Abstract:
:Electron cryo-microscopy (cryoEM) has advanced dramatically to become a viable tool for high-resolution structural biology research. The ultimate outcome of a cryoEM study is an atomic model of a macromolecule or its complex with interacting partners. This chapter describes a variety of algorithms and software to build a de novo model based on the cryoEM 3D density map, to optimize the model with the best stereochemistry restraints and finally to validate the model with proper protocols. The full process of atomic structure determination from a cryoEM map is described. The tools outlined in this chapter should prove extremely valuable in revealing atomic interactions guided by cryoEM data.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
DiMaio F,Chiu Wdoi
10.1016/bs.mie.2016.06.003subject
Has Abstractpub_date
2016-01-01 00:00:00pages
255-76eissn
0076-6879issn
1557-7988pii
S0076-6879(16)30113-6journal_volume
579pub_type
杂志文章,评审abstract::Growing appreciation for the biogeochemical significance of uncultured microorganisms is changing the focus of environmental microbiology. Techniques designed to investigate microbial metabolism in situ are increasingly popular, from mRNA-targeted fluorescence in situ hybridization (FISH) to the "-omics" revolution, i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381294-0.00012-2
更新日期:2011-01-01 00:00:00
abstract::Complex I is the major entry point in the bacterial and mitochondrial respiratory chain. Structural knowledge of the enzyme is still limited because of its large size and complicated architecture. Only the structure of the hydrophilic domain of a bacterial Complex I has been solved to high resolution by X-ray crystall...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)04401-7
更新日期:2009-01-01 00:00:00
abstract::Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabol...
journal_title:Methods in enzymology
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abstract::Microarrays of defined glycans represent a high throughput approach to determining the specificity of lectins, or more generally glycan-binding proteins (GBPs). The utility of a glycan microarray is directly related to the number and variety of the glycans available on the printed surface for interrogation by GBPs. Th...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(10)80033-3
更新日期:2010-01-01 00:00:00
abstract::Pulsed electron double resonance technique, also known as double electron-electron resonance, jointly with site-directed spin labeling (SDSL) have been used extensively for studying structures and structural change. During the last decades, significant enhancements have been made by optimization of the experimental pr...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.05.027
更新日期:2015-01-01 00:00:00
abstract::Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS) where human immunodeficiency virus (HIV-1) is sequestered in patients with HIV-1-associated neurological disorders (HAND). HAND is a spectrum of neurocognitive disorders that continue to persist in HIV-...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391858-1.00003-4
更新日期:2012-01-01 00:00:00
abstract::An overview of resolution enhancement of conventional, field-swept, continuous-wave electron paramagnetic resonance spectra using Fourier transform-based deconvolution methods is presented. Basic steps that are involved in resolution enhancement of calculated spectra using an implementation based on complex discrete F...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.06.009
更新日期:2015-01-01 00:00:00
abstract::During the past several years, major progress has been accomplished in the production of "designer cellulosomes," artificial enzymatic complexes that were demonstrated to efficiently degrade crystalline cellulose. This progress is part of a global attempt to promote biomass waste solutions and biofuel production. In d...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-415931-0.00023-9
更新日期:2012-01-01 00:00:00
abstract::Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2015.06.021
更新日期:2015-01-01 00:00:00
abstract::gamma-Glutamyltranspeptidase is a heterodimeric glycoprotein that catalyzes the transpeptidation and hydrolysis of the gamma-glutamyl group of glutathione and related compounds. It is known that the enzyme plays a role in the metabolism of glutathione and in salvaging constituents of glutathione. In the adult animal, ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01025-6
更新日期:2005-01-01 00:00:00
abstract::Endosomes are isolated from rat liver using high-speed centrifugation through sucrose density gradients. They are distinguishable from Golgi elements, with which they coisolate, by their capacity to concentrate internalized protein ligands (viz., insulin and epidermal growth factor (EGF)) in receptor-bound intact form...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-397925-4.00017-1
更新日期:2014-01-01 00:00:00
abstract::Many methods have been developed for the cloning of PCR products. These methods include blunt-end cloning, TA cloning, and using restriction sites incorporated into the PCR primers. The restrictionless cloning technique allows efficient directional cloning of PCR products into any cloning site within a vector regardle...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-418687-3.00009-4
更新日期:2013-01-01 00:00:00
