Cryogenic electron microscopy

With emerging new methods of cryo-TEM imaging and image reconstruction the new software solutions appear that help to automate the process. After the empirical Bayesian approach have been implemented in the open source computer program RELION (REgularized LIkelihood OptimizatioN) for 3D reconstruction,{{Cite journal |last=Scheres |first=Sjors H. W. |date=2012-12-01 |title=RELION: Implementation of a Bayesian approach to cryo-EM structure determination |journal=Journal of Structural Biology |language=en |volume=180 |issue=3 |pages=519–530 |doi=10.1016/j.jsb.2012.09.006 |issn=1047-8477 |pmc=3690530 |pmid=23000701}}{{cite web |date=27 October 2023 |title=RELION: Image-processing software for cryo-electron microscopy |url=https://github.com/3dem/relion |access-date=27 October 2023 |website=GitHub |publisher=3dem}} the program became widespread in the cryo-TEM field. It offers a range of corrections that improve the resolution of reconstructed images, allows implementing versatile scripts using [[Python (programming language)|python]] language and executes the usual tasks of 2D/3D model classifications or creating ''[[De novo protein structure prediction|de novo]]'' models.{{Cite journal |last1=Bai |first1=Xiao-chen |last2=McMullan |first2=Greg |last3=Scheres |first3=Sjors H.W |date=January 2015 |title=How cryo-EM is revolutionizing structural biology |journal=Trends in Biochemical Sciences |volume=40 |issue=1 |pages=49–57 |doi=10.1016/j.tibs.2014.10.005 |issn=0968-0004 |pmid=25544475 |s2cid=19727349}}{{Cite journal |last1=Zivanov |first1=Jasenko |last2=Nakane |first2=Takanori |last3=Forsberg |first3=Björn O |last4=Kimanius |first4=Dari |last5=Hagen |first5=Wim JH |last6=Lindahl |first6=Erik |last7=Scheres |first7=Sjors HW |date=2018-11-09 |editor-last=Egelman |editor-first=Edward H |editor2-last=Kuriyan |editor2-first=John |title=New tools for automated high-resolution cryo-EM structure determination in RELION-3 |journal=eLife |volume=7 |article-number=e42166 |doi=10.7554/eLife.42166 |issn=2050-084X |pmc=6250425 |pmid=30412051 |doi-access=free}}

With emerging new methods of cryo-TEM imaging and image reconstruction the new software solutions appear that help to automate the process. After the empirical Bayesian approach have been implemented in the open source computer program RELION (REgularized LIkelihood OptimizatioN) for 3D reconstruction,{{Cite journal |last=Scheres |first=Sjors H. W. |date=2012-12-01 |title=RELION: Implementation of a Bayesian approach to cryo-EM structure determination |journal=Journal of Structural Biology |language=en |volume=180 |issue=3 |pages=519–530 |doi=10.1016/j.jsb.2012.09.006 |issn=1047-8477 |pmc=3690530 |pmid=23000701}}{{cite web |date=27 October 2023 |title=RELION: Image-processing software for cryo-electron microscopy |url=https://github.com/3dem/relion |access-date=27 October 2023 |website=GitHub |publisher=3dem}} the program became widespread in the cryo-TEM field. It offers a range of corrections that improve the resolution of reconstructed images, allows implementing versatile scripts using [[Python (programming language)|python]] language and executes the usual tasks of 2D/3D model classifications or creating ''[[De novo protein structure prediction|de novo]]'' models.{{Cite journal |last1=Bai |first1=Xiao-chen |last2=McMullan |first2=Greg |last3=Scheres |first3=Sjors H.W |date=January 2015 |title=How cryo-EM is revolutionizing structural biology |journal=Trends in Biochemical Sciences |volume=40 |issue=1 |pages=49–57 |doi=10.1016/j.tibs.2014.10.005 |issn=0968-0004 |pmid=25544475 |s2cid=19727349}}{{Cite journal |last1=Zivanov |first1=Jasenko |last2=Nakane |first2=Takanori |last3=Forsberg |first3=Björn O |last4=Kimanius |first4=Dari |last5=Hagen |first5=Wim JH |last6=Lindahl |first6=Erik |last7=Scheres |first7=Sjors HW |date=2018-11-09 |editor-last=Egelman |editor-first=Edward H |editor2-last=Kuriyan |editor2-first=John |title=New tools for automated high-resolution cryo-EM structure determination in RELION-3 |journal=eLife |volume=7 |article-number=e42166 |doi=10.7554/eLife.42166 |issn=2050-084X |pmc=6250425 |pmid=30412051 |doi-access=free}}

For samples with [[symmetry]] mismatches or flexible components, advanced computational methods have been developed to reconstruct specific subunits independently. [[Localized reconstruction]] methods allow extraction and refinement of sub-particles from larger complexes, enabling detailed analysis of components that have a different symmetry than the overall particle.{{cite journal |last1=Ilca |first1=Serban L |last2=Kotecha |first2=Abhay |last3=Sun |first3=Xiaoyu |last4=Poranen |first4=Minna M |last5=Stuart |first5=David I |last6=Huiskonen |first6=Juha T |title=Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes |journal=Nature Communications |date=2015 |volume=6 |pages=8843 |doi=10.1038/ncomms9843}} These approaches have been particularly valuable for studying [[virus]]es and large protein complexes where individual components may exhibit conformational flexibility.{{cite journal |last1=Huiskonen |first1=Juha T |title=Image processing for cryogenic transmission electron microscopy of symmetry-mismatched complexes |journal=Bioscience Reports |date=2018 |volume=38 |issue=2 |doi=10.1042/BSR20170203}}

The rapid growth of cryo-EM has led to community-wide efforts to establish standards for data validation and archiving. Guidelines for data deposition to the [[Electron Microscopy Data Bank]] (EMDB) and validation procedures have been developed through collaborative workshops.{{cite journal |last1=Kleywegt |first1=Gerard J |last2=Adams |first2=Paul D |last3=Butcher |first3=Sarah J |display-authors=3 |title=Community recommendations on cryoEM data archiving and validation |journal=IUCrJ |date=2024 |volume=11 |issue=2 |pages=140-151 |doi=10.1107/S2052252524001246}} These standards ensure reproducibility and enable proper assessment of reconstruction quality, particularly important as the method has become routine in structural biology.

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