Kay Diederichs

Linking Crystallographic Model and Data Quality

Finessing Crystal Analysis Protein crystallography has revolutionized our understanding of a whole variety of biological processes (see the Perspective by Evans). In crystallography, the measure of agreement between the data and the calculated model is not on the same scale as the measure of data quality, making it challenging to choose an optimal high resolution limit beyond which the data should be discarded. Now, Karplus and Diederichs (p. 1030) introduce a statistical model that assesses agreement of model and data accuracy on the same scale. Determining the structures of biological macromolecules by x-ray crystallography requires solving the phase problem. The two techniques that dominate phase evaluation (multi- and single-wavelength anomalous diffraction) rely on element-specific scattering from incorporated heavy atoms. Liu et al. (p. 1033) present procedures for routine structure determination of native proteins with no heavy atom incorporation. The technique, which relies on combining data from multiple crystals, was used to determine the structures of four native proteins, including a 1200-residue complex. A statistical method places model and data quality on the same scale and indicates how far one can model. In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, Rmerge values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, Rmerge values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.

Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism

The AcrA/AcrB/TolC complex spans the inner and outer membranes of Escherichia coli and serves as its major drug-resistance pump. Driven by the proton motive force, it mediates the efflux of bile salts, detergents, organic solvents, and many structurally unrelated antibiotics. Here, we report a crystallographic structure of trimeric AcrB determined at 2.9 and 3.0 angstrom resolution in space groups that allow asymmetry of the monomers. This structure reveals three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating access mechanism and a novel peristaltic mode of drug transport by this type of transporter.

Structural Basis of Light Harvesting by Carotenoids: Peridinin-Chlorophyll-Protein from Amphidinium carterae

Peridinin-chlorophyll-protein, a water-soluble light-harvesting complex that has a blue-green absorbing carotenoid as its main pigment, is present in most photosynthetic dinoflagellates. Its high-resolution (2.0 angstrom) x-ray structure reveals a noncrystallographic trimer in which each polypeptide contains an unusual jellyroll fold of the α-helical amino- and carboxyl-terminal domains. These domains constitute a scaffold with pseudo-twofold symmetry surrounding a hydrophobic cavity filled by two lipid, eight peridinin, and two chlorophyll a molecules. The structural basis for efficient excitonic energy transfer from peridinin to chlorophyll is found in the clustering of peridinins around the chlorophylls at van der Waals distances.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Crystal structure of human β2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome

The high affinity of human plasma β2‐glycoprotein I (β2GPI), also known as apolipoprotein‐H (ApoH), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. β2GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the β2GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C‐terminal domain arranged like beads on a string to form an elongated J‐shaped molecule. Domain V folds into a central β‐spiral of four antiparallel β‐sheets with two small helices and an extended C‐terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313–316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The β2GPI structure reveals potential autoantibody‐binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid‐binding capacity of β2GPI.

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate‐binding subunits or domains in order to couple the energy‐providing reaction with transport. In the maltose transporter of several Gram‐negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate‐binding subunit contains a C‐terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 Å resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2‐fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

Structure of the sucrose-specific porin ScrY from Salmonella typhimurium and its complex with sucrose.

The X-ray structure of a sucrose-specific porin (ScrY) from Salmonella typhimurium has been determined by multiple isomorphous replacement at 2.4 Å resolution both in its uncomplexed form and with bound sucrose. ScrY is a noncrystallographic trimer of identical subunits, each with 413 structurally well-defined amino acids. A monomer is built up of 18 anti-parallel β-strands surrounding a hydrophilic pore, with a topology closely similar to that of maltoporin. Two non-overlapping sucrose-binding sites were identified in difference Fourier maps. The higher permeability for sucrose of ScrY as compared to maltoporin is mainly accounted for by differences in their pore-lining residues.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins

BACKGROUND Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. RESULTS The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. CONCLUSIONS These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Better models by discarding data

Making the most of hard-won data in protein crystallography: to keep or not to keep, that is the question.

The Crystal Structure of the Ligand Binding Module of Axonin-1/TAG-1 Suggests a Zipper Mechanism for Neural Cell Adhesion

We have determined the crystal structure of the ligand binding fragment of the neural cell adhesion molecule axonin-1/TAG-1 comprising the first four immunoglobulin (Ig) domains. The overall structure of axonin-1(Ig1-4) is U-shaped due to contacts between domains 1 and 4 and domains 2 and 3. In the crystals, these molecules are aligned in a string with adjacent molecules oriented in an anti-parallel fashion and their C termini perpendicular to the string. This arrangement suggests that cell adhesion by homophilic axonin-1 interaction occurs by the formation of a linear zipper-like array in which the axonin-1 molecules are alternately provided by the two apposed membranes. In accordance with this model, mutations in a loop critical for the formation of the zipper resulted in the loss of the homophilic binding capacity of axonin-1.