Heat-shock protein 90 (HSP90) is a highly active molecular chaperone that plays a crucial role in cellular function. It facilitates the folding, assembly and stability of various oncogenic proteins, particularly kinases and transcription factors involved in regulating tumor growth and maintenance signaling pathways. Consequently, HSP90 inhibitors are being explored as drugs for cancer therapy. Crystallographic fragment screening is a novel screening method that has been developed in recent years for fragment-based drug discovery and is known for its high hit rate and its ability to provide direct insights into the complex structures of proteins and compounds. In this paper, high-diffraction-resolution crystals of the N-terminal domain of human HSP90α were employed in crystallographic fragment screening to discover binding fragments and binding sites. A diverse library of 800 structurally distinct fragments was screened, yielding 91 starting points for the fragment-based drug design of new HSP90α N-terminal inhibitors. Nearly a thousand crystals were measured, with 738 being processed and phased using a highly automated data-processing pipeline including data reduction and phasing, refinement and hit identification via PanDDA multi-data-set analysis. The 91 identified compounds bind to eight distinct regions of the HSP90α N-terminus, with 63 fragments located in the ATP-binding pocket and its surroundings, thus demonstrating the potential for the development of HSP90α- and ATP-binding inhibitors. This study emphasizes crystallographic fragment screening as a powerful method that can effectively identify fragment molecules and inhibitors that bind to HSP90α, contributing to ongoing efforts in cancer drug discovery.

MMACHC is an essential protein for the body to metabolise vitamin B₁₂, and its deficiency will cause cblC-type methylmalonic aciduria and homocystinuria. MMACHC can interact with cyanocobalamin (a type of vitamin B₁₂) cofactor and plays an important role in targeting cyanocobalamin to the enzyme of interest. In this paper, the GST-tag fusion-tagged MMACHC protein was successfully expressed by Escherichia coli (E. coli) low-temperature induction, and the high-purity MMACHC protein was successfully purified by affinity chromatography and gel filtration. Further, the crystal structure of MMACHC and cyanocobalamin complex was obtained with a resolution of 1.93 Å using X-ray diffraction. By analysing the complex structure of MMACHC and cyanocobalamin, we revealed the reasons for the diversity of MMACHC substrates and explained the reasons for the differences in disease conditions caused by different MMACHC site mutations. The acquisition of the complex structure of MMACHC and cyanocobalamin will play a significant role in promoting research on the metabolic pathway of vitamin B₁₂.

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods.