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Article ; Online: Square beams for optimal tiling in transmission electron microscopy.

Chua, Eugene Y D / Alink, Lambertus M / Kopylov, Mykhailo / Johnston, Jake D / Eisenstein, Fabian / de Marco, Alex

2024 Volume 21, Issue 4, Page(s) 562–565

Abstract: Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose- ... ...

Abstract	Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose-sensitive samples. Here, we introduce a square electron beam that can easily be retrofitted in existing microscopes, and demonstrate its application, showing that it can tile nearly perfectly and deliver cryo-electron microscopy imaging with a resolution comparable to conventional set-ups.
MeSH term(s)	Cryoelectron Microscopy/methods ; Microscopy, Electron, Transmission
Language	English
Publishing date	2024-01-18
Publishing country	United States
Document type	Journal Article
ZDB-ID	2169522-2
ISSN	1548-7105 ; 1548-7091
ISSN (online)	1548-7105
ISSN	1548-7091
DOI	10.1038/s41592-023-02161-x
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Article ; Online: Measuring the Effect of Ice Thickness and Microscope Configuration on Resolution in Single Particle Cryo-EM.

Chua, Eugene Y D / Neselu, Kasahun / Wang, Bing / Rice, William J / Potter, Clinton S / Carragher, Bridget

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada

2023 Volume 29, Issue 29 Suppl 1, Page(s) 1040

Language	English
Publishing date	2023-08-23
Publishing country	England
Document type	Journal Article
ZDB-ID	1385710-1
ISSN	1435-8115 ; 1431-9276
ISSN (online)	1435-8115
ISSN	1431-9276
DOI	10.1093/micmic/ozad067.531
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Article ; Online: Automated pipelines for rapid evaluation during cryoEM data acquisition.

Mendez, Joshua H / Chua, Eugene Y D / Paraan, Mohammadreza / Potter, Clinton S / Carragher, Bridget

Current opinion in structural biology

2023 Volume 83, Page(s) 102729

Abstract: Cryo-electron microscopy (cryoEM) has become a popular method for determining high-resolution structures of biomolecules. However, data processing can be time-consuming, particularly for new researchers entering the field. To improve data quality and ... ...

Abstract	Cryo-electron microscopy (cryoEM) has become a popular method for determining high-resolution structures of biomolecules. However, data processing can be time-consuming, particularly for new researchers entering the field. To improve data quality and increase data collection efficiency, several software packages have been developed for on-the-fly data processing with various degrees of automation. These software packages allow researchers to perform tasks such as motion correction, CTF estimation, 2D classification, and 3D reconstruction in real-time, with minimal human input. On-the-fly data processing can not only improve data collection efficiency but also increase the productivity of instrumentation in high demand. However, the various software packages available differ in their performance, computational requirements, and levels of automation. In this review, we describe the minimal metrics used to assess data quality during data collection, outline the features of an ideal on-the-fly data processing software systems, and provide results from using three of these systems.
MeSH term(s)	Humans ; Cryoelectron Microscopy/methods ; Image Processing, Computer-Assisted/methods ; Software ; Automation
Language	English
Publishing date	2023-11-21
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	1068353-7
ISSN	1879-033X ; 0959-440X
ISSN (online)	1879-033X
ISSN	0959-440X
DOI	10.1016/j.sbi.2023.102729
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Zs.A 3206: Show issues

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Article ; Online: Measuring the effects of ice thickness on resolution in single particle cryo-EM.

Neselu, Kasahun / Wang, Bing / Rice, William J / Potter, Clinton S / Carragher, Bridget / Chua, Eugene Y D

Journal of structural biology: X

2023 Volume 7, Page(s) 100085

Abstract: Ice thickness is a critical parameter in single particle cryo-EM - too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution ... ...

Abstract	Ice thickness is a critical parameter in single particle cryo-EM - too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50-150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.
Language	English
Publishing date	2023-01-24
Publishing country	United States
Document type	Journal Article
ISSN	2590-1524
ISSN (online)	2590-1524
DOI	10.1016/j.yjsbx.2023.100085
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Article ; Online: Structure of a 28.5 kDa duplex-embedded G-quadruplex system resolved to 7.4 Å resolution with cryo-EM.

Monsen, Robert C / Chua, Eugene Y D / Hopkins, Jesse B / Chaires, Jonathan B / Trent, John O

Nucleic acids research

2023 Volume 51, Issue 4, Page(s) 1943–1959

Abstract: Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information ... ...

