Routine single particle CryoEM sample and grid characterization by tomography

ELife
Alex J NobleBridget Carragher

Abstract

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

References

Jun 1, 1976·Journal of Ultrastructure Research·K A Taylor, R M Glaeser
Jan 1, 1989·Journal of Microscopy·P M FrederikW M Busing
May 1, 1988·Quarterly Reviews of Biophysics·J DubochetP Schultz
Dec 13, 1974·Science·K A Taylor, R M Glaeser
Mar 1, 1984·Nature·M AdrianA W McDowall
Apr 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·S ZhangA Rich
Jan 1, 1996·Journal of Structural Biology·J R KremerJ R McIntosh
Nov 10, 1996·Journal of Colloid and Interface Science·A P GunningP A Gunning
Jan 30, 1999·Journal of Colloid and Interface Science·A R MackieV J Morris
Dec 22, 1999·Journal of Structural Biology·S J LudtkeW Chiu
Nov 13, 2003·Journal of Colloid and Interface Science·Yanrong TiePaul R Van Tassel
Apr 10, 2004·Advances in Colloid and Interface Science·Peter WildeVictor Morris
May 11, 2004·Biomacromolecules·Paul A GunningVictor J Morris
May 12, 2004·Biopolymers·Harmen H J de JonghPeter A Wierenga
Jul 21, 2004·Journal of Computational Chemistry·Eric F PettersenThomas E Ferrin
Jan 22, 2005·Protein Science : a Publication of the Protein Society·Elena V KudryashovaHarmen H J De Jongh
Apr 20, 2005·Langmuir : the ACS Journal of Surfaces and Colloids·Anneke H MartinTon van Vliet
May 14, 2005·Journal of Structural Biology·Christian SulowayBridget Carragher
Jun 6, 2006·Physical Chemistry Chemical Physics : PCCP·Mitaben D LadRebecca J Green
Dec 21, 2006·Nature Methods·Sjors H W ScheresJose-Maria Carazo
Sep 29, 2007·Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada·Joel QuispeClinton S Potter
Oct 20, 2007·Journal of Colloid and Interface Science·Renate A GanzevlesHarmen H J de Jongh
Jun 10, 2008·Nature Protocols·Sjors H W ScheresRoberto Marabini
Mar 6, 2009·Journal of Structural Biology·Gabriel C LanderBridget Carragher
Apr 14, 2009·Journal of Structural Biology·Christian SulowayGrant J Jensen
Dec 22, 2010·Bioinformatics·J I Agulleiro, J J Fernandez
Feb 8, 2011·Advances in Colloid and Interface Science·Michael RabeStefan Seeger
Aug 11, 2011·Structure·Steven J LudtkeIrina I Serysheva
Mar 1, 2012·Journal of Structural Biology·Axel F BrilotNikolaus Grigorieff
May 10, 2012·Journal of Structural Biology·Tilak JainClinton S Potter
Aug 21, 2012·Journal of Structural Biology·Dominika Elmlund, Hans Elmlund
Sep 25, 2012·Journal of Structural Biology·Sjors H W Scheres
Nov 21, 2012·Journal of Physics. Condensed Matter : an Institute of Physics Journal·Yohko F Yano
Jun 21, 2013·Journal of the Royal Society, Interface·Steven J VanceMalcolm W Kennedy
Dec 17, 2014·Science·Christopher J Russo, Lori A Passmore
Dec 22, 2014·Journal of Structural Biology·Jose-Ignacio Agulleiro, Jose-Jesus Fernandez
Sep 21, 2015·Biophysical Journal·Robert M GlaeserJamie H D Cate
Oct 13, 2015·Journal of Structural Biology·Alex J Noble, Scott M Stagg

