Long-wavelength macromolecular crystallography - First successful native SAD experiment close to the sulfur edge.

Phasing of novel macromolecular crystal structures has been challenging since the start of structural biology. Making use of anomalous diffraction of natively present elements, such as sulfur and phosphorus, for phasing has been possible for some systems, but hindered by the necessity to access longer X-ray wavelengths in order to make most use of the anomalous scattering contributions of these elements. Presented here are the results from a first successful experimental phasing study of a macromolecular crystal structure at a wavelength close to the sulfur K edge. This has been made possible by the in-vacuum setup and the long-wavelength optimised experimental setup at the I23 beamline at Diamond Light Source. In these early commissioning experiments only standard data collection and processing procedures have been applied, in particular no dedicated absorption correction has been used. Nevertheless the success of the experiment demonstrates that the capability to extract phase information can be even further improved once data collection protocols and data processing have been optimised.

Production and crystallization of the C-propeptide trimer from human procollagen III.

The C-propeptide domains of the fibrillar procollagens, which are present throughout the Metazoa in the form of ∼90 kDa trimers, play crucial roles in both intracellular molecular assembly and extracellular formation of collagen fibrils. The first crystallization of a C-propeptide domain, that from human procollagen III, is described. Following transient expression in mammalian 293T cells of both the native protein and a selenomethionine derivative, two crystal forms of the homotrimer were obtained: an orthorhombic form (P2(1)2(1)2(1)) that diffracted to 1.7 Šresolution and a trigonal form (P321) that diffracted to 3.5 Šresolution. Characterization by MALDI-TOF mass spectrometry allowed the efficiency of selenomethionine incorporation to be determined.

Structures of respiratory syncytial virus nucleocapsid protein from two crystal forms: details of potential packing interactions in the native helical form.

Respiratory syncytial virus (RSV) is a frequent cause of respiratory illness in infants, but there is currently no vaccine nor effective drug treatment against this virus. The RSV RNA genome is encapsidated and protected by a nucleocapsid protein; this RNA-nucleocapsid complex serves as a template for viral replication. Interest in the nucleocapsid protein has increased owing to its recent identification as the target site for novel anti-RSV compounds. The crystal structure of human respiratory syncytial virus nucleocapsid (HRSVN) was determined to 3.6 Å resolution from two crystal forms belonging to space groups P2(1)2(1)2(1) and P1, with one and four decameric rings per asymmetric unit, respectively. In contrast to a previous structure of HRSVN, the addition of phosphoprotein was not required to obtain diffraction-quality crystals. The HRSVN structures reported here, although similar to the recently published structure, present different molecular packing which may have some biological implications. The positions of the monomers are slightly shifted in the decamer, confirming the adaptability of the ring structure. The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions.

First Lebanese Antibiotic Awareness Week campaign: knowledge, attitudes and practices towards antibiotics.

Antibiotic resistance (ABR) is a major global health threat that increases the risk of treatment failure and increases medical costs. One of the most common factors contributing to the spread of ABR is self-medication. The public, as well as workers in clinical and veterinary sectors, commit false practices towards appropriate antibiotic use, favouring the spread of resistance. As such, the first Lebanese Antibiotic Awareness Week campaign was initiated with a human-centred and interactive approach. The data showed a strikingly low level of antibiotic awareness. Cooperation between relevant stakeholders, policy-makers and health actors is crucial to control and overcome the problem of ABR.

Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein.

Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.

Timescales of water accumulation in magmas and implications for short warning times of explosive eruptions.

Water plays a key role in magma genesis, differentiation, ascent and, finally, eruption. Despite the recognized crucial function of water, there are still several issues that continue to blur our view about its role in magmatic systems. What are the timescales of H2O accumulation in crystallizing magmas? What are the ascent rates of water-rich residual melts leading to explosive eruptions? Here, we track the timescale of water accumulation in a residual melt resulting from crystallization of a hydrous CO2-bearing magmatic mass stored at mid- to deep-crustal levels in a subduction-related geodynamic setting. Our results indicate that, after a repose period ranging from few to several thousand years, water-rich melts with water concentrations larger than 6-9 wt.% can migrate towards the Earth surface in very short timescales, on the order of days or even hours, possibly triggering explosive eruptions with short warning times and devoid of long-term geophysical precursors.

[Ocular manifestation of Behçet disease (study of 50 patients consulting in dermatology department)]. / Les manifestations oculaires de la maladie de Behçet (étude de 50 patients consultant en dermatologie).

