Single particle analysis: Difference between revisions

Revision as of 01:53, 6 December 2023

Revision as of 15:06, 8 December 2023

Line 36:

===Tilt methods===

The specimen stage of the microscope can be tilted (typically along a single axis), allowing the single particle technique known as [[wikibooks:Three_Dimensional_Electron_Microscopy/Initial_model#Random_Conical_Tilt|random conical tilt.]]<ref name="RCT">{{Cite journal|vauthors=Radermacher M, Wagenknecht T, Verschoor A, Frank J |title=Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli |journal=Journal of Microscopy |volume=146 |issue=Pt 2 |pages=113–36 |date=May 1987 |pmid=3302267 |doi=10.1111/j.1365-2818.1987.tb01333.x|doi-access=free }}</ref> An area of the specimen is imaged at both zero and at high angle (~60-70 degrees) tilts, or in the case of the related method of orthogonal tilt reconstruction,<ref>{{cite book |last1=Leschziner |first1=A |titlechapter=The orthogonalOrthogonal Tilt Reconstruction Method |title=Cryo-EM, Part tiltB: reconstruction3-D method.Reconstruction |series=Methods in Enzymology |date=2010 |volume=482 |pages=237–62 |doi=10.1016/S0076-6879(10)82010-5 |pmid=20888964|isbn=9780123849915 }}</ref> +45 and −45 degrees. Pairs of particles corresponding to the same object at two different tilts (tilt pairs) are selected, and by following the parameters used in subsequent alignment and classification steps a three-dimensional reconstruction can be generated relatively easily. This is because the viewing angle (defined as three [[Euler angles]]) of each particle is known from the tilt geometry.

3D reconstructions from random conical tilt suffer from missing information resulting from a restricted range of orientations. Known as the missing cone<ref>{{cite web |url=https://www.c-cina.org/research/algorithms/missing-cone/}}</ref> (due to the shape in reciprocal space), this causes distortions in the 3D maps. However, the missing cone problem can often be overcome by combining several tilt reconstructions. Tilt methods are best suited to [[Negative stain|negatively stained]] samples, and can be used for particles that adsorb to the carbon support film in preferred orientations. The phenomenon known as charging or beam-induced movement<ref>{{cite journal |last1=Li |first1=Xueming |last2=Mooney |first2=Paul |last3=Zheng |first3=Shawn |last4=Booth |first4=Christopher R. |last5=Braunfeld |first5=Michael B. |last6=Gubbens |first6=Sander |last7=Agard |first7=David A. |last8=Cheng |first8=Yifan |title=Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM |journal=Nature Methods |date=June 2013 |volume=10 |issue=6 |pages=584–590 |doi=10.1038/nmeth.2472 |pmid=23644547 |language=en |issn=1548-7105|pmc=3684049 }}</ref> makes collecting high-tilt images of samples in vitreous ice challenging.