❮ Previous
Next ❯

Citations

Feb 2, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Hendrik SielaffMichael Börsch
Dec 26, 2018·ELife·Jillian ChaseDavid Jeruzalmi
Apr 2, 2019·ELife·Edoardo D'ImprimaWerner Kühlbrandt
Sep 4, 2018·Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada·Rebecca S DillardElizabeth R Wright
Feb 21, 2019·Annual Review of Biophysics·Ahmed-Noor A AgipJudy Hirst
Sep 4, 2019·Acta Crystallographica. Section D, Structural Biology·Max E WilkinsonAna Casañal
Nov 28, 2019·Nature·Yang LiuKarolin Luger
Jul 28, 2020·FEMS Microbiology Reviews·Alba Herrero Del Valle, C Axel Innis
Nov 18, 2018·The Journal of General Physiology·Farooq A KianiMichael J Rynkiewicz
May 28, 2019·Proceedings of the National Academy of Sciences of the United States of America·Katerina NaydenovaChristopher J Russo
Oct 9, 2019·Nature Methods·Dimitry Tegunov, Patrick Cramer
Nov 20, 2019·Nature Structural & Molecular Biology·Anthony H NguyenRobert J Lefkowitz
Jan 30, 2020·Nature Communications·Liming ZhengHailin Peng
May 24, 2020·Nature Communications·Raimond B G RavelliPeter J Peters
Sep 12, 2020·Proceedings of the National Academy of Sciences of the United States of America·Feng WangDavid A Agard
Sep 27, 2018·Nature Methods·Alex J NobleBridget Carragher
Feb 26, 2019·The Journal of Biological Chemistry·Dmitry Lyumkis
Dec 4, 2019·Acta Crystallographica. Section D, Structural Biology·John L RubinsteinTimothy Kwok
Aug 12, 2020·Nature Methods·Venkata P DandeyBridget Carragher
Apr 8, 2020·Acta Crystallographica. Section D, Structural Biology·David P KleblStephen P Muench
Jul 18, 2020·F1000Research·Michael C ThompsonJose A Rodriguez
Dec 7, 2018·Nature Methods·Mingxu HuXueming Li
Oct 10, 2020·Science·Katerina NaydenovaChristopher J Russo
Nov 3, 2020·Acta Crystallographica. Section D, Structural Biology·Yong Zi Tan, John L Rubinstein
Jan 10, 2021·Journal of Structural Biology·C O S SorzanoJ M Carazo
Dec 7, 2018·Journal of Pharmaceutical Sciences·Estelle J A SuysDavid K Chalmers
Jan 12, 2021·Computational and Structural Biotechnology Journal·Samantha J ZieglerYannick J Bomble
Feb 16, 2021·ACS Nano·Nesrine AissaouiGaetan Bellot
Mar 12, 2021·Quarterly Reviews of Biophysics·Edoardo D'Imprima, Werner Kühlbrandt
Dec 12, 2020·Journal of Structural Biology·Natàlia de Martín GarridoChristopher H S Aylett
Jun 25, 2020·Progress in Biophysics and Molecular Biology·Jan Böhning, Tanmay A M Bharat
Jul 10, 2020·Progress in Biophysics and Molecular Biology·Philip R Baldwin, Dmitry Lyumkis
Apr 8, 2021·Acta Crystallographica. Section D, Structural Biology·Joseph Atherton, Carolyn A Moores
Apr 29, 2021·Médecine sciences : M/S·Daniel LévyManuela Dezi
May 6, 2021·Acta Crystallographica. Section D, Structural Biology·Jan RheinbergerCristina Paulino
May 9, 2021·Nature Methods·Giulia WeissenbergerPeter J Peters

❮ Previous
Next ❯

Methods Mentioned

BETA
atomic force microscopy
AFM

Software Mentioned

SerialEM
Appion
CryoET
Dynamo
Leginon
Xmipp
- Protomo
Relion
UCSF Chimera
IMOD Chimera

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
Joel QuispeClinton S Potter
Journal of Structural Biology
Ivan RazinkovBridget Carragher
© 2021 Meta ULC. All rights reserved