Pub Date : 2023-07-07DOI: 10.1107/s2053273323099187
Kunio Hirata, Hiroaki Matsuura, Y. Kawano, Naoki Sakai, K. Hasegawa, T. Kumasaka, M. Yamamoto
In this decade, we have advanced the efficient high-resolution crystal structure analysis of membrane proteins using microcrystals at the beamline BL32XU at SPring - 8 in Japan, by developing beamline hardware and software. Our approach has revealed that the signal-to-noise ratio can be significantly improved using dozens or hundreds of such crystals, even they are low - diffracting or challenging. Consequently, we have successfully determined the numerous high-impact crystal structures of a wide range of membrane proteins. The developed ‘serial’ data collec tion protocol had been implemented our automated data collection system, ZOO, and enabled un-attended experiments at MX beamlines. Recently, we achieved 1.8Å resolution crystal structure determination using 600 nm sized polyhedra crystals grown in cell - fre e protein crystallization technique. The serial synchrotron rotation crystallography ( SSROX) implemented in ZOO opened a new window for protein nano-crystallography with synchrotron radiation. We will discuss the experimental limitation coming from crystal size and dose for the synchrotron experiments by comparing results in some literature from SR and XFEL projects.
在这十年中，我们通过开发光束线硬件和软件，在日本 SPring - 8 的 BL32XU 光束线利用微晶体推进了对膜蛋白的高分辨率晶体结构分析。我们的方法表明，使用数十或数百个这样的晶体，即使它们的吸引力很低或具有挑战性，也能显著提高信噪比。因此，我们已经成功测定了大量膜蛋白的高影响力晶体结构。我们开发的 "串行 "数据收集协议已在我们的自动数据收集系统ZOO中实施，并在MX光束线实现了无人值守实验。最近，我们利用细胞自由蛋白质结晶技术中生长的 600 nm 大小的多面体晶体，实现了 1.8 Å 分辨率的晶体结构测定。ZOO 实施的串行同步辐射旋转晶体学（SSROX）为利用同步辐射进行蛋白质纳米晶体学研究打开了一扇新窗口。我们将通过比较 SR 和 XFEL 项目的一些文献结果，讨论同步辐射实验中晶体尺寸和剂量带来的实验限制。
{"title":"Current status of serial crystallography on Spring-8 MX beamlines","authors":"Kunio Hirata, Hiroaki Matsuura, Y. Kawano, Naoki Sakai, K. Hasegawa, T. Kumasaka, M. Yamamoto","doi":"10.1107/s2053273323099187","DOIUrl":"https://doi.org/10.1107/s2053273323099187","url":null,"abstract":"In this decade, we have advanced the efficient high-resolution crystal structure analysis of membrane proteins using microcrystals at the beamline BL32XU at SPring - 8 in Japan, by developing beamline hardware and software. Our approach has revealed that the signal-to-noise ratio can be significantly improved using dozens or hundreds of such crystals, even they are low - diffracting or challenging. Consequently, we have successfully determined the numerous high-impact crystal structures of a wide range of membrane proteins. The developed ‘serial’ data collec tion protocol had been implemented our automated data collection system, ZOO, and enabled un-attended experiments at MX beamlines. Recently, we achieved 1.8Å resolution crystal structure determination using 600 nm sized polyhedra crystals grown in cell - fre e protein crystallization technique. The serial synchrotron rotation crystallography ( SSROX) implemented in ZOO opened a new window for protein nano-crystallography with synchrotron radiation. We will discuss the experimental limitation coming from crystal size and dose for the synchrotron experiments by comparing results in some literature from SR and XFEL projects.","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139362019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323097000
Robert E. Thorne
Serial synchrotron crystallography (SSX) enables use of small crystals for structure – function studies of biomolecules and for drug discovery. Many SSX approaches require large numbers of crystals having similar (small) size and shape, and allow data collection from only a fraction of available crystals. However, crystals of non -model proteins generated in crystallization trials are typically modest in number and heterogeneous in size and shape. Despite some striking successes and impressive technical achievements, the impact of serial crystallographic methods on general practice has so far been limited. An integrated SSX system has been developed with the goal of getting all available crystals – from 1 to 105 – into the X -ray beam with the least effort and in the best possible condition. The system consists of ultra - low background -scatter large area sample holders suitable for room and cryogenic temperature data collection and a humidified sample-loading workstation. The sample holders incorporate thin - film supports with a variety of designs optimized for different crystal - loading challenges. These holders facilitate dispersion of crystals across the support and removal of excess liquid, can be cooled at extremely high rates, generate little background scatter, allow data collection over >90° of oscillation without obstruction or risk of generating saturating Bragg peaks, are resusable, and are compatible with existing infrastructure for high - throughput cryocrystallography. The sample-loading workstation allows sample preparation and loading onto the support film; application of time - varying suction for optimal removal of excess liquid, for crystal repositioning and for crystal cryoprotection; and application of sealing films for room - temperature data collection. The workstation provides a near saturating humidity (>95% r.h.) environment, eliminating dehydration of even the smallest crystals and drying of open crystallization drops, while allowing observation of all operations via a microscope. This integrated system addresses common problems in obtaining properly dispersed, properly hydrated and isomorphous microcrystals for fixed -orientation and oscillation data collection. Its ease of use, flexibility,
{"title":"A complete, versatile and cost-effective solution for routine serial and 'conventional' synchrotron crystallography","authors":"Robert E. Thorne","doi":"10.1107/s2053273323097000","DOIUrl":"https://doi.org/10.1107/s2053273323097000","url":null,"abstract":"Serial synchrotron crystallography (SSX) enables use of small crystals for structure – function studies of biomolecules and for drug discovery. Many SSX approaches require large numbers of crystals having similar (small) size and shape, and allow data collection from only a fraction of available crystals. However, crystals of non -model proteins generated in crystallization trials are typically modest in number and heterogeneous in size and shape. Despite some striking successes and impressive technical achievements, the impact of serial crystallographic methods on general practice has so far been limited. An integrated SSX system has been developed with the goal of getting all available crystals – from 1 to 105 – into the X -ray beam with the least effort and in the best possible condition. The system consists of ultra - low background -scatter large area sample holders suitable for room and cryogenic temperature data collection and a humidified sample-loading workstation. The sample holders incorporate thin - film supports with a variety of designs optimized for different crystal - loading challenges. These holders facilitate dispersion of crystals across the support and removal of excess liquid, can be cooled at extremely high rates, generate little background scatter, allow data collection over >90° of oscillation without obstruction or risk of generating saturating Bragg peaks, are resusable, and are compatible with existing infrastructure for high - throughput cryocrystallography. The sample-loading workstation allows sample preparation and loading onto the support film; application of time - varying suction for optimal removal of excess liquid, for crystal repositioning and for crystal cryoprotection; and application of sealing films for room - temperature data collection. The workstation provides a near saturating humidity (>95% r.h.) environment, eliminating dehydration of even the smallest crystals and drying of open crystallization drops, while allowing observation of all operations via a microscope. This integrated system addresses common problems in obtaining properly dispersed, properly hydrated and isomorphous microcrystals for fixed -orientation and oscillation data collection. Its ease of use, flexibility,","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139362022","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323098960
H. Choe
Na 0.5Bi0.5TiO 3 (NBT) is interesting as a potential lead-free piezoelectric and intriguing as the model system for the crystallography of perovskites. The average / local structures of NBT are frequently debated, while their relationship to the physical properties is unclear. The previous reports [1] of the monoclinic (Cc) average symmetry of NBT structure motivate for the in-depth studies of the processes, which are triggered to the polarization rotation and its implication for the physical properties. It is therefore particularly interesting to investigate the response of NBT to such external influences as electric field, change of a temperature or high-pressure, where polarization rotation may be observed and described. The aim of this work is to probe the lattice response of NBT single crystal to an external electric field. We implemented stroboscopic time-resolved X -ray diffraction (descripted in [2]) to acquire high -resolution reciprocal space maps as a functi on of time and triangular-shaped 100 Hz cyclic electric field. We focused at the reciprocal space region around {004} reflection and carefully mapped it in -situ using state-of -the- art X - ray optics and synchrotron radiation source (P08 beamline at PETRA III and KMC-3 XPP at BESSY II). We observed that reciprocal space maps are separated into two peak components, where the separation magnitude follows electric field (see the figure). The similar radial separation of {00l} was used as the evidence of non - rhombohe dral (monoclinic) average symmetry in NBT [1]. We analysed this separation by fi tting the observed two -dimensional intensity maps with a pair of two -dimensional Pseudo-Voigt functions to track the individual peaks independently. The results are discussed in the framework of the proposed monoclinic symmetry and allowed polarization rotation.
Na 0.5Bi0.5TiO 3（NBT）作为一种潜在的无铅压电体非常有趣，而作为包晶石晶体学的模型系统也非常引人入胜。NBT 的平均/局部结构经常引起争议，而它们与物理性质的关系却不清楚。之前关于 NBT 结构的单斜（Cc）平均对称性的报道 [1] 促使人们深入研究引发极化旋转的过程及其对物理性质的影响。因此，研究 NBT 对电场、温度变化或高压等外部因素的反应尤为有趣，因为在这种情况下可以观察和描述极化旋转。这项工作的目的是探测 NBT 单晶对外部电场的晶格响应。我们采用频闪时间分辨 X 射线二折射技术（见文献 [2]）来获取高分辨率的倒易空间图，并将其作为时间和三角形 100 Hz 循环电场的函数。我们聚焦于{004}重射周围的倒易空间区域，并使用最先进的 X 射线光学仪器和同步辐射源（佩特拉三期的 P08 光束线和 BESSY 二期的 KMC-3 XPP）对其进行了仔细的原位测绘。我们观察到，倒易空间图被分成两个峰值成分，其分离程度与电场有关（见图）。{00l}的类似径向分离被用作 NBT 中非斜方晶系（单斜晶系）平均对称性的证据[1]。我们用一对二维伪伏依格特函数对观察到的二维强度图进行了分析，以独立跟踪各个峰值。我们在所提出的单斜对称性和允许偏振旋转的框架内对结果进行了讨论。
{"title":"Time-resolved reciprocal-space mapping of ferroelectric perovskites under an alternating electric field","authors":"H. Choe","doi":"10.1107/s2053273323098960","DOIUrl":"https://doi.org/10.1107/s2053273323098960","url":null,"abstract":"Na 0.5Bi0.5TiO 3 (NBT) is interesting as a potential lead-free piezoelectric and intriguing as the model system for the crystallography of perovskites. The average / local structures of NBT are frequently debated, while their relationship to the physical properties is unclear. The previous reports [1] of the monoclinic (Cc) average symmetry of NBT structure motivate for the in-depth studies of the processes, which are triggered to the polarization rotation and its implication for the physical properties. It is therefore particularly interesting to investigate the response of NBT to such external influences as electric field, change of a temperature or high-pressure, where polarization rotation may be observed and described. The aim of this work is to probe the lattice response of NBT single crystal to an external electric field. We implemented stroboscopic time-resolved X -ray diffraction (descripted in [2]) to acquire high -resolution reciprocal space maps as a functi on of time and triangular-shaped 100 Hz cyclic electric field. We focused at the reciprocal space region around {004} reflection and carefully mapped it in -situ using state-of -the- art X - ray optics and synchrotron radiation source (P08 beamline at PETRA III and KMC-3 XPP at BESSY II). We observed that reciprocal space maps are separated into two peak components, where the separation magnitude follows electric field (see the figure). The similar radial separation of {00l} was used as the evidence of non - rhombohe dral (monoclinic) average symmetry in NBT [1]. We analysed this separation by fi tting the observed two -dimensional intensity maps with a pair of two -dimensional Pseudo-Voigt functions to track the individual peaks independently. The results are discussed in the framework of the proposed monoclinic symmetry and allowed polarization rotation.","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139361513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323099230
Kent Griffith
{"title":"Complex host structures and phase transitions in fast-charging lithium-ion battery anodes from diffraction and complementary techniques","authors":"Kent Griffith","doi":"10.1107/s2053273323099230","DOIUrl":"https://doi.org/10.1107/s2053273323099230","url":null,"abstract":"","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139361577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323096730
Yimin Mao
{"title":"Hierarchical structures of polysaccharides","authors":"Yimin Mao","doi":"10.1107/s2053273323096730","DOIUrl":"https://doi.org/10.1107/s2053273323096730","url":null,"abstract":"","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"199 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139361589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323097036
Joseph Davis, Barratt Powell, S. Mosalaganti
Compositional and conformational dynamics are integral to the assembly and function of macromolecular complexes. Fueled by deep learning, new single -particle cryo-EM image analysis tools have revealed these structural dynamics in isolated samples. However, a key goal of structural biology is to interrogate these dynamic structures in their native cellular environment, which would reveal how distinct structural states are partitioned throughout the cell, how they uniquely interact with other cellular components, and how they respond to genetic and environmental perturbations. Cryo-electron tomography (cryo-ET), which has the potential for high -resolution imaging directly in flash - frozen cells, represents a promising path toward achieving this goal. Indeed, modern cryo-ET workflows have revealed molecularly interpretable, sub-nm structures of key complexes, including the ribosome. To date, most cryo - ET processing algorithms aim to increase resolution by relying on expert-guided classification of structures into a discrete set of approximately homogeneous classes. Such discrete classification models scale poorly to highly heterogeneous ensembles and are inherently ill-match to molecules undergoing continuous motion. To analyze such complex structural ensembles in situ, we developed tomoDRGN, which employs a modified variational autoencoder to embed individual particles in a continuous latent space and to reconstruct unique volumes informed by the latent. Here, we describe the tomoDRGN model architecture, which was purpose - built for tomographic datasets; we detail its performance on simulated and exemplar experimental datasets, and we highlight tools built to aid in interpreting tomoDRGN outputs in the context of a cellular tomogram. Additionally, we showcase its application to the process of bacterial ribosome biogenesis - specifically comparing the structural ensembles observed in situ with those observed in isolated samples.
{"title":"TomoDRGN: resolving structural heterogeneity in situ","authors":"Joseph Davis, Barratt Powell, S. Mosalaganti","doi":"10.1107/s2053273323097036","DOIUrl":"https://doi.org/10.1107/s2053273323097036","url":null,"abstract":"Compositional and conformational dynamics are integral to the assembly and function of macromolecular complexes. Fueled by deep learning, new single -particle cryo-EM image analysis tools have revealed these structural dynamics in isolated samples. However, a key goal of structural biology is to interrogate these dynamic structures in their native cellular environment, which would reveal how distinct structural states are partitioned throughout the cell, how they uniquely interact with other cellular components, and how they respond to genetic and environmental perturbations. Cryo-electron tomography (cryo-ET), which has the potential for high -resolution imaging directly in flash - frozen cells, represents a promising path toward achieving this goal. Indeed, modern cryo-ET workflows have revealed molecularly interpretable, sub-nm structures of key complexes, including the ribosome. To date, most cryo - ET processing algorithms aim to increase resolution by relying on expert-guided classification of structures into a discrete set of approximately homogeneous classes. Such discrete classification models scale poorly to highly heterogeneous ensembles and are inherently ill-match to molecules undergoing continuous motion. To analyze such complex structural ensembles in situ, we developed tomoDRGN, which employs a modified variational autoencoder to embed individual particles in a continuous latent space and to reconstruct unique volumes informed by the latent. Here, we describe the tomoDRGN model architecture, which was purpose - built for tomographic datasets; we detail its performance on simulated and exemplar experimental datasets, and we highlight tools built to aid in interpreting tomoDRGN outputs in the context of a cellular tomogram. Additionally, we showcase its application to the process of bacterial ribosome biogenesis - specifically comparing the structural ensembles observed in situ with those observed in isolated samples.","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139361791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323099047
Martina Maritan