INTRODUCTION: Behçet disease is a multisystemic vascularitis. Ocular affection is one of the major criteria of this disease. The aim of this study is to specify the clinical, therapeutical characteristics and the prognosis factors of the ocular affection in patients having BD and admitted to the dermatology department. PATIENTS AND METHODS: It is a retrospective investigation carried out in the dermatology department of Ibn Rochd university hospital center of Casablanca, Morroco, from Jannuary 1990 until December 2003. Two patient groups have been distinguished. The first one involved 50 patients (44,2%) having BD with ocular affection, and the second group involved 63 patients having BD without ocular affection. RESULTS: The mean age was 29 +/- 8 years in the first group VS 30 +/- 7,9 years in the 2(nd) group. The ocular affection was more frequent in males than in females (P < 0.05). The ocular manifestations were marked by uveitis and retinal periphlebitis. The frequency of the cutaneomucosal and joint manifestations was similar in both groups, whereas neurologic and vascular with ocular affections. The choice of the treatment depended on the type of ocular affection. Evolution was marked by blindness in 6 patients (12%). DISCUSSION: The ocular affection comes second after the cutanous mucuous affection. Males are more clearly affected than females. This allows saying that there is a marked effect of the sexual hormones on the ocular affection. Age is not predictive of this ocular affection. The ocular affection was severe in our series and was dominated by uveitis and vascularitis. We insist on the severity of ocular Behçet and its evolution? Risk toward blindness especially concerning young man. Currently, the treatment is not codified; however, the encouraging outcome obtained with some immunosuppressive therapies would be better if this treatment was set up early. CONCLUSION: This study enebed us to re-examine the ocular manifestations of the Behçet disease in the Maroccan population by the means of a consultation of Dermatology. Il should be noted that it is worse forecast because on the one hand of its frequent association to vascular and neurological affections and other share of the delay of consultation noted at the majority of our patients.

[Vulvar pathology]. / La pathologie vulvaire.

We evaluated the main causes of vulvar dermatoses; in 785 patients with a vulvar diseases who visited Ibn Rochd Hospital Centre, Casablanca between January 1991 and December 2002. The average age was 31 years (range 2 months to 81 years); 362 patients (41.52%) had vulvar pruritus, 273 patients (34.77%) had warts and 157 (20%) had one or more vulva ulcers. The most common infectious pathology was papillomavirus infection, found in 273 cases (34.77%), followed by vulvovaginal candidiases in 102 cases (12.99 %), syphilitic chancre in 18 cases (2.29%) and herpes genitalis in 17 cases (2.16%). The most common non-infectious pathology was vulvar dermatosis: 259 cases (32.99%); idiopathic pruritus vulvae: 61 cases (7.7%); and tumour-related conditions: 45 cases (5.6%). The frequency of infectious conditions was over 50% and these were generally sexually transmitted infections.

Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex.

LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular protein function and the structural contusion implies a model in which newly synthesized, intrinsically disordered LMO2 binds to a partner protein nucleating further interactions and suggests approaches for therapeutic targeting of LMO2.

Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex.

YgjD from COG0533 is amongst a small group of highly conserved proteins present in all three domains of life. Various roles and biochemical functions (including sialoprotease and endonuclease activities) have been ascribed to YgjD and orthologs, the most recent, however, is involvement in the post transcriptional modification of certain tRNAs by formation of N6-threonyl-adenosine (t6A) at position 37. In bacteria, YgjD is essential and along with YeaZ, YjeE, and YrdC has been shown to be 'necessary and sufficient' for the tRNA modification. To further define interactions and possible roles for some of this set of proteins we have undertaken structural and biochemical studies. We show that formation of the previously reported heterodimer of YgjD-YeaZ involves ordering of the C-terminal region of YeaZ which extends along the surface of YgjD in the crystal structure. ATPγS or AMP is observed in YgjD while no nucleotide is bound on YeaZ. ITC experiments reveal previously unreported binary and ternary complexes which can be nucleotide dependent. The stoichiometry of the YeaZ-YgjD complex is 1:1 with a K(D) of 0.3 µM. YgjD and YjeE interact only in the presence of ATP, while YjeE binds to YgjD-YeaZ in the presence of ATP or ADP with a K(D) of 6 µM. YgjD doesn't bind the precursors of t6A, threonine, and bicarbonate. These results show a more complex set of interactions than previously thought, which may have a regulatory role. The understanding gained should help in deriving inhibitors of these essential proteins that might have potential as antibacterial drugs.