abstract::EPR long-range distance measurements on spin-labeled macromolecules have recently become a popular tool in structural biology. The method can be used to obtain coarse-grained structures of biomolecules, to track conformational changes and dynamics, to dock macromolecular complexes, or to localize spin centers within m...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.06.006
更新日期:2015-01-01 00:00:00
abstract::Isothermal Titration Calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of RNA folding, RNA binding to small molecules, and RNA-protein interactions. The advent of extremely sensitive instrumentation and the increasing availability of ITC in shared facilities have made it in...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)68019-8
更新日期:2009-01-01 00:00:00
abstract::Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2017.07.004
更新日期:2017-01-01 00:00:00
abstract::Live cell microscopy using fluorescent proteins and small fluorescent probes is a well-established and essential tool for cell biology; however, there is a considerable need for noninvasive techniques able to study tissue and cell dynamics without the need to introduce chemical or genetically encoded probes. Coherent ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/B978-0-12-391857-4.00014-8
更新日期:2012-01-01 00:00:00
abstract::Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)05003-2
更新日期:2005-01-01 00:00:00
abstract::Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.12.037
更新日期:2019-01-01 00:00:00
abstract::We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly t...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.03.013
更新日期:2020-01-01 00:00:00
abstract::Gene therapy vectors based on murine retroviruses have now been in clinical trials for over 20 years. During that time, a variety of novel vector pseudotypes were developed in an effort to improve gene transfer. Lentiviral vectors are now in clinical trials and a similar evolution of vector technology is anticipated. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-386509-0.00005-3
更新日期:2012-01-01 00:00:00
abstract::The microbial production of commodity, fine, and specialty chemicals is a driving force in biotechnology. An essential requirement is to introduce biosynthetic pathways to the target compound(s) into chassis organisms. First suitable enzymes must be selected and characterized, and then genetic pathways must be designe...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.04.011
更新日期:2018-01-01 00:00:00
abstract::This chapter provides the methodologies employed to study the polymorphism of human apoE. These and other related studies have advanced our understanding of the structure and function of this protein as follows: The complex array of human apoE observed by two-dimensional gel electrophoresis results from genetic variat...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(86)28109-4
更新日期:1986-01-01 00:00:00
abstract::ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stres...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.04.007
更新日期:2015-01-01 00:00:00
abstract::We describe here the methods we have used to generate selective peptide inhibitors and activators of PKC-mediated signaling. These approaches should be applicable to any signaling event that is dependent on protein-protein interaction. Furthermore, targeting downstream enzymes in signal transduction has been notorious...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)45039-2
更新日期:2002-01-01 00:00:00
abstract::The nitrogen (N) cycle is the only global biogeochemical cycle that is driven by biological functions involving the interaction of many microorganisms. The N cycle has evolved over geological time and its interaction with the oxygen cycle has had profound effects on the evolution and timing of Earth's atmosphere oxyge...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381294-0.00022-5
更新日期:2011-01-01 00:00:00
abstract::Autophagy is the major cellular process of degradation and is modulated by several signaling pathways. Phosphatidylinositol 3-kinase (PtdIns3K) class III (Vps34) and PtdIns3K class I regulate the autophagy pathway positively and negatively, respectively. Both classes of PtdIns3K participate in the synthesis of phospha...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.060
更新日期:2017-01-01 00:00:00
abstract::The diverse structures of polyketide natural products are reflected by the equally diverse polyketide biosynthetic enzymes, namely polyketide synthases (PKSs). Three major classes of PKSs are known-noniterative type I PKSs, iterative type II PKSs and acyl carrier protein-independent type III PKSs, each of which consis...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)04608-4
更新日期:2009-01-01 00:00:00
abstract::Rab7, a member of the Rab family of small G proteins, has been shown to regulate late endocytic traffic and lysosome biogenesis, but the exact roles and the mode of actions of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins and works together with them to ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03059-4
更新日期:2005-01-01 00:00:00
abstract::kappaB-Ras1 and kappaB-Ras2 are two small proteins that display similarity at the amino acid level to Ras-like small GTPases. Although little is known about the function of the kappaB-Ras proteins, they have been shown to interfere with activation of transcription factor NF-kappaB. They accomplish this by binding to I...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)07042-4
更新日期:2006-01-01 00:00:00
abstract::Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metallopro...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2016.09.044
更新日期:2017-01-01 00:00:00