Chia-Ying Huang

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

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing.

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

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase at cryogenic temperatures (100 K) is described. It works with nanogram to single-digit microgram quantities of protein and lipid (and ligand when present), and is compatible with both high-resolution native data collection and experimental phasing without the need for crystal harvesting.

Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals

This article reports the incorporation of a fast continuous grid scan with both still and oscillation images into the Swiss Light Source macromolecular crystallography beamlines and its application in visualization of protein crystals with scanning transmission X-ray microscopy.

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.

Crystal structure of the human 5-HT 1B serotonin receptor bound to an inverse agonist

Abstract5-hydroxytryptamine (5-HT, also known as serotonin) regulates many physiological processes through the 5-HT receptor family. Here we report the crystal structure of 5-HT1B subtype receptor (5-HT1BR) bound to the psychotropic serotonin receptor inverse agonist methiothepin (MT). Crystallization was facilitated by replacing ICL3 with a novel optimized variant of BRIL (OB1) that enhances the formation of intermolecular polar interactions, making OB1 a potential useful tool for structural studies of membrane proteins. Unlike the agonist ergotamine (ERG), MT occupies only the conserved orthosteric binding pocket, explaining the wide spectrum effect of MT on serotonin receptors. Compared with ERG, MT shifts toward TM6 and sterically pushes residues W3276.48, F3306.50 and F3316.51 from inside the orthosteric binding pocket, leading to an outward movement of the extracellular end and a corresponding inward shift of the intracellular end of TM6, a feature shared by other reported inactive G protein-coupled receptor (GPCR) structures. Together with the previous agonist-bound serotonin receptor structures, the inverse agonist-bound 5-HT1BR structure identifies a basis for the ligand-mediated switch of 5-HT1BR activity and provides a structural understanding of the inactivation mechanism of 5-HT1BR and some other class A GPCRs, characterized by ligand-induced outward movement of the extracellular end of TM6 that is coupled with inward movement of the cytoplasmic end of this helix.

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.Bacterial cell wall components are assembled in a transmembrane cycle that involves the membrane integral pyrophosphorylase, BacA. Here the authors solve the crystal structure of BacA which shows an interdigitated inverted topology repeat that hints towards a flippase function for BacA.

Challenges and strategies for n-SAD phasing at longer X-ray wavelength

Single-wavelength Anomalous Dispersion (SAD) is the most popular experimental phasing technique to determine X-ray structure in the field of structural biology. The data collection, in this method, is performed at the absorption edge of anomalous scatterers, which are either introduced in the crystal or natively present in the macromolecules. In case of native SAD phasing, which uses the weak anomalous scattering signals from light elements – such as sulfur, phosphorous, or any ions, naturally present in the macromolecules, the most suitable energy would be around 2.5 keV (or λ = 5 Å), above the sulfur K-edge. However, such low X-ray energy is not attainable at most of the current operational macromolecular crystallography beamlines. In addition, native SAD at such low energy comes with more challenges, caused by x-ray absorption due to crystal thickness, cryo-loop, solvent around the crystal, air, as well as detector efficiency. Thereby, an Xray energy around 6 keV is considered as a good “compromise” between anomalous diffraction signal and absorption effect [1, 2, 3]. Here, we present the promises and challenges associated with native-SAD data collection at X-ray energy below 6 keV, in particular for X-ray absorption effects and optimum crystal size, using both test proteins and real-life examples. [1] Liu et al. (2012) Science, 336, 1033-1037. [2] Olieric et al. (2015) Acta Cryst, D72, 421-429. [3] Weinert et al. (2015) Nat. Methods, 12, 131-133.