Structural biology enables scientists to examine molecular structures in exceptional detail, including at the atomic level. This knowledge of molecular anatomy is crucial for understanding how molecules function and for guiding structure-based drug discovery. Visualizing and manipulating molecular structures is an essential step in this process, and advances in technology are providing increasingly sophisticated methods for doing so. The very process of visual exploration can be a moment for creativity and lead to unexpected ideas. Nanome has developed a platform that utilizes virtual and mixed reality to enable scientists to brainstorm in front of 3D structures and use the platform as a sandbox for visually testing hypotheses. The intuitive interaction with molecules offered by the virtual reality environment makes it a powerful tool for promoting creativity and unlocking unforeseen inspirations. Research groups have used this virtual environment to freely explore structures and molecular designs in real-time, leading to the ideation of completely novel compounds and gaining new structural insights.
{"title":"Virtual reality as a thinking tool for structural investigation","authors":"Martina Maritan","doi":"10.1107/s2053273323099047","DOIUrl":"https://doi.org/10.1107/s2053273323099047","url":null,"abstract":"Structural biology enables scientists to examine molecular structures in exceptional detail, including at the atomic level. This knowledge of molecular anatomy is crucial for understanding how molecules function and for guiding structure-based drug discovery. Visualizing and manipulating molecular structures is an essential step in this process, and advances in technology are providing increasingly sophisticated methods for doing so. The very process of visual exploration can be a moment for creativity and lead to unexpected ideas. Nanome has developed a platform that utilizes virtual and mixed reality to enable scientists to brainstorm in front of 3D structures and use the platform as a sandbox for visually testing hypotheses. The intuitive interaction with molecules offered by the virtual reality environment makes it a powerful tool for promoting creativity and unlocking unforeseen inspirations. Research groups have used this virtual environment to freely explore structures and molecular designs in real-time, leading to the ideation of completely novel compounds and gaining new structural insights.","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"47 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139361857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323098145
Hideki Shigematsu, Christoph Gerle, Chai Gopalasingam
Since October 2021, we have started public use of CryoTEM as an ancillary facility for structural biology beamlines at SPring-8. We have set up a facility with two CryoTEMs, EM01CT for high -resolution data collection and EM02CT for screening, user training, and general purpose. EM01CT produces high-resolution data in a high-throughput manner for single particle analysis by using a CRYO ARM 300 (JEM - Z300FSC, JEOL) which has a cold - field emission gun, an in-column energy filter, a cryo -supporter system and is coupled with a K3 camera (Gatan). EM02CT is a CRYO ARM 200 (JEM-Z200FSC, JEOL) equipped with a K2 summit camera (Gatan). We provide a training course for all new users of the facility to enable them to have an easy start with structural analysis projects by using our CryoTEMs and continue to provide advice throughout their projects to provide the best possible environment for the successful completion of projects. We have been able to continuously provide productive machine time to the users of SPring-8 resulting in many high-resolution solution structures in the range of 2 ~ 2.5 Å. The first paper was published by one of our users in June 2022[1]. The authors tried to obtain structures of the gastric proton pump with its known inhibitors to understand its inhibitory mechanism by using X -ray crystallography. In this paper, they succeeded in obtaining the crystal structures with three compounds but could not obtain good crystals with another compound. It was crucial to obtain structures with compounds to comp are the interactions between compounds and the protein, therefore they switched to using CryoEM for the complex with the other compound, for which it was difficult to obtain good crystals. This is exactly the situation in which we set up our CryoTEMs as an ancillary facility for structural biology beamlines. In addition to the CryoTEMs
