Max Planck Institute for Medical Research

The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic determination of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the number of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.

Abstract For a successful analysis of the relation between amino acid sequence and protein structure, an unambiguous and physically meaningful definition of secondary structure is essential. We have developed a set of simple and physically motivated criteria for secondary structure, programmed as a pattern‐recognition process of hydrogen‐bonded and geometrical features extracted from x‐ray coordinates. Cooperative secondary structure is recognized as repeats of the elementary hydrogen‐bonding patterns “turn” and “bridge.” Repeating turns are “helices,” repeating bridges are “ladders,” connected ladders are “sheets.” Geometric structure is defined in terms of the concepts torsion and curvature of differential geometry. Local chain “chirality” is the torsional handedness of four consecutive C α positions and is positive for right‐handed helices and negative for ideal twisted β‐sheets. Curved pieces are defined as “bends.” Solvent “exposure” is given as the number of water molecules in possible contact with a residue. The end result is a compilation of the primary structure, including SS bonds, secondary structure, and solvent exposure of 62 different globular proteins. The presentation is in linear form: strip graphs for an overall view and strip tables for the details of each of 10.925 residues. The dictionary is also available in computer‐readable form for protein structure prediction work.

Microglial cells represent the immune system of the mammalian brain and therefore are critically involved in various injuries and diseases. Little is known about their role in the healthy brain and their immediate reaction to brain damage. By using in vivo two-photon imaging in neocortex, we found that microglial cells are highly active in their presumed resting state, continually surveying their microenvironment with extremely motile processes and protrusions. Furthermore, blood-brain barrier disruption provoked immediate and focal activation of microglia, switching their behavior from patroling to shielding of the injured site. Microglia thus are busy and vigilant housekeepers in the adult brain.

Activity-driven modifications in synaptic connections between neurons in the neocortex may occur during development and learning. In dual whole-cell voltage recordings from pyramidal neurons, the coincidence of postsynaptic action potentials (APs) and unitary excitatory postsynaptic potentials (EPSPs) was found to induce changes in EPSPs. Their average amplitudes were differentially up- or down-regulated, depending on the precise timing of postsynaptic APs relative to EPSPs. These observations suggest that APs propagating back into dendrites serve to modify single active synaptic connections, depending on the pattern of electrical activity in the pre- and postsynaptic neurons.

An algorithm has been developed for the automatic interpretation of a given set of observed reciprocal-lattice points. It extracts a reduced cell and assigns indices to each reflection by a graph-theoretical implementation of the local indexing method. All possible symmetries of the observed lattice compatible with the metric of the reduced cell are recognized and reported, together with the unit-cell constants and the linear index transformation relating the conventional to the reduced cell. This algorithm has been incorporated into the program XDS [Kabsch (1988). J. Appl. Cryst. 21 , 916–924], which is now able to process single-crystal area-detector data without prior knowledge of the symmetry and the unit-cell constants.

A simple procedure is derived which determines a best rotation of a given vector set into a second vector set by minimizing the weighted sum of squared deviations. The method is generalized for any given metric constraint on the transformation.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.

Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-reduction package XDS are explained, including the new features of automatic determination of spot size and reflecting range, recognition and assignment of crystal symmetry and a highly efficient algorithm for the determination of correction/scaling factors.

The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

A method for obtaining sequence information directly from plasmid DNA is presented. The procedure involves the rapid preparation of clean supercoiled plasmid DNA from small bacterial cultures, its complete denaturation by alkali, and sequence determination using oligodeoxyribonucleotide-primed enzymatic DNA synthesis in the presence of dideoxynucleoside triphosphates. The advantages of the method include speed, simplicity, avoidance of additional cloning steps into single-stranded phage M13 vectors, and hence applicability to sequencing large numbers of samples.

