The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.

• MeSH
• Endosomal Sorting Complexes Required for Transport chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray methods   MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy methods   MeSH
• Mitogen-Activated Protein Kinases chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Scattering, Radiation * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Many prokaryotic genomes comprise Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) offering defense against foreign nucleic acids. These immune systems are conditioned by the production of small CRISPR-derived RNAs matured from long RNA precursors. This often requires a Csy4 endoribonuclease cleaving the RNA 3'-end. METHODS: We report extended explicit solvent molecular dynamic (MD) simulations of Csy4/RNA complex in precursor and product states, based on X-ray structures of product and inactivated precursor (55 simulations; ~3.7μs in total). RESULTS: The simulations identify double-protonated His29 and deprotonated terminal phosphate as the likely dominant protonation states consistent with the product structure. We revealed potential substates consistent with Ser148 and His29 acting as the general base and acid, respectively. The Ser148 could be straightforwardly deprotonated through solvent and could without further structural rearrangements deprotonate the nucleophile, contrasting similar studies investigating the general base role of nucleobases in ribozymes. We could not locate geometries consistent with His29 acting as general base. However, we caution that the X-ray structures do not always capture the catalytically active geometries and then the reactive structures may be unreachable by the simulation technique. CONCLUSIONS: We identified potential catalytic arrangement of the Csy4/RNA complex but we also report limitations of the simulation technique. Even for the dominant protonation state we could not achieve full agreement between the simulations and the structural data. GENERAL SIGNIFICANCE: Potential catalytic arrangement of the Csy4/RNA complex is found. Further, we provide unique insights into limitations of simulations of protein/RNA complexes, namely, the influence of the starting experimental structures and force field limitations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

• MeSH
• CRISPR-Associated Proteins chemistry   metabolism   MeSH
• Time Factors MeSH
• CRISPR-Cas Systems * MeSH
• Endoribonucleases chemistry   metabolism   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Clustered Regularly Interspaced Short Palindromic Repeats * MeSH
• Molecular Dynamics Simulation * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Electrophysiologic Techniques, Cardiac methods   instrumentation   MeSH
• Electrocardiography, Ambulatory MeSH
• Catheter Ablation methods   MeSH
• Ventricular Premature Complexes diagnosis   therapy   MeSH
• Humans MeSH
• Treatment Outcome MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH

Understanding the RNA binding specificity of protein is of primary interest to decipher their function in the cell. Here, we review the methodology used to solve the structures of protein-RNA complexes using solution-state NMR spectroscopy: from sample preparation to structure calculation procedures. We also describe how molecular dynamics simulations can help providing additional information on the role of key amino acid side chains and of water molecules in protein-RNA recognition.

• MeSH
• CELF Proteins chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy methods   MeSH
• RNA chemistry   genetics   metabolism   MeSH
• RNA Splicing Factors chemistry   metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Thermodynamics MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Hydrogen Bonding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Fibrilace síní (FS) je nejčastější srdeční arytmií u dospělých a příčinou značné morbidity a mortality. Ve srovnání s běžnou populací zvyšuje FS riziko cévních mozkových příhod pětinásobně, přičemž základem prevence vzniku cévních mozkových příhod je perorální antikoagulační léčba. Aby bylo možno vyhledávat pacienty s vysokou pravděpodobností vzniku FS, je třeba zjistit faktory predikující vznik FS. Popisujeme případ 69letého pacienta s asymptomatickou palpitací, u něhož 24hodinové holterovské monitorování prokázalo několik nepřevedených předčasných síňových komplexů (non-conducted premature atrial complex, ncPAC) vedoucích ke vzniku FS.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in adults with substantial morbidity and mortality. AF increases the risk of stroke 5-fold compared to the general population and oral anticoagulant therapy represents the cornerstone for stroke prevention. Therefore, it is necessary to identify predictive factors for the onset of AF in order to screen patients with a high probability of AF development. We report the case of a 69-year-old patient asymptomatic for palpitation with a 24-hour Holter ECG showing several ncPACs (non-conducted premature atrial complexes) leading to AF onset.

• MeSH
• Stroke prevention & control   MeSH
• Electrocardiography, Ambulatory methods   MeSH
• Atrial Fibrillation diagnosis   etiology   prevention & control   MeSH
• Humans MeSH
• Heart Disease Risk Factors MeSH
• Aged MeSH
• Atrial Premature Complexes * diagnostic imaging   prevention & control   MeSH
• Arrhythmias, Cardiac diagnostic imaging   prevention & control   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Publication type
• Case Reports MeSH

• MeSH
• Central Nervous System physiology   MeSH
• Electroencephalography MeSH
• Epilepsy MeSH
• Brain physiology   MeSH
• Cerebral Cortex MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• neurologie
• neurovědy