Abstract	Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment. Here, we report the 7.4 Å resolution structure and dynamics of a 28.5 kDa duplex-G4-duplex (DGD) model system using cryo-EM, molecular dynamics, and small-angle X-ray scattering (SAXS) studies. The DGD cryo-EM refined model features a 53° bend induced by a stacked duplex-G4 interaction at the 5' G-tetrad interface with a persistently unstacked 3' duplex. The surrogate complement poly dT loop preferably stacks onto the 3' G-tetrad interface resulting in occlusion of both 5' and 3' tetrad interfaces. Structural analysis shows that the DGD model is quantifiably more druggable than the monomeric G4 structure alone and represents a new structural drug target. Our results illustrate how the integration of cryo-EM, MD, and SAXS can reveal complementary detailed static and dynamic structural information on DNA G4 systems.
MeSH term(s)	G-Quadruplexes ; Scattering, Small Angle ; Cryoelectron Microscopy ; X-Ray Diffraction ; DNA/chemistry
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2023-01-03
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkad014
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Zs.A 1067: Show issues

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Article ; Online: Correction to article 'The mechanics behind DNA sequence-dependent properties of the nucleosome'.

Chua, Eugene Y D / Vasudevan, Dileep / Davey, Gabriela E / Wu, Bin / Davey, Curt A

Nucleic acids research

2021 Volume 49, Issue 10, Page(s) 5999

Language	English
Publishing date	2021-05-25
Publishing country	England
Document type	Published Erratum
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkab464
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Article ; Online: The National Center for CryoEM Access and Training - Establishing a Cross-Facility-Honored Training Curriculum.

Eng, Edward T / Zimanyi, Christina / Aragon, Mahira / Chua, Eugene Y D / Kopylov, Elina / Dubbeldam, Charlie / Castello, Cathleen / Kieft, Jeffrey S / de Marco, Alex

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada

2023 Volume 29, Issue 29 Suppl 1, Page(s) 1042–1043

Language	English
Publishing date	2023-08-23
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	1385710-1
ISSN	1435-8115 ; 1431-9276
ISSN (online)	1435-8115
ISSN	1431-9276
DOI	10.1093/micmic/ozad067.533
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Article ; Online: Cryo-Electron Microscopy Screening Automation across Multiple Grids using Smart Leginon.

Sawh-Gopal, Anjelique / Ishemgulova, Aygul / Chua, Eugene Y D / Aragon, Mahira F / Mendez, Joshua H / Eng, Edward T / Noble, Alex J

Journal of visualized experiments : JoVE

2023 , Issue 202

Abstract: Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline ... ...

Abstract	Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to ~10 min with Autoscreen compared to ~6 h using previous methods which are hampered by their inability to account for high variability between grids. This protocol first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12-grid automated screening session.
MeSH term(s)	Cryoelectron Microscopy ; Ice ; Computer Systems ; Automation ; Algorithms
Chemical Substances	Ice
Language	English
Publishing date	2023-12-01
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/66007
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Advances in phase plate cryo-EM imaging of DNA and nucleosomes.

Chua, Eugene Y D / Sandin, Sara

Nucleus (Austin, Tex.)

2017 Volume 8, Issue 3, Page(s) 275–278

Abstract: Contrast in electron cryo-microscopy (cryo-EM) is limited by the weak phase and radiation sensitive nature of biologic samples embedded in vitrified ice. We have recently shown that a new contrast enhancement technique utilizing the Volta phase plate can ...

Abstract	Contrast in electron cryo-microscopy (cryo-EM) is limited by the weak phase and radiation sensitive nature of biologic samples embedded in vitrified ice. We have recently shown that a new contrast enhancement technique utilizing the Volta phase plate can be combined with single particle analysis to determine the structure of a small chromatin complex, the nucleosome core particle, at near-atomic resolution. Here, we discuss advantages and limitations of the technique in terms of data collection, particle detection, and visualization of individual DNA molecules and higher-order chromatin structure.
MeSH term(s)	Cryoelectron Microscopy/methods ; DNA/chemistry ; DNA/metabolism ; Nucleosomes/chemistry ; Nucleosomes/metabolism
Chemical Substances	Nucleosomes ; DNA (9007-49-2)
Language	English
Publishing date	2017-02-07
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	2619626-8
ISSN	1949-1042 ; 1949-1042
ISSN (online)	1949-1042
ISSN	1949-1042
DOI	10.1080/19491034.2017.1287643
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Measuring the effects of ice thickness on resolution in single particle cryo-EM

Kasahun Neselu / Bing Wang / William J. Rice / Clinton S. Potter / Bridget Carragher / Eugene Y.D. Chua

Journal of Structural Biology: X, Vol 7, Iss , Pp 100085- (2023)

2023

Abstract: Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution ... ...

Abstract	Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50–150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.
Keywords	Cryo-EM ; Ice thickness ; Single particle analysis ; Energy filter ; High tension ; Resolution ; Biology (General) ; QH301-705.5
Subject code	290
Language	English
Publishing date	2023-01-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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