Electron cryomicroscopy: Difference between revisions
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==Resolution== | ==Resolution== | ||
<StructureSection load='' size='350' side='right' caption='' scene='80/805038/Insulin_receptor/6'> | <StructureSection load='' size='350' side='right' caption='' scene='80/805038/Insulin_receptor/6'> | ||
The median | The median resolution of cryo-EM structures deposited in '''2020''' in the [[Protein Data Bank]] was 3.5 Å (improved from 3.8 Å in 2018, and 4.2 Å in 2016)<ref name="mvr">See cryo-EM Resolution compared with X-ray diffraction resolution: | ||
[http://tinyurl.com/method-vs-resolution tinyurl.com/method-vs-resolution].</ref>. For comparison, the median resolution of X-ray crystallographic entries in the PDB has been 2.0 Å for many years<ref name="mvr" />. When resolution improves by a factor of 2, the available data (to support the coordinate model) goes up by a factor of 8. For example, a 2.4 Å resolution structure is a great improvement over a 3.0 Å resolution structure because the number of available measurements doubles. | [http://tinyurl.com/method-vs-resolution tinyurl.com/method-vs-resolution].</ref>. For comparison, the median [[resolution]] of X-ray crystallographic entries in the PDB has been 2.0 Å for many years<ref name="mvr" />. | ||
In 2015, Cheng, Grigorieff, Penczek & Walz<ref name="primer" /> concluded: <blockquote> | |||
"''Resolution'' in single-particle EM is ... a somewhat arbitrarily chosen cut-off level ...." "... the resolution of a density map remains subject to controversies." "... it is the opinion of the authors that there is currently no real ''gold standard'' procedure for structure refinement and resolution estimation of an EM map." "The problematic issue with single-particle EM, however, is that there is still no objective quality criterion that is simple and easy to use, such as the [[Free R|R-free value in X-ray crystallography]], that would allow one to assess whether the determined density map is accurate or not."<ref name="primer" /> | |||
</blockquote> | |||
When resolution improves by a factor of 2, the available data (to support the coordinate model) goes up by a factor of 8. For example, a 2.4 Å resolution structure is a great improvement over a 3.0 Å resolution structure because the number of available measurements doubles. | |||
A direct comparison between the quality of cryo-EM structures and crystal structures is not possible. In crystal structures, the electron density is calculated from measured structure factors and from calculated phases (in the most extreme case, half of the information is not available from experiment). Even in cases where there are experimental phases (e.g. from multiple isomorphous replacement), these are typically not available to the full resolution. Especially at resolutions below 4 Å, X-ray structures are prone to model bias in the absence of experimental phases. Cryo-EM does not have this limitation, so low resolution structures can still carry reliable information, for instance about conformational changes of known structures. For example, a 4 Å crystal structure solved by molecular replacement (i.e. no experimental phases) is not as reliable as a 4 Å cryo-EM structure. If atomic models of the components of a structure are available, like in the structure of the insulin receptor depicted at right, it is possible to build an atomic model from 4.3 Å resolution data (with some patience, <jmol><jmollink><text>the EM density</text><script>isosurface ID s_one "/wiki/scripts/80/805038/Insulin_receptor/2.iso"</script></jmollink></jmol> will be displayed). | A direct comparison between the quality of cryo-EM structures and crystal structures is not possible. In crystal structures, the electron density is calculated from measured structure factors and from calculated phases (in the most extreme case, half of the information is not available from experiment). Even in cases where there are experimental phases (e.g. from multiple isomorphous replacement), these are typically not available to the full resolution. Especially at resolutions below 4 Å, X-ray structures are prone to model bias in the absence of experimental phases. Cryo-EM does not have this limitation, so low resolution structures can still carry reliable information, for instance about conformational changes of known structures. For example, a 4 Å crystal structure solved by molecular replacement (i.e. no experimental phases) is not as reliable as a 4 Å cryo-EM structure. If atomic models of the components of a structure are available, like in the structure of the insulin receptor depicted at right, it is possible to build an atomic model from 4.3 Å resolution data (with some patience, <jmol><jmollink><text>the EM density</text><script>isosurface ID s_one "/wiki/scripts/80/805038/Insulin_receptor/2.iso"</script></jmollink></jmol> will be displayed). | ||