Jednostranná statická záťaž, minimálna pohybová aktivita a zvýšené psychické napätie zohrávajú významnú úlohu pri vzniku bolesti chrbta. V rámci komplexnej rehabilitácie je doporučované striedanie ergonomických sedov a relaxačných sedov. Ide však len o statické pozície s predpokladaným minimalizujúcim negatívnym vplyvom na pohybový systém. V našom príspevku prezentujeme perspektívny a účinnejší terapeuticko - preventívny prístup, ktorým je dynamizácia ergonomického sedu. Z pohľadu kinezioterapie predstavuje dynamizácia sedu koaktiváciu svalových skupín prostredníctvom pohybových synergii typu uzavretých reťazcov, facilitujúc posturálny systém. Testovali sme hypotézu, či dynamizácia ergonomického sedu môže byť parciálnou prevenciou funkčnej patológie pohybového systému u pacientov so sedavvm zamestnaním. Vyšetrili a sledovali sme 25 subjektov. Sledovanie pozostávalo z hodnotenia bolestí pomocou vizuálnej analógovej škály (VAS) a z objektívneho klinického a posturografického vyšetrenia počas sedu a stoja. Pacientov sme vyšetrili pred rehabilitáciou, po rehabilitácii a po mesiaci sedenia na vzduchom plnenom PC vankúši. Vyhodnotenie VAS poukázalo na významný ústup pocitu bolesti po rehabilitácii ako aj po mesiaci sedenia na PC vankúši. Posturografické vyšetrenie oporných síl pri sede na molitane so zatvoreným očami ukázalo významné zlepšenie stability sedu po rehabilitácii a po mesiaci sedenia na PC vankúši. Po rehabilitácii ako aj po dynamizácii sedu sme zaznamenali tendenciu k úprave postúry v stoji so zatvorenými očami a extenziou hlavy. Naše výsledky podporujú predpoklad, že dynamizácia ergonomického sedu na PC vankúši, by mohla byť dôležitou zložkou prevencie a liečby funkčnej patológie pohybového systému.

Stereotype in static load, minimal motor activity and psychic tension results often in low back pain. Based on present knowledge, for complex motor rehabilitation is supposed an alternation of ergonomic sitting condition with decreased static load and also relaxed sitting position. These rehabilitation approaches are of course only static positions where minimal negative influence on motor system is predicted. In our contribution we presents the perspective approach for effective therapy and prevention - an dynamic condition in the ergonomic sitting position. From point of view of kinesiotherapy the dynamic sitting means an coactivation of muscle groups through motion synergies of type closed up chains facilitating posture system. We tested hypothesis that the increase of dynamic condition during the ergonomic sitting position could be a partial prevention of the functional pathology of locomotor system in patients whose sitting position is the most common working posture. We observed 25 patients. Our observation was based on: patientës evaluation of pain by visual analog scale, physical examination, and postural activity measurement on posturography during sitting and in the upright posture. Patient was evaluated: before rehabilitation after rehabilitation and after one-month period of dynamic sitting in ergonomic position on air pillow. Assessment of pain showed significant decrease of pain after rehabilitation and after dynamic condition of seating on air pillow. During seating on soft support with eyes closed results showed significant improvement of sitting posture after rehabilitation and after dynamic sitting. Influence of rehabilitation and dynamic condition of seating on postural stability showed a tendency of balance improvement in upright posture with eyes closed and head extension. Our results support a view that the dynamic condition of seating on air pillow might be an important part of therapy and prevention of functional pathology of locomotors system

• MeSH
• Back Pain MeSH
• Kinesiology, Applied methods   MeSH
• Humans MeSH
• Recovery of Function MeSH
• Posture MeSH
• Rehabilitation methods   MeSH
• Check Tag
• Humans MeSH

Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.

• MeSH
• Bacteriochlorophylls chemistry   radiation effects   MeSH
• Time MeSH
• Spectrometry, Fluorescence methods   MeSH
• Microscopy, Confocal MeSH
• Lasers MeSH
• Statistics, Nonparametric MeSH
• Normal Distribution MeSH
• Energy Transfer * MeSH
• Rhodopseudomonas chemistry   MeSH
• Light MeSH
• Light-Harvesting Protein Complexes chemistry   radiation effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Comparative Study MeSH

• MeSH
• Deafness MeSH
• Humans MeSH
• Motor Skills MeSH
• Neuropsychological Tests MeSH
• Hearing Disorders MeSH
• Child, Preschool MeSH
• Check Tag
• Humans MeSH
• Child, Preschool MeSH

We provide a critical assessment of explicit-solvent atomistic molecular dynamics (MD) simulations of RNA and protein/RNA complexes, written primarily for non-specialists with an emphasis to explain the limitations of MD. MD simulations can be likened to hypothetical single-molecule experiments starting from single atomistic conformations and investigating genuine thermal sampling of the biomolecules. The main advantage of MD is the unlimited temporal and spatial resolution of positions of all atoms in the simulated systems. Fundamental limitations are the short physical time-scale of simulations, which can be partially alleviated by enhanced-sampling techniques, and the highly approximate atomistic force fields describing the simulated molecules. The applicability and present limitations of MD are demonstrated on studies of tetranucleotides, tetraloops, ribozymes, riboswitches and protein/RNA complexes. Wisely applied simulations respecting the approximations of the model can successfully complement structural and biochemical experiments. WIREs RNA 2017, 8:e1405. doi: 10.1002/wrna.1405 For further resources related to this article, please visit the WIREs website.

• MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• RNA chemistry   metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Computational Biology methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH