Cryo-EM structure of ABCG5/G8 in complex with modulating antibodies.

The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution.

Automated buffer preparation using quaternary valve in fast performance liquid chromatography for protein purification from a cell membrane.

There is a great need for high-throughput protein purification to produce protein molecules for research and therapeutics. Although there have been significant advancements made in automated multi-step chromatography and preparative in-process design-of-experiment (DOE) capabilities in commercial fast performance liquid chromatography (FPLC) instruments, almost all commercial FPLCs rely on a binary buffer mixing system, which hinders automated buffer preparation. Nevertheless, current-generation FPLCs are equipped with a quaternary mixer designed for limited in-line buffer preparation and preparative pH scouting DOE experiments.

Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression.

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets.

A Brief Clinical Update on Hepatitis C--The Essentials.

Infection with the hepatitis C virus (HCV) is a common cause of cirrhosis and liver failure and the most common indication for liver transplant in the United States. Based on the prevalence of HCV infection at 1.3% of the US population, there are an estimated 74,000 people living with HCV infection in the state of Wisconsin, the majority of whom are undiagnosed.

The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold.

Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold.

Structural basis for heavy metal detoxification by an Atm1-type ABC exporter.

Although substantial progress has been achieved in the structural analysis of exporters from the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, much less is known about how they selectively recognize substrates and how substrate binding is coupled to ATP hydrolysis. We have addressed these questions through crystallographic analysis of the Atm1/ABCB7/HMT1/ABCB6 ortholog from Novosphingobium aromaticivorans DSM 12444, NaAtm1, at 2.4 angstrom resolution.

Self-assembled lipid and membrane protein polyhedral nanoparticles.

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼ 20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry.

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40.

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and d-galactose complex determined at 2.

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40.

Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl-(1-->3)-D-galactose] using various beta-agarases. Neoagarobiose hydrolase is an enzyme that acts on the alpha-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production.

Water polygons in high-resolution protein crystal structures.

We have analyzed the interstitial water (ISW) structures in 1500 protein crystal structures deposited in the Protein Data Bank that have greater than 1.5 A resolution with less than 90% sequence similarity with each other. We observed varieties of polygonal water structures composed of three to eight water molecules.

PDB Editor: a user-friendly Java-based Protein Data Bank file editor with a GUI.

The Protein Data Bank file format is the format most widely used by protein crystallographers and biologists to disseminate and manipulate protein structures. Despite this, there are few user-friendly software packages available to efficiently edit and extract raw information from PDB files. This limitation often leads to many protein crystallographers wasting significant time manually editing PDB files.

High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences.

Immobilized metal ion affinity chromatography (IMAC) has become one of the most popular protein purification methods for recombinant proteins with a hexa-histidine tag (His-tag) placed at the C- or N-terminus of proteins. Nevertheless, there are always difficult proteins that show weak binding to the metal chelating resin and thus low purity. These difficulties are often overcome by increasing the His-tag to 8 or 10 histidines.

Crystal structure of the multidrug efflux transporter AcrB at 3.1A resolution reveals the N-terminal region with conserved amino acids.

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies, respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114-kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined.