Conference Agenda

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Session Overview
Session
Poster - 18 CryoEM: Cryo-EM
Time:
Tuesday, 17/Aug/2021:
4:40pm - 5:40pm

Session Chair: Jose-Maria Carazo
Session Chair: Jiri Novacek

 


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Presentations

Poster session abstracts

Radomír Kužel



Structural analysis of transcription related complexes and operation of 200kV cryo-EM in KEK

Naruhiko Adachi, Masato Kawasaki, Toshio Moriya, Akira Shinoda, Yusuke Yamada, Toshiya Senda

High Energy Accelerator Research Organization (KEK), Ibaraki, Japan

Transcription is fundamental process for withdrawing genetic information stored in the genome. In eukarya, multi-subunit complexes, known as RNA polymerase II, general transcription initiation factors –i.e. TFIIA, TFIIB, TBP/TFIID, TFIIE, TFIIF, TFIIH–, mediator, and chromatin factors, carry out this reaction. To elucidate the detailed mechanisms of the reaction, their tertiary structural information is indispensable. So far, we determined crystal structures of subunit/domain of TFIID [1,2] and examined molecular evolution of TBP and TFIIB [3,4]. We have also performed large-scale purification of eukaryotic transcription-related complexes for structural analysis [5]. Preliminary cryo-EM analysis showed that these complexes seemed to be disrupted due to the harsh condition during cryo-grid preparation. Further optimization for cryo-grid preparation is required.

March 2018, our institute, KEK, obtained 200kV cryo-EM (Talos Arctica with Falcon3EC) and prepared pipeline for solving high resolution structures of protein complexes. From October 2018, the cryo-EM facility in KEK is open to academic and industrial users for scientific research. Our mission is twofold: to provide cryo-EM machine time for external users, and to assist users in acquiring cryo-EM skills. Until now, we have provided machine time for 36 academic and 15 industrial users. We also have held an initial training for cryo-grid preparation and EPU operation 12 times and a RELION workshop for beginners 3 times. In the last two years, our facility obtained 25 cryo-EM maps whose resolution is higher than 5 angstrom. Here we show two representative results: single particle analyses of 110kDa enzyme at 2.85 angstrom resolution and 860kDa enzymes at 2.24 angstrom resolution. These results suggest that we established a proper protocol for cryo-grid preparation, cryo-EM data collection, and single particle analysis. We would like to keep supporting external users and carry out cryo-EM analysis of transcription-related complexes.

[1] Adachi, N., Senda, M., Natsume, R., Senda, T. & Horikoshi, M. (2008). Crystal structure of Methanococcus jannaschii TATA box-binding protein. Genes Cells 13, 1127-1140.

[2] Akai, Y., Adachi, N., Hayashi, Y., Eitoku, M., Sano, N., Natsume, R., Kudo, N., Tanokura, M., Senda, T. & Horikoshi, M. (2010). Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction. Proc. Natl. Acad. Sci. USA 107, 8153-8158.

[3] Adachi, N., Senda, T. & Horikoshi, M. (2016). Uncovering ancient transcription systems with a novel evolutionary indicator. Sci. Rep. 6, 27922.

[4] Kawakami, E., Adachi, N., Senda, T. & Horikoshi, M. (2017). Leading role of TBP in the Establishment of Complexity in Eukaryotic Transcription Initiation Systems. Cell Rep. 21, 3941-3956.

[5] Adachi, N., Aizawa, K., Kratzer, Y., Saijo, S., Shimizu, N. & Senda, T. (2017). Improved method for soluble expression and rapid purification of yeast TFIIA. Protein Expr. Purif. 133, 50-56.

Keywords: transcription; TFIID; cryo-EM



SAMase of bacteriophage T3 inactivates E. coli’s methionine Sadenosyltransferase by forming hetero-polymers

Hadas Simon-Baram1, Daniel Kleiner1, Fannia Shmulevich1, Raz Zarivach1,2, Ran Zalk2, Huayuan Tang3, Feng Ding3, Shimon Bershtein1

1Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; 2Macromolecular Crystallography and Cryo-EM Research Center, The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel; 33Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, USA

ABSTRACT

S-adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host E. coli cells, thus inactivating a crucial metabolite involved in plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy ─ it allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle Cryo-EM and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM, but also by interacting with and efficiently inhibiting the host’s methionine S-adenosyltransferase (MAT) ─ the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined together by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency high-amplitude active site-deforming modes. The amplification of uncorrelated motions between active site residues weakens MAT's ability to withhold substrates, explaining the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools.

