Subtraction of liposome signals in cryo-EM structural determination of protein-liposome complexes

Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins.Here,we propose a liposome signal subtraction method based on single-particle two-dimensional(2D)classification average images,aimed at enhancing the reconstruction resolution of membrane proteins.We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain.Based on this analysis,we designed a method to subtract the liposome signals from the original particle images.After the subtraction,the accuracy of single-particle three-dimensional(3D)alignment was improved,enhancing the resolution of the final 3D reconstruction.We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

Structures of SdrD from Staphylococcus aureus reveal the molecular mechanism of how the cell surface receptors recognize their ligands

Staphylococcus aureus is the most important Gram-positive colonizer of human skin and nasal passage,causing high morbidity and mortality.SD-repeat containing protein D(SdrD),an MSCRAMM(Microbial Surface Components Recognizing Adhesive Matrix Molecules)family surface protein,plays an important role in S.aureus adhesion and pathogenesis,while its binding target and molecular mechanism remain largely unknown.Here we solved the crystal structures of SdrD N2-N3 domain and N2-N3-B1 domain.Through structural analysis and comparisons,we characterized the ligand binding site of SdrD,and proposed a featured sequence motif of its potential ligands.In addition,the structures revealed for the first time the interactions between B1 domain and N2-N3 domain among B domain-containing MSCRAMMs.Our results may help in understanding the roles SdrD plays in S.aureus adhesion and shed light on the development of novel antibiotics.

Crystal structures of Bbp from Staphylococcus aureus reveal the ligand binding mechanism with Fibrinogen a

Bone sialoprotein-binding protein （Bbp）, a MSCRAMMs （Microbial Surface Components Recognizing Adhesive Matrix Molecules） family protein expressed on the surface of Staphylococcus aureus （S. aureus）, mediates adherence to fibrinogen a （Fg a）, a component in the extracellular matrix of the host cell and is important for infection and pathogenesis. In this study, we solved the crystal structures of apo-Bbp273-598 and Bbp273-598-Fg a561-575 complex at a resolution of 2.03 A and 1.45 A, respectively. Apo-Bbp273-598 contained the ligand binding region N2 and N3 domains, both of which followed a DE variant IgG fold characterized by an additional DI strand in N2 domain and D1＇ and D2＇ strands in N3 domain. The peptide mapped to the Fg o561-575 bond to Bbp273-sgs on the open groove between the N2 and N3 domains. Strikingly, the disordered C-terminus in the apo-form reorganized into a highly-ordered loop and a β-strand G＂ covering the ligand upon ligand binding. BbpAla298-Gly301 in the N2 domain of the Bbp273-598-Fg a561-575 complex, which is a loop in the apo-form, formed a short a-helix to interact tightly with the peptide. In addition, Bbpser547-Glns61 in the N3 domain moved toward the binding groove to make contact directly with the peptide, while BbpAsp338-Gly355 and BbpThr365-Tyr387 in N2 domain shifted their configurations to stabilize the reorganized C-terminus mainly through strong hydrogen bonds. Altogether, our results revealed the molecular basis for Bbp-ligand interaction and advanced our understanding of S. aureus infection process.