beam fluxes of ~10 13 ph/sec, allow data to be collected from µm-sized crystals in under a minute. This high flux, combined with the ability for remote operation, has made synchrotron facilities the go-to for biological macromolecular determination.…”

Molecular determinant deciphering of MIC-guided RND efflux substrates in E. coli

“…State-of-the-art cryo-electron microscopy technologies, as employed by various research teams, have unveiled the intricate details of asymmetric trimeric structures characterizing AcrB. These studies span from seminal investigations (Murakami et al, 2006) to the latest advancements (El Omari et al, 2023), providing insights into a dynamic cyclic mechanism facilitated by a proton gradient. In this mechanism, each protomer assumes distinct conformations, aligning with the functional states of the transport cycle-Loose (L), Tight (T), and Open (O).…”