The title to a seminar presentation by I. C. Gunsalus in 1973 was Oxygen: An essential toxin, referring to the complex \nrole that atmospheric dioxygen has in biology. The relatively simple function as terminal oxidant for aerobic life was dramatically \naugmented by Osamu Hayaishi with his identification of an enzyme that catalyzes the conversion of catechol to muconic acid by \noxidative cleavage.1 He named this biological catalyst pyrocatechase, which proved to be the landmark discovery of an enzyme \nthat incorporated atmospheric dioxygen into the carbon chain of the substrate, thereby initiating cleavage of the benzene ring. \nThis review of the oxygenase cytochrome P450 is dedicated to Dr. Hayaishi and his pioneering discovery in what is now the 50th anniversary of his work!\n\nWe now realize that Nature has found many ways to utilize atmospheric dioxygen to functionalize molecules through the use of a diverse \nset of cofactors. Flavin, non−heme iron, copper, and metalloporphyrin complexes have all been conscripted to metabolize atmospheric \ndioxygen in an oxygenase catalytic cycle, resulting in the incorporation of one or both oxygen atoms into a substrate. This review focuses \non one of the heme−containing classes, termed cytochrome P450s and abbreviated CYP. Although but one member in the large group of \noxygenases, the cytochrome P450s play a variety of critical roles in biology.\n\nMany members of the cytochrome P450 superfamily of hemoproteins are currently known, and the numbers continue to grow as more genomes \nare sequenced. There are almost 4000 identified P450 genes at the date of this writing, and they are collected and annotated in a variety \nof web sites, such as that maintained by Nelson (http://drnelson. utmem.edu/CytochromeP450.html). The cytochrome P450s have been found in \nall branches of the tree of life that catalogs the diversity of life forms. In the broadest terms, there are two main functional roles for \nthese oxygenases. One is the metabolism of xenobiotics (compounds exogenous to the organism) as a protective role of degradation or provision \nof polar handles for solubilization in preparation for excretion. A second broad functional role is in the biosynthesis of critical signaling \nmolecules used for control of development and homeostasis. In mammalian tissues the P450s play these roles through the metabolism of drugs \nand xenobiotics and the synthesis of steroid hormones and fat−soluble vitamin metabolism and the conversion of polyunsaturated fatty \nacids to biologically active molecules, respectively. Similar roles are fulfilled in plants (hormone biosynthesis and herbicide degradation) \nand insects (control of development via hormone biosynthesis or provision of insecticide resistance). For instance, plants have an unusually \nlarge number of P450 genes. A reason is their sessile nature: for example, plants defend themselves through breakdown of herbicides by \ncatalyzing the synthesis of a large number of secondary metabolites or by synthesizing defense molecules such as DIMBOA.2,3 In addition, \nthe biosynthesis of critical metabolic regulators is also often carried out by the cytochrome P450s.\n\nThe important metabolic role together with the unique chemistry and physical properties of the cytochrome P450s provide a strong attraction \nfor scientists in many disciplines. Relevance to human health was the initial focus of pharmacologists and toxicologists. The role of metal \ncenters and their associated unique spectral properties in the cytochrome P450s is a magnet for bioinorganic chemists and biophysicists. The \ndifficult conversion of unactivated hydrocarbons attracted the bioorganic chemist. With the genome revolution and insights into the complex \nprocess of transcriptional and translational regulation, biochemists and molecular biologists found exciting problems in the study of CYPs.\n\nA continuing challenge is to understand how the diverse set of substrate specificities and metabolic transformations are determined by the \nprecise nature of the heme−iron oxygen and protein structure. The structure and electronic configuration of the active oxygen \nintermediates which serve as efficient catalysts remains an area of active research. Complicating this richness in metabolic potential \nis the importance of genetic differences, including single nucleotide polymorphisms, which can alter the physiological responses of the \ncytochrome P450s. Thus, over the past five−plus decades one has seen the evolution from a whole−organ and animal pharmacology \napproach to a quest for the molecular details necessary for precise understanding of structure and function of the P450 systems in \nmaintaining cellular homeostasis. The P450s are now recognized to occupy a great variety of phylogenetically distributed isoform \nactivities, and these variations in metabolic profile and substrate specificity are ultimately dictated by the bioinorganic chemistry \nof heme iron and oxygen as controlled by the protein environment.\n\nWith the elucidation of precise structures for many P450 hemoproteins as well as the application of varied biochemical and biophysical \nmethodologies, this diverse class of oxygenases is beginning to yield its secrets. Much remains to be learned, however, as many of the \nfundamental chemical entities and catalytic details, though perhaps described in textbooks, are in fact still poorly understood. The \nfocus of this review is to place the current knowledge base of cytochrome P450 structure−function in context with the general \naspects of metalloenzyme function. In 2006 Dr. Hayaishi, the founder of this broad field of oxygen metabolism, will celebrate an \nimportant birthday. Hopefully, in reading this review, he will be struck with the outstanding progress that has been realized with \nthis one particular oxygenase and at the same time perhaps provide some important suggestions as to pathways for solving the remaining problems.4\n\nCytochrome P450 has benefited from the attention of inorganic, organic, and physical chemists since its discovery due to its unique \nspectral properties as well as its ability to efficiently catalyze a variety of difficult biotransformations. With the discovery of \nP450 involvement in steroid biosynthesis in the 1970s, joined with its central function in drug metabolism, with its role in a variety \nof other pharmaceutical applications, P450 became one of the most intensively investigated biochemical systems. Multiple monographs, \nprinted conference proceedings, and thematic books have been published as well as special Methods in Enzymology volumes, only a few \nof which can be referenced here.5−12\n\nThe cytochrome P450s became most known for their efficiency in hydroxylation of unactivated alkanes as only a select few oxygenases \npossess the requisite active oxygen state. With equal efficacy, P450s can carry out a wide variety of biotransformations. The list \nin ref 13 includes more than 20 different chemical reactions. Some more unusual reactions catalyzed by P450 were recently reviewed by Guengerich.14\n\nThe mechanism of P450 is a complex cascade of individual steps involving the interaction of protein redox partners and consumption of \nreducing equivalents, most commonly in the form of NAD(P)H. It is somewhat humbling that the earliest versions of the enzymatic cycle \npublished over 30 years ago had much of the important steps characterized by physical and chemical methods.15 Continual refinement has \nled to more detailed versions and the direct observation and structural characterization of new adducts of iron and oxygen. The current \nversion contains eight intermediates, including highly transient caged radical pairs, and has been reviewed from various perspectives.11,12,16−19\n\nWhile the basic concepts central to P450 catalysis were appreciated by early 1970, notable progress in the detailed understanding of these \nmechanisms has been made in the past decade. This has been possible due to the accumulation of exciting data generated through application \nof a wide set of new methodologies, including systematic directed mutagenesis, high−resolution X−ray crystal structure \ndetermination, multiparametric spectroscopic characterization of intermediates, isolation of critical steps using cryogenic or fast \nkinetic techniques, and many excellent quantum chemical and molecular dynamics computational studies. The current view of the oxygen \nactivation mechanisms, catalyzed by metal centers in heme enzymes (as well as in non−heme enzymes, which lie outside the scope \nof this review), ensures one with a much better opportunity to see the common mechanistic picture than was possible earlier.20 \nSuccessful mechanistic studies of other heme enzymes which use different forms of so−called 'active oxygen \nintermediates', such as peroxidases,21,22 heme oxygenases (HO),23−25 catalases,26 nitric oxide synthases \n(NOS),27,28 peroxygenases,29,30 provide a vision of a highly diverse cofactor. Mechanistic insight from each of these various \nsystems has provided important complementary insight into cytochrome P450 mechanism. A fundamental question remaining is how the \nprotein controls efficient performance of such different functions using similar highly reactive heme−oxygen complexes. The \ncomparison of similar reactive intermediates in different enzymes helps to distinguish between the essential features of each of the \nenzymes and so provides additional clues to the revelation of the active role of the protein in heme−enzyme catalysis. The \nrecent progress in isolation and cryogenic stabilization of some of these intermediates makes possible direct spectroscopic and \nstructural studies of this type.\n\nAn exhaustive review of all achievements in oxygen activation chemistry is clearly difficult, even if the field is limited to \nthe processes directly relevant to P450 catalysis. Discussion of the P450 cat

