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1.  Crystal Structure of Escherichia coli-Expressed Haloarcula marismortui Bacteriorhodopsin I in the Trimeric Form 
PLoS ONE  2014;9(12):e112873.
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.
PMCID: PMC4257550  PMID: 25479443
2.  Mesh–related and intraoperative complications of pelvic organ prolapse repair 
To evaluate the rates of complications of pelvic organ prolapse repair and to determine their risk factors.
Material and methods
The study included 677 patients operated for pelvic organ prolapse with trocar guided Prolift mesh. Patients were followed up within 1 and 3 months. A phone interview was conducted and patients with complaints were invited and evaluated in office settings.
Mean age was 60 years. For the phone interview, 86.5% of patients were available. Overall complication rates were 22.5% (152/677). Fifteen patients (2.2%) developed bleeding over 500 cc; pelvic hematomas – 5.5%; perineal hematomas – 2.5%; urethral injuries – 0.3%; bladder injury in 1.6%; rectal damage in 0.7% and ureteral trauma in 0.2%.
Mesh related complications included: erosions in 4.8%; vaginal synechiae – 0.3%; protrusion of mesh into the bladder – 0.15%; vesicovaginal fistula with mesh protrusion – 0.3%; mesh shrinkage – 1%; dyspareunia and pain in 2.4% cases. Pelvic abscess was found in 0.6% including one case of lethal necrotizing fasciitis. The risk factors of complications were assessed via logistic regression analysis.
Younger age, less prominent prolapse, hematomas and concomitant hysterectomies are associated with higher risk of complications.
PMCID: PMC4165670  PMID: 25247091
pelvic organ prolapse; complications; mesh related; risk factors
3.  Structural insights into lipid-dependent reversible dimerization of human GLTP 
It is shown that dimerization is promoted by glycolipid binding to human GLTP. The importance of dimer flexibility in wild-type protein is manifested by point mutation that ‘locks’ the dimer while diversifying ligand/protein adaptations.
Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the ‘portal entrance’ of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP–SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a ‘locked’ dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the ‘flexible’ and ‘locked’ dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP.
PMCID: PMC3606038  PMID: 23519669
glycolipid transfer protein; selectivity; sulfatides; lipid-mediated homodimerization; GLTP fold
4.  A survey of global radiation damage to 15 different protein crystal types at room temperature: a new decay model 
Journal of Synchrotron Radiation  2012;20(Pt 1):14-22.
A systematic study of the sensitivity to radiation damage of crystals held at room temperature for a large set of model macromolecular structures is presented.
The radiation damage rates to crystals of 15 model macromolecular structures were studied using an automated radiation sensitivity characterization procedure. The diffracted intensity variation with dose is described by a two-parameter model. This model includes a strong resolution-independent decay specific to room-temperature measurements along with a linear increase in overall Debye–Waller factors. An equivalent representation of sensitivity via a single parameter, normalized half-dose, is introduced. This parameter varies by an order of magnitude between the different structures studied. The data show a correlation of crystal radiation sensitivity with crystal solvent content but no dose-rate dependency was detected in the range 0.05–300 kGy s−1. The results of the crystal characterization are suitable for either optimal planning of room-temperature data collection or in situ crystallization plate screening experiments.
PMCID: PMC3943537  PMID: 23254652
radiation damage; room temperature; X-ray diffraction; data collection
5.  Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein 
Structure (London, England : 1993)  2011;19(11):1644-1654.
Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) which are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D||D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of ‘portal entrance’ residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homo-dimerization. ‘Door-opening’ conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of ‘engineered GLTPs’ with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.
PMCID: PMC3220887  PMID: 22078563
GLTP-fold; sulfatide-binding pocket; structure-based protein design; lipid chain re-arrangement; dimer interface of ligand-bound human GLTP
6.  The use of workflows in the design and implementation of complex experiments in macromolecular crystallography 
A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines. This tool provides the possibility of automating complex experiments without the need for expertise in instrumentation control and programming, but rather by accessing defined beamline services.
The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal re­orientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE.
PMCID: PMC3413211  PMID: 22868763
workflows; automation; data processing; macromolecular crystallography; experimental protocols; characterization; reorientation; radiation damage
7.  Experimental procedure for the characterization of radiation damage in macromolecular crystals 
Journal of Synchrotron Radiation  2011;18(Pt 3):381-386.
A novel automatic procedure to determine the sensitivity of macromolecular crystals to radiation damage is presented. The information extracted from this procedure can be directly used for optimal planning of data collection or/and beamline calibration.
A reliable and reproducible method to automatically characterize the radiation sensitivity of macromolecular crystals at the ESRF beamlines has been developed. This new approach uses the slope of the linear dependence of the overall isotropic B-factor with absorbed dose as the damage metric. The method has been implemented through an automated procedure using the EDNA on-line data analysis framework and the MxCuBE data collection control interface. The outcome of the procedure can be directly used to design an optimal data collection strategy. The results of tests carried out on a number of model and real-life crystal systems are presented.
PMCID: PMC3268693  PMID: 21525646
BEST; EDNA; radiation damage
8.  Optimization of data collection taking radiation damage into account 
Software implementing a new method for the optimal choice of data-collection parameters, accounting for the effects of radiation damage, is presented.
To take into account the effects of radiation damage, new algorithms for the optimization of data-collection strategies have been implemented in the software package BEST. The intensity variation related to radiation damage is approximated by log-linear functions of resolution and cumulative X-­ray dose. Based on an accurate prediction of the basic characteristics of data yet to be collected, BEST establishes objective relationships between the accessible data completeness, resolution and signal-to-noise statistics that can be achieved in an experiment and designs an optimal plan for data collection.
PMCID: PMC2852305  PMID: 20382994
X-ray data collection; protein crystals; BEST software; radiation damage

Results 1-8 (8)