IMPORTANCE

Self-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy ─ silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes might be a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.

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Reciprocal space refinement and map calculation for cryo-EM single particle structures

Keitaro Yamashita, Rangana Warshamanage, Garib N. Murshudov

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

The number of high resolution structure determination by cryo-EM single particle analysis (SPA) is growing rapidly. Focusing on maps having better resolution than 3 Å deposited in the Electron Microscopy Data Bank (EMDB), there were 316 depositions in 2019 while it was 82 in 2018. It increases the importance of method developments for accurate determination of atomic coordinates and thus the model validation, where not only the geometric quality but also the fitness to the map is of great importance.

Here we present a new program Servalcat for the refinement and map calculation of cryo-EM SPA structures. Servalcat implements a refinement pipeline using REFMAC5, which uses a dedicated likelihood function for SPA [1]. It takes as inputs unsharpened and unweighted half maps from independent reconstructions. The variance of noise in Fourier coefficients is estimated using the half maps. A weighted and sharpened Fo-Fc map is calculated after the refinement. The Fourier coefficients for the difference maps are derived as expectation values of unknown Fourier coefficients using their posterior distribution given observations and model parameters. Refinement of atomic displacement parameters is crucial for calculation of a sensible Fo-Fc map. It was shown to be useful for visualization of weak features like hydrogen atoms and model errors as it is done routinely in crystallography. Although hydrogen densities are weaker than heavier atoms (e.g. C, N, O), they are stronger than in the electron density maps produced by X-ray crystallography, and some hydrogen atoms are even visible at ~1.8 Å.

About half of the EMDB-deposited SPA maps have non-C1 point group symmetry. If the map has been symmetrised during reconstruction then all downstream programs should be aware of it and the atomic structure model must follow the symmetry. A user can give an asymmetric unit model and a point group symbol to Servalcat for refinement. The NCS constraint function in REFMAC5 was updated to consider non-bonded interactions and ADP similarity restraints between symmetry copies. The MTRIX records in the PDB format and _struct_ncs_oper in the mmCIF format are used to encode the symmetry information. Currently, there are only few asymmetric unit model depositions to the PDB except viruses. We think that refining and depositing asymmetric unit models with annotations of symmetry will be a common practice in future.

We are also developing a new program EMDA, for cryo-EM map and model manipulation with the main focus on validation. EMDA offers several metrics for map validation including FSC combined with mask correction by high resolution noise substitution [2], local correlation using half maps, optimal alignment between maps and magnification scaling using maximum-likelihood method. Also, EMDA includes metrics for map-model validation such as local correlation between map-and-model, which can be used to investigate the quality of the map-to-model fit.

Both EMDA [3] and Servalcat [4] are freely available under an open source licence. They are also available within the CCP-EM package.

[1] Murshudov (2016). Methods in Enzymology 579, 277-305.
[2] Scheres and Chen (2012). Nature Methods 9, 853-854
[3] EMDA https://emda.readthedocs.io
[4] Servalcat https://github.com/keitaroyam/servalcat

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Cryo-EM structure of a functional monomeric Photosystem I from Thermosynechococcus elongatus reveals red chlorophyll cluster

Christoph Gerle1, Orkun Coruh2, Anna Frank3, Hideaki Tanaka2, Akihiro Kawamoto2, Eithar El-Mohsnawy4, Takayuki Kato2, Keiichi Namba2, Marc Nowaczyk3, Genji Kurisu2

1Riken, Sayo, Japan; 2Osaka University, Japan; 3Ruhr Universität Bochum, Germany; 4Kafrelsheikh University, Egypt

A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from T. elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700.

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Structural Comparation of heterotrimer PCNA from Crenarchaeon Aeropyrum pernix by solution scattering, Cryo-EM, and Crystallography

Takahiro Yamauchi1,2, Tsubasa Takemori3, Makiko Kikuchi4, Yasuhito Iizuka1,3, Satoshi Ishikawa3, Masaru Tsunoda1,3,4

1Grad. Sch. of Life Sci. and Tech., Iryo Sosei Univ.; 2Dept. of Pharm., Fukushima Rosai Hosp.; 3Fac. of Pharm., Iryo Sosei Univ.; 4Grad. Sch. of Sci. and Eng., Iryo Sosei Univ.

