Multi-crystal anomalous diffraction for low-resolution macromolecular phasing

Abstract

Multiwavelength anomalous diffraction (MAD) and singlewavelength anomalous diffraction (SAD) are the two most commonly used methods for de novo determination of macromolecular structures. Both methods rely on the accurate extraction of anomalous signals; however, because of factors such as poor intrinsic order, radiation damage, inadequate anomalous scatterers, poor diffraction quality and other noisecausing factors, the anomalous signal from a single crystal is not always good enough for structure solution. In this study, procedures for extracting more accurate anomalous signals by merging data from multiple crystals are devised and tested. SAD phasing tests were made with a relatively large (1456 ordered residues) poorly diffracting (d min = 3.5 Å ) selenomethionyl protein (20 Se). It is quantified that the anomalous signal, success in substructure determination and accuracy of phases and electron-density maps all improve with an increase in the number of crystals used in merging. Structure solutions are possible when no single crystal can support structural analysis. It is proposed that such multi-crystal strategies may be broadly useful when only weak anomalous signals are available.

Structural basis for PECAM-1 homophilic binding

“…Multi-wavelength anomalous diffraction or single-wavelength anomalous diffraction (SAD) data from single SeMet-labeled crystals gave weak anomalous signals (supplemental Figure 3) owing to incomplete incorporation of SeMet (supplemental Figure 2), thus multicrystal anomalous diffraction was used for phasing. 34 Selenium SAD data sets collected from 16 single crystals at the same wavelength were processed separately by XDS (supplemental Table 1) and then combined at a resolution cutoff of 3.0Å using POINTLESS and SCALA 35 within the CCP4 package 36 (supplemental Figure 3; Table 1). The combined multicrystal data set analyzed using Phenix.Xtriage showed anomalous measurability up to 0.6 at low resolution, extending to ;5-Å resolution (supplemental Figure 3).…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…Many of these challenges have been addressed through serial data collection strategies, 3,4 which extend the concept of combining data from multiple crystals [5][6][7][8][9][10][11][12][13] with the limit of a single frame of data per crystal. This type of single-frame-per-crystal data collection has been critical for structural biology efforts at X-ray free-electron laser (XFEL) sources where radiation damage may require a "diffraction before destruction" approach 3,14 and has subsequently been extended for use at synchrotron sources.…”

“…These issues are then further compounded by the need to deliver those samples efficiently to the X-ray beam. 5,[7][8][9][10][11]18,[37][38][39][40][41][42] Microfluidic and microscale technologies have played a critical role in facilitating both protein crystallization and structure determination, with the breadth and variety of reported solutions demonstrating the challenging nature of the field. In this review, we discuss the utility of microfluidic technologies for addressing the challenges of crystal growth and sample manipulation for applications in serial crystallography and time-resolved structure determination.…”

Crystal structures of apparent saccharide sensors from histidine kinase receptors prevalent in a human gut symbiont

“…We previously described an alternative structure determination for one of these [32], and a structure of the BT4663 sensor domain was reported subsequently [21]. The folded chains from these HK3 sensors comprise three domains each, including two seven-bladed β-propeller domains and one C-terminal β-sandwich domain.…”