The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses.

Muscle contraction consists of a cyclical interaction between myosin and actin driven by the concomitant hydrolysis of adenosine triphosphate (ATP). A model for the rigor complex of F actin and the myosin head was obtained by combining the molecular structures of the individual proteins with the low-resolution electron density maps of the complex derived by cryo-electron microscopy and image analysis. The spatial relation between the ATP binding pocket on myosin and the major contact area on actin suggests a working hypothesis for the crossbridge cycle that is consistent with previous independent structural and biochemical studies.

Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20-60 Pa H(2)O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50-70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.

The expression patterns of 13 GABAA receptor subunit encoding genes (alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta) were determined in adult rat brain by in situ hybridization. Each mRNA displayed a unique distribution, ranging from ubiquitous (alpha 1 mRNA) to narrowly confined (alpha 6 mRNA was present only in cerebellar granule cells). Some neuronal populations coexpressed large numbers of subunit mRNAs, whereas in others only a few GABAA receptor-specific mRNAs were found. Neocortex, hippocampus, and caudate-putamen displayed complex expression patterns, and these areas probably contain a large diversity of GABAA receptors. In many areas, a consistent coexpression was observed for alpha 1 and beta 2 mRNAs, which often colocalized with gamma 2 mRNA. The alpha 1 beta 2 combination was abundant in olfactory bulb, globus pallidus, inferior colliculus, substantia nigra pars reticulata, globus pallidus, zona incerta, subthalamic nucleus, medial septum, and cerebellum. Colocalization was also apparent for the alpha 2 and beta 3 mRNAs, and these predominated in areas such as amygdala and hypothalamus. The alpha 3 mRNA occurred in layers V and VI of neocortex and in the reticular thalamic nucleus. In much of the forebrain, with the exception of hippocampal pyramidal cells, the alpha 4 and delta transcripts appeared to codistribute. In thalamic nuclei, the only abundant GABAA receptor mRNAs were those of alpha 1, alpha 4, beta 2, and delta. In the medial geniculate thalamic nucleus, alpha 1, alpha 4, beta 2, delta, and gamma 3 mRNAs were the principal GABAA receptor transcripts. The alpha 5 and beta 1 mRNAs generally colocalized and may encode predominantly hippocampal forms of the GABAA receptor. These anatomical observations support the hypothesis that alpha 1 beta 2 gamma 2 receptors are responsible for benzodiazepine I (BZ I) binding, whereas receptors containing alpha 2, alpha 3, and alpha 5 contribute to subtypes of the BZ II site. Based on significant mismatches between alpha 4/delta and gamma mRNAs, we suggest that in vivo, the alpha 4 subunit contributes to GABAA receptors that lack BZ modulation.