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. In bacteria, the β-clamp protein forms a homodimer, whereas in eukaryotes or euryarchaeotes, proliferating cell nuclear antigen (PCNA) proteins form homotrimers. However, PCNA from Aeropyrum pernix (ApPCNA), a crenarchaeote species, forms a heterotrimer. The actual structure of ApPCNA-mediated sliding clamps and the mechanism by which they slide along DNA is unknown. Previously, we have analysed the crystal structure of ApPCNA1 from the APE_0162 gene[1], ApPCNA2 from the APE_0441.1, and ApPCNA3 from the APE_2182 genes[2]. The present study aimed to analyse the crystal, solution structure and cryo-electron microscopy (cryo-EM) of the heterotrimeric ring of ApPCNA, examine its interaction with DNA and other proteins, and elucidate the mechanism of PCNA function.

Each ApPCNA molecule, which constitutes a heterotrimer, was expressed using the Escherichia coli expression system. The proteins were purified using heat treatment, ammonium sulfate precipitation, and column chromatography. The purified proteins were crystallized using the vapor-diffusion method and the crystals were analysed by X-ray diffraction. To verify the ring shape of ApPCNA2 in solution, the solution structure was analysed using size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). A mixture of ApPCNA1-2-3 and ApPCNA2-3 were analysed by SEC-multi-angle light scattering for the presence of a complex, and the solution structure was analysed by SEC-SAXS. The mixture was analysed by cryo-EM, after purified with gel filtration chromatography.

The solution structure of the ApPCNA1-2-3 complex is similar to shape of the British Isles islands. ApPCNA2 and ApPCNA3 interact in a similar manner as the PCNA rings of other organisms; however, ApPCNA1 is located such that it did not form a perfect ring-shaped structure. The scattering curves of the complex and those of the model edited trimeric ring are almost similar with minor differences. The solution structure of ApPCNA2-3 complex was similar to shape of a naan. This particle contains four subunits rather than trimer. The electron density from cryo-EM forms hexagon.

The solution structure was not trimeric ring, containing ApPCNA1-2-3. The N-terminus of ApPCNA1 is approximately 10 residues longer than that of ApPCNA2 and ApPCNA3. This could be why the tripartite complex is not ring shaped. Moreover, Met16 is present downstream of the N-terminal of ApPCNA1. In the future, the effect of N-terminus deletion and binding of the DNA duplex on ApPCNA1 structure should be evaluated. The solution structure of ApPCNA2-3 complex was not trimeric ring too. In crystal structure of ApPCNA3, the C-terminus interacts between adjacent subunits, probably PIP-Box binding site. This interaction may cause ApPCNA2-3 Complex dose not form ring shape. Generally, PCNA rings that consists of homotrimer have 3-fold symmetry, comprise six edges from concave edge between subunits and flat edge that formed PIP-Box binding site. This hexagonal electron density suggests ApPCNA1-2-3 forms trimeric ring in cryo-EM structure. Interestingly, one of the three edges is completely separated. The Fitting model containing ApPCNA1-2-3 hetero subunits suggests, the long N-terminus of ApPCNA1 cause this separated edge.

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Structural analysis of a tetradecameric HSP100 chaperone by cryo-EM and SAXS

Hyunwoo Cho, Gyuhee Kim, Sangho Lee

SungKyunKwan University, Suwon, Korea, Republic of (South Korea)

Heat shock protein 100 (HSP100) family members serve as ATPases associated with diverse cellular activities (AAA+) chaperones. HSP100s possess three enzymatic activities: ATPase, refoldase and disaggregase/holdase. With these activities, HSP100s play key roles in cellular stress responses and protein homeostasis. The functional oligomeric state of HSP100 is mostly modulated by their hexameric/dodecameric quaternary structures as exemplified by studies on ClpB and ClpC. However, little is known about non-hexameric/dodecameric HSP100 chaperones. We found that ClpL from Streptococcus pneumoniae forms tetradecamers in solution. Here we report the structural characterizations of the tetradecameric ClpL to reveal key residues responsible for the tetradecameric assembly. Cryo-EM structure of ClpL reveals a striking tetradecameric arrangement where two heptameric rings are connected by vertical middle domains. Non-conserved residues, Q321 and R670, are crucial in the heptameric ring assembly of ClpL while hydrophobic F350 contributes to the interface among the middle domains. Site-directed mutagenesis analysis supported the differential roles of the aforementioned residues. Mutations in Q321 and R670 abrogated ATPase and refolase activities, supporting that these residues are critical in the integrity of the heptameric ring arrangement. Mutations in hydrophobic residues of the middle domain deteriorated refoldase and disaggregase/holdase activities. Solution structures of ClpL derived from small-angle X-ray scattering (SAXS) data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric HSP100 chaperone structures. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric HSP100 chaperones.

