Single particle analysis: Difference between revisions

Revision as of 13:14, 30 September 2019

Revision as of 00:10, 1 December 2019

Line 1:

[[File:SingleParticleAnalysis.png|thumb|right|Single particle analysis segments and averages many particles from a sample, allowing for computer algorithms to process the individual images into a combined "representative" image. This allows for improvements in signal to noise, and can be combined with [[deconvolution]] to provide limited improvements to spatial resolution in the image.]]

'''Single particle analysis''' is a group of related computerized image processing techniques used to analyze images from [[Transmission electron microscope|transmission electron microscopy]] (TEM).<ref name="Frank">{{Cite book|first=Joachim |last=Frank |title=Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state |publisher=Oxford University Press |___location=Oxford |year=2006 |pages= |isbn=978-0-19-518218-7 |url=https://books.google.com/books?id=vWaSRUjicbgC}}{{Page needed|date=August 2010}}</ref> These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically [[proteins]] or other large biological entities such as [[virus]]es. Individual images of stained or unstained particles are very [[Signal noise|noisy]], and so hard to interpret. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a [[Transmission electron microscopy#Three dimensional imaging|three-dimensional reconstruction]] of the particle. Using [[cryogenic transmission electron microscopy|cryo-electron microscopy]] it has become possible to generate reconstructions with sub-nanometer [[Resolution (electron density)|resolution]] and near-atomic resolution <ref name="Zhou">{{Cite journal|author=Zhou ZH |title=Towards atomic resolution structural determination by single-particle cryo-electron microscopy |journal=Current Opinion in Structural Biology |volume=18 |issue=2 |pages=218–28 |date=April 2008 |pmid=18403197 |pmc=2714865 |doi=10.1016/j.sbi.2008.03.004}}</ref><ref name="Dynamics">{{Cite journal|vauthors=Wang Q, Matsui T, Domitrovic T, Zheng Y, Doerschuk PC, Johnson JE |title=Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps |journal=Journal of Structural Biology|volume=181|issue=3 |pages=195–206 |date=March 2013 |doi=10.1016/j.jsb.2012.11.005|pmid=23246781 |pmc=3870017}}</ref> first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well.<ref name="Bartesaghi">{{Cite journal| doi = 10.1126/science.aab1576| issn = 1095-9203| volume = 348| issue = 6239| pages = 1147–1151| last1 = Bartesaghi| first1 = Alberto| last2 = Merk| first2 = Alan| last3 = Banerjee| first3 = Soojay| last4 = Matthies| first4 = Doreen| last5 = Wu| first5 = Xiongwu| last6 = Milne| first6 = Jacqueline L. S.| last7 = Subramaniam| first7 = Sriram| title = 2.2 Å resolution CryoTEM structure of β-galactosidase in complex with a cell-permeant inhibitor| journal = Science| accessdate = 2016-03-28| date = 2015-06-05| urlpmid = http://science.sciencemag.org/content/348/6239/114725953817| pmidpmc = 259538176512338| pmcbibcode = 65123382015Sci...348.1147B}}</ref>

==Techniques==

Single particle analysis can be done on both [[negative stain|negatively stained]] and vitreous ice-embedded [[Cryogenic transmission electron microscopy|CryoTEM]] samples. Single particle analysis methods are, in general, reliant on the sample being homogeneous, although techniques for dealing with conformational heterogeneity are being developed.

Images (micrographs), in the past, were collected on film are digitized using high-quality scanners or using built-in [[charge-coupled device|CCD]] detectors coupled to a phosphorescent layer. Now it is common to use direct electron detectors to collect images. The image processing is carried out using specialized software [[Software tools for molecular microscopy|programs]] (for instance <ref>{{Cite web | url=http://www.femtoscanonline.com/wiki/en/processing/%D0%B0%D0%BD%D0%B0%D0%BB%D0%B8%D0%B7_%D0%B2%D1%8B%D0%B4%D0%B5%D0%BB%D0%B5%D0%BD%D0%BD%D1%8B%D1%85_%D0%BE%D0%B1%D0%BB%D0%B0%D1%81%D1%82%D0%B5%D0%B9 |title = 送彩金38满100提现_无需申请自动送彩金【官网】}}</ref>), often run on multi-processor computer clusters. Depending on the sample or the desired results, various steps of two- or three-dimensional processing can be done.

===Alignment and classification===

Line 43:

==Examples==

* Important information on protein synthesis, ligand binding and RNA interaction can be obtained using this novel technique at medium resolutions of 7.5 to 25Å.<ref>{{cite journal |vauthors=Arias-Palomo E, Recuero-Checa MA, Bustelo XR, Llorca O |title=3D structure of Syk kinase determined by single-particle electron microscopy |journal=Biochim. Biophys. Acta |volume=1774 |issue=12 |pages=1493–9 |date=December 2007 |pmid=18021750 |pmc=2186377 |doi=10.1016/j.bbapap.2007.10.008 |url=}}</ref>

* ''Methanococcus maripaludis'' chaperonin,<ref>Japanese Protein databank http://www.pdbj.org/emnavi/emnavi_movie.php?id=5137</ref> reconstructed to 0.43 nanometer resolution.<ref name="Zhang J">{{Cite journal |vauthors=Zhang J, Baker ML, Schröder GF, etal |title=Mechanism of folding chamber closure in a group II chaperonin |journal=Nature |volume=463 |issue=7279 |pages=379–83 |date=January 2010 |pmid=20090755 |pmc=2834796 |doi=10.1038/nature08701|bibcode=2010Natur.463..379Z }}</ref> This bacterial protein complex is a machine for folding other proteins, which get trapped within the shell.

* Fatty acid synthase<ref>Japanese Protein databank http://www.pdbj.org/emnavi/emnavi_movie.php?id=1623</ref> from yeast at 0.59 nanometer resolution.<ref name="Gipson">{{Cite journal|vauthors=Gipson P, Mills DJ, Wouts R, Grininger M, Vonck J, Kühlbrandt W |title=Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy |journal=Proceedings of the National Academy of Sciences of the United States of America |volume=107 |issue=20 |pages=9164–9 |date=May 2010 |pmid=20231485 |pmc=2889056 |doi=10.1073/pnas.0913547107|bibcode=2010PNAS..107.9164G }}</ref> This huge enzyme complex is responsible for building the long chain fatty acids essential for cellular life.

* A 0.33 nanometer reconstruction of Aquareovirus.<ref>Japanese Protein databank http://www.pdbj.org/emnavi/emnavi_movie.php?id=5160</ref><ref name="Zhang X">{{Cite journal|vauthors=Zhang X, Jin L, Fang Q, Hui WH, Zhou ZH |title=3.3 A cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry |journal=Cell |volume=141 |issue=3 |pages=472–82 |date=April 2010 |pmid=20398923 |doi=10.1016/j.cell.2010.03.041 |pmc=3422562}}</ref> These viruses infect fish and other aquatic animals. The reconstruction has high enough resolution to have amino acid side chain densities easily visible.