Four cloned cDNAs encoding 900-amino acid putative glutamate receptors with approximately 70 percent sequence identity were isolated from a rat brain cDNA library. In situ hybridization revealed differential expression patterns of the cognate mRNAs throughout the brain. Functional expression of the cDNAs in cultured mammalian cells generated receptors displaying alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective binding pharmacology (AMPA = quisqualate greater than glutamate greater than kainate) as well as cation channels gated by glutamate, AMPA, and kainate and blocked by 6,7-dinitroquinoxaline-2,3-dione (CNQX).

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

We report data from infrared absorption (FTIR) and X-ray photoelectron spectroscopies that correlate the molecular conformation of oligo(ethylene glycol) (OEG)-terminated self-assembled alkanethiolate monolayers (SAMs) with the ability of these films to resist protein adsorption. We studied three different SAMs of alkanethiolates on both evaporated Au and Ag surfaces. The SAMs were formed from substituted 1-undecanethiols with either a hydroxyl-terminated hexa(ethylene glycol) (EG6-OH) or a methoxy-terminated tri(ethylene glycol) (EG3-OMe) end group, or a substituted 1-tridecanethiol chain with a methoxy-terminated tri(ethylene glycol) end group and a −CH2OCH3 side chain at the C-12 atom (EG[3,1]-OMe). The infrared data of EG6-OH-terminated SAMs on both Au and Ag surfaces reveal the presence of a crystalline helical OEG phase, coexisting with amorphous OEG moieties; the EG[3,1]-OMe-terminated alkanethiolates on Au and Ag show a lower absolute coverage and greater disorder than the two other compounds. The molecular conformation of the methoxy-terminated tri(ethylene glycol) (EG3-OMe) is different on Au and Ag surfaces due to the different lateral densities of SAMs on these substrates: on Au we find a conformation similar to that of EG6-OH alkanethiolates, whereas on Ag the infrared spectra indicate a densely packed film with trans conformation around the C−C bonds of the glycol units. The resistance of these OEG-functionalized alkanethiolate SAMs to adsorption of fibrinogen from a buffered solution correlates with the molecular conformation of the OEG moieties. The predominantly crystalline helical and the amorphous forms of OEG on gold substrates are resistant to adsorption of proteins, while a densely packed “all-trans” form of EG3-OMe present on silver surfaces adsorbs protein. The experimental observations are compatible with the hypothesis that binding of interfacial water by the OEG moieties is important in their ability to resist protein adsorption.

Superresolution imaging in sharper focus An optical microscope cannot distinguish objects separated by less than half the wavelength of light. Superresolution techniques have broken this “diffraction limit” and provided exciting new insights into cell biology. Still, such techniques hit a limit at a resolution of about 10 nm. Balzarotti et al. describe another way of localizing single molecules called MINFLUX (see the Perspective by Xiao and Ha). As in photoactivated localization microscopy and stochastic optical reconstruction microscopy, fluorophores are stochastically switched on and off, but the emitter is located using an excitation beam that is doughnut-shaped, as in stimulated emission depletion. Finding the point where emission is minimal reduces the number of photons needed to localize an emitter. MINFLUX attained ∼1-nanometer precision, and, in single-particle tracking, achieved a 100-fold enhancement in temporal resolution. Science , this issue p. 606 ; see also p. 582

In the central nervous system (CNS), the principal mediators of fast synaptic excitatory neurotransmission are L-glutamate-gated ion channels that are responsive to the glutamate agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). In each member of a family of four abundant AMPA receptors, a small segment preceding the predicted fourth transmembrane region has been shown to exist in two versions with different amino acid sequences. These modules, designated "flip" and "flop," are encoded by adjacent exons of the receptor genes and impart different pharmacological and kinetic properties on currents evoked by L-glutamate or AMPA, but not those evoked by kainate. For each receptor, the alternatively spliced messenger RNAs show distinct expression patterns in rat brain, particularly in the CA1 and CA3 fields of the hippocampus. These results identify a switch in the molecular and functional properties of glutamate receptors operated by alternative splicing.