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==Density Maps== | ==Density Maps== | ||
<table align="right" class="wikitable"><tr><td> | |||
[[Image:6nef-em-map-hec503-his169-360.gif]] | |||
</td></tr><tr><td> | |||
Density map for heme and two histidines in the 3.4<br>Å cryo-EM structure of a cytochrome ([[6nef]]). | |||
</td></tr></table> | |||
The result of a cryo-EM experiment is a density map. Just as for [[X-ray diffraction]], it is then necessary to fit an atomic model optimally into the map<ref name="primer" />. | The result of a cryo-EM experiment is a density map. Just as for [[X-ray diffraction]], it is then necessary to fit an atomic model optimally into the map<ref name="primer" />. | ||
Cryo-EM "has the advantage of recording images containing both amplitude and phase information, so there is no phase problem as in [X-ray] crystallography"<ref name="rosenthal2019">PMID: 30713698</ref>. | Cryo-EM "has the advantage of recording images containing both amplitude and phase information, so there is no phase problem as in [X-ray] crystallography"<ref name="rosenthal2019">PMID: 30713698</ref>. | ||
Electrons are diffracted by the charges in the sample, in contrast to X-rays that are diffracted by electron density, producing [[electron density maps]]. Consequently, EM maps may be termed "electron potential maps"<ref name="rosenthal2019" />, "Coulomb potential maps"<ref name="marques2019">PMID: 31400843</ref>, or "electric potential maps"<ref name="wang2016">PMID: 27706888</ref>. Electron densities are all positive, while electron potential maps can be positive or negative<ref name="wang2016" /><ref name="marques2019" />. Wang provided a way to convert electron potential maps to <i>charge density maps</i> (using [[Chimera]]), where densities have better resolution and better reflect the positions of atomic nuclei | Electrons are diffracted by the charges in the sample, in contrast to X-rays that are diffracted by electron density, producing [[electron density maps]]. Consequently, EM maps may be termed "electron potential maps"<ref name="rosenthal2019" />, "Coulomb potential maps"<ref name="marques2019">PMID: 31400843</ref>, or "electric potential maps"<ref name="wang2016">PMID: 27706888</ref>. Electron densities are all positive, while electron potential maps can be positive or negative<ref name="wang2016" /><ref name="marques2019" />. Wang (2017)<ref name="wang2017">PMID: 28543856</ref> provided a way to convert electron potential maps to <i>charge density maps</i> (using [[Chimera]]), where densities have better resolution and better reflect the positions of atomic nuclei. | ||
===Visualizing EM Maps=== | |||
[http://firstglance.jmol.org FirstGlance in Jmol] makes it easy to visualize EM density maps, as shown in the example at right. Load your [[PDB ID]] (or [[6nef]]). Then, use '''Find..''' (in the [https://proteopedia.org/wiki/fgij/where.htm#focusbox Focus Box]) to locate the residues of interest (or "503,169,360" for 6nef -- In 6nef, these happen to be unambiguous sequence numbers for HEC503, HIS169, and HIS360.) Click on "EM Density Map". After adjusting the view as you wish, click "Save Image or Animation for Powerpoint". An example of an animation saved from FirstGlance is above in this page. | |||
{{Template:PDBMapViewers}} | |||
===The Electron Microscopy Data Bank=== | ===The Electron Microscopy Data Bank=== | ||
The [[Electron Microscopy Data Bank]] (EMDB) archives EM density maps. Only "all features" maps are deposited, as cryo-EM analysis has no equivalent to the [[Electron_density_maps#Fo-Fc_Difference_Map|difference map]] of X-ray diffraction. | The [[Electron Microscopy Data Bank]] (EMDB) archives EM density maps. Only "all features" maps are deposited, as '''cryo-EM analysis has no equivalent to the [[Electron_density_maps#Fo-Fc_Difference_Map|difference map]]''' of X-ray diffraction. | ||
==Temperatures (B factors)== | ==Temperatures (B factors)== | ||