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Developments in Advanced Handling, Storage, Transport, and Tracking of Cryo-EM Samples

Benjamin Apker, David Closs, Robert Thorne, Rich Jayne

MiTeGen LLC, Ithaca, United States of America

Interest in cryoelectron microscopy (cryoEM) is growing rapidly as technical advances in electron detectors and optics dramatically improve imaging capabilities and sample throughput. The federal government, via the NIH, has established multiple national centers that provide user access to this technology. Efficient use of these centers requires improved tools and methods for sample management. Building on experience gained in the high-throughput revolution in cryocrystallography, systems for the advanced storage, transport, and tracking of cryo-EM samples are being developed. We report on our current and planned developments in this area.

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Cryo-electron tomography study of detonation nanodiamonds hydrosols

Alexey A. Mikhutkin1, Roman A. Kamyshinsky1,2, Nikita M. Kuznetsov1, Sergei N. Chvalun1, Alexander L. Vasiliev1,2

1National Research Center «Kurchatov Institute», Moscow, Russian Federation; 2Shubnikov Institute of Crystallography of FSRC “Crystallography and Photonics” RAS, Moscow, Russian Federation

In recent years serious attention has been focused on various applications of nanodiamonds obtained by detonation synthesis of carbon-containing explosives (detonation nanodiamonds - DNDs) [1]. DNDs find applications in biomedicine, development of novel liquid heat carriers and magnetic liquids. Thus, study of their rheological properties in various liquid media is of great importance [1]. Two rheological features of hydrosols have been recently discovered: the sharp increase in viscosity and the phase sol-gel transition at a relatively low filler concentration [2]. Such behaviour has been explained in the frame of percolation model based on network formation from the chains of faceted DND particles due to electrostatic interaction of facets [3]. The comprehension of DNDs structural organisation in various media is crucial for practical applications and it would be important to obtain a direct experimental evidence of DND chains existence in sol and network formation at the sol-gel transition. Such direct observation has been successfully performed by Cryo-Electron Tomography (Cyo-ET): the processes of bonding and agglomeration of the DND particles were observed, and the 3D spatial distribution and quantitative analysis, including fractal analysis, were accomplished on the basis of Cyo-ET data. The obtained results explain the rheological properties of the DNDs hydrosols.

The study was focused on two types of DNDs hydrosols with positive and negative electrokinetic potential (ζ-potential) in the concentration range from 1.0 to 7.0 wt%. Cryo-ET study was performed on Titan Krios 60-300 TEM/STEM (FEI, USA) at acceleration voltage of 300 kV.

The 3D models of positive and negative ζ-potential DNDs were obtained and the 3D spatial distribution of DNDs was revealed [4]. The data demonstrate the formation of extended fractal structures and chains of individual faceted DND particles. The skeletonization procedure was applied in order to evaluate the bonding of objects and percolation. It was observed that DNDs with positive ζ-potential form a percolation network. However, such network was not observed in DNDs hydrosols with negative ζ-potential. This explains the differences in the rheological behavior at low concentrations of samples with different sign of ζ-potential. The 2D and 3D fractal dimensions were calculated for the mass-fractal and fractal surface from the Cryo-ET data. The fractal dimensions are in good correlation with the small angle X-ray scattering (SAXS) data for the fractal dimension of a DNDs cluster. Moreover, the distances between DND particles were estimated after the 3D reconstruction of the specimen volume. Interparticle distance distributions showed, that the secondary maximum position qualitatively matches the interplanar distance between fractals at 1 wt% contain of DND particles in hydrosol measured by SAXS.

The results of 3D reconstruction and analysis based on Cryo-ET data allowed to explain observed features of the rheological behavior associated with DNDs agglomeration and chain formation.

[1] Shvidchenko, A.V., Eidelman, E.D., Vul', A.Ya. et al. (2019). Adv. Colloid Interface Sci. 268, 64.

[2] Vul', A.Ya., Eidelman, E.D., Aleksenskiy, A.E. et al. (2017). Carbon. 114, 242.

[3] Kuznetsov, N.M., Belousov, S.I., Stolyarova, D.Yu. et al. (2018). Diam. Relat. Mater. 83, 141.

[4] Kuznetsov, N.M., Belousov, S.I., Bakirov, A.V. et al. (2020). Carbon. 161, 486.

The detonation nanodiamonds hydrosols were kindly provided by Prof. Vul' A.Ya. and coworkers from Laboratory Physics for Cluster Structures of Ioffe Institute. This work was partially supported by Russian Foundation for Basic Researches, project 18-29-19117 mk.

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