Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.

• MeSH
• algoritmy * MeSH
• elektronová kryomikroskopie metody   MeSH
• makromolekulární látky ultrastruktura   MeSH
• molekulární biologie metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• průběh práce MeSH
• výpočetní biologie MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Realising the importance of assessing the quality of the biomolecular structures deposited in the Protein Data Bank (PDB), the Worldwide Protein Data Bank (wwPDB) partners established Validation Task Forces to obtain advice on the methods and standards to be used to validate structures determined by X-ray crystallography, nuclear magnetic resonance spectroscopy and three-dimensional electron cryo-microscopy. The resulting wwPDB validation pipeline is an integral part of the wwPDB OneDep deposition, biocuration and validation system. The wwPDB Validation Service webserver (https://validate.wwpdb.org) can be used to perform checks prior to deposition. Here, it is shown how validation metrics can be combined to produce an overall score that allows the ranking of macromolecular structures and domains in search results. The ValTrendsDB database provides users with a convenient way to access and analyse validation information and other properties of X-ray crystal structures in the PDB, including investigating trends in and correlations between different structure properties and validation metrics.

• MeSH
• databáze proteinů normy   MeSH
• datové kurátorství MeSH
• elektronová kryomikroskopie MeSH
• internet * MeSH
• konformace proteinů * MeSH
• lidé MeSH
• makromolekulární látky chemie   MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny analýza   chemie   MeSH
• uživatelské rozhraní počítače * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

• MeSH
• adenosintrifosfát biosyntéza   MeSH
• ATP-synthetasa (komplexy) metabolismus   MeSH
• buňky Hep G2 MeSH
• down regulace MeSH
• fyziologická adaptace fyziologie   MeSH
• interakční proteinové domény a motivy MeSH
• kyslík * MeSH
• lidé MeSH
• mitochondriální dynamika fyziologie   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie fyziologie   MeSH
• multiproteinové komplexy fyziologie   MeSH
• podjednotky proteinů MeSH
• regulace genové exprese fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery.

• MeSH
• databáze proteinů * MeSH
• internet MeSH
• konformace proteinů MeSH
• lektiny chemie   MeSH
• makromolekulární látky chemie   MeSH
• molekulární konformace * MeSH
• molekulární modely MeSH
• software * MeSH
• vazebná místa MeSH
• zinkové prsty MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cryo Electron Tomography (cryoET) plays an essential role in Structural Biology, as it is the only technique that allows to study the structure of large macromolecular complexes in their close to native environment in situ. The reconstruction methods currently in use, such as Weighted Back Projection (WBP) or Simultaneous Iterative Reconstruction Technique (SIRT), deliver noisy and low-contrast reconstructions, which complicates the application of high-resolution protocols, such as Subtomogram Averaging (SA). We propose a Progressive Stochastic Reconstruction Technique (PSRT) - a novel iterative approach to tomographic reconstruction in cryoET based on Monte Carlo random walks guided by Metropolis-Hastings sampling strategy. We design a progressive reconstruction scheme to suit the conditions present in cryoET and apply it successfully to reconstructions of macromolecular complexes from both synthetic and experimental datasets. We show how to integrate PSRT into SA, where it provides an elegant solution to the region-of-interest problem and delivers high-contrast reconstructions that significantly improve template-based localization without any loss of high-resolution structural information. Furthermore, the locality of SA is exploited to design an importance sampling scheme which significantly speeds up the otherwise slow Monte Carlo approach. Finally, we design a new memory efficient solution for the specimen-level interior problem of cryoET, removing all associated artifacts.

• MeSH
• algoritmy MeSH
• elektronová kryomikroskopie metody   MeSH
• makromolekulární látky chemie   MeSH
• metoda Monte Carlo MeSH
• počítačové zpracování obrazu metody   MeSH
• reprodukovatelnost výsledků MeSH
• ribozomy chemie   MeSH
• stochastické procesy * MeSH
• tomografie elektronová metody   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH

We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number N(aggr) of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer-cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.

• MeSH
• akrylamidy chemie   MeSH
• algoritmy MeSH
• anizotropie MeSH
• antibiotika antitumorózní chemie   MeSH
• cholesterol MeSH
• difrakce rentgenového záření MeSH
• doxorubicin analogy a deriváty   chemie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• makromolekulární látky chemie   MeSH
• maloúhlový rozptyl MeSH
• micely MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• nanokapsle chemie   MeSH
• neutronová difrakce MeSH
• světlo MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The extracellular matrix plays an important role in the biomechanical behaviour of the lung parenchyma. The matrix is composed of a three-dimensional fibre mesh filled with different macromolecules, including proteoglycans which have important functions in many lung pathophysiological processes, as they regulate tissue hydration, macromolecular structure and function, response to inflammatory agents, and tissue repair and remodelling. The aim of this review is to describe the role of mechanical ventilation on pulmonary extracellular matrix structure and function. RECENT FINDINGS: Recent experimental and clinical data suggest that in healthy lungs, mechanical ventilation with tidal volume ranging between 7 and 12 ml/kg in the absence of positive end-expiratory pressure may lead to endothelial, extracellular matrix and peripheral airways damage without major inflammatory response. Several mechanisms may explain damage to the lung structure induced by mechanical ventilation: regional overdistension, 'low lung volume' associated with tidal airway closure, and inactivation of surfactant. SUMMARY: Tidal volume reduction to 6 ml/kg may be useful during mechanical ventilation of healthy lungs. The study of the extracellular matrix may be useful to better understand the pathophysiology of ventilator-induced lung injury in healthy and diseased lungs.

• MeSH
• barotrauma patofyziologie   prevence a kontrola   MeSH
• dechový objem MeSH
• extracelulární matrix fyziologie   ultrastruktura   MeSH
• klinické zkoušky jako téma MeSH
• lidé MeSH
• plíce fyziologie   MeSH
• proteoglykany metabolismus   MeSH
• syndrom dechové tísně etiologie   patologie   prevence a kontrola   MeSH
• umělé dýchání škodlivé účinky   MeSH
• ventilace umělá s výdechovým přetlakem MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

The knowledge of the access paths connecting interior of molecular systems with surrounding environment is important for the understanding of structurefunction relationships and engineering of molecules for biotechnological applications. CAVER is a computer program developed for calculations of tunnels, channels or pores in the biomolecules, inorganic materials and molecular ensembles. The algorithm performs a skeleton search based on a reciprocal distance function grid. The algorithm is implemented in the stand-alone version, web version and as plug-in for PyMol. CAVER is available from the website http://loschmidt.chemi.muni.cz/caver

• MeSH
• algoritmy MeSH
• anorganické látky chemie   MeSH
• chemické modely MeSH
• financování organizované MeSH
• makromolekulární látky chemie   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• nukleové kyseliny chemie   ultrastruktura   MeSH
• počítačová grafika MeSH
• počítačová simulace MeSH
• proteiny chemie   ultrastruktura   MeSH
• software MeSH
• uživatelské rozhraní počítače MeSH

• MeSH
• hydroxybutyráty chemie   MeSH
• mechanický stres MeSH
• molekulární konformace MeSH
• polyestery chemie   MeSH
• pružnost MeSH
• testování materiálů MeSH

• MeSH
• makromolekulární látky MeSH
• molekulární konformace MeSH
• molekulární struktura MeSH

• Konspekt
• Biologické vědy
• NLK Obory
• biologie
• NLK Publikační typ
• elektronické časopisy