{"title":"Public use cryo-EM at Spring-8","authors":"Hideki Shigematsu, Christoph Gerle, Chai Gopalasingam","doi":"10.1107/s2053273323098145","DOIUrl":"https://doi.org/10.1107/s2053273323098145","url":null,"abstract":"Since October 2021, we have started public use of CryoTEM as an ancillary facility for structural biology beamlines at SPring-8. We have set up a facility with two CryoTEMs, EM01CT for high -resolution data collection and EM02CT for screening, user training, and general purpose. EM01CT produces high-resolution data in a high-throughput manner for single particle analysis by using a CRYO ARM 300 (JEM - Z300FSC, JEOL) which has a cold - field emission gun, an in-column energy filter, a cryo -supporter system and is coupled with a K3 camera (Gatan). EM02CT is a CRYO ARM 200 (JEM-Z200FSC, JEOL) equipped with a K2 summit camera (Gatan). We provide a training course for all new users of the facility to enable them to have an easy start with structural analysis projects by using our CryoTEMs and continue to provide advice throughout their projects to provide the best possible environment for the successful completion of projects. We have been able to continuously provide productive machine time to the users of SPring-8 resulting in many high-resolution solution structures in the range of 2 ~ 2.5 Å. The first paper was published by one of our users in June 2022[1]. The authors tried to obtain structures of the gastric proton pump with its known inhibitors to understand its inhibitory mechanism by using X -ray crystallography. In this paper, they succeeded in obtaining the crystal structures with three compounds but could not obtain good crystals with another compound. It was crucial to obtain structures with compounds to comp are the interactions between compounds and the protein, therefore they switched to using CryoEM for the complex with the other compound, for which it was difficult to obtain good crystals. This is exactly the situation in which we set up our CryoTEMs as an ancillary facility for structural biology beamlines. In addition to the CryoTEMs","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139361869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-07DOI: 10.1107/s2053273323098303
Estella F. Yee, Kriti Chopra, Nicolas Grosjean, D. Kumaran, Macon Abernathy, James Byrnes, Lin Yang
A large family of domain of unknown function (DUF) -containing proteins was recently identified by phylogenomic studies to bind to heme. DUF2470 and related subfamilies of phototroph-specific homologs have diverse heme -related functions, but the structure-function link of DUF2470 itself had yet to be determined. In Synechocystis, DUF2470 forms single domain proteins and were discovered to bind heme and zinc ions, generating a unique two - fold symmetric, zinc-bound bis-histidine heme site. Structural and spectroscopic characterizations of the wild -type and variants lacking conserved histidine residues elucidate the importance of zinc-binding and histidine residues fo r heme-binding activity. Results here supplement in vivo experiments and observed phenotypes that implicate DUF2470 in heme-dependent regulation of electron transport chains.
{"title":"Structural characterization of a zinc-coordinated bis-histidine heme-binding site in the DUF2470 cyanobacterial protein","authors":"Estella F. Yee, Kriti Chopra, Nicolas Grosjean, D. Kumaran, Macon Abernathy, James Byrnes, Lin Yang","doi":"10.1107/s2053273323098303","DOIUrl":"https://doi.org/10.1107/s2053273323098303","url":null,"abstract":"A large family of domain of unknown function (DUF) -containing proteins was recently identified by phylogenomic studies to bind to heme. DUF2470 and related subfamilies of phototroph-specific homologs have diverse heme -related functions, but the structure-function link of DUF2470 itself had yet to be determined. In Synechocystis, DUF2470 forms single domain proteins and were discovered to bind heme and zinc ions, generating a unique two - fold symmetric, zinc-bound bis-histidine heme site. Structural and spectroscopic characterizations of the wild -type and variants lacking conserved histidine residues elucidate the importance of zinc-binding and histidine residues fo r heme-binding activity. Results here supplement in vivo experiments and observed phenotypes that implicate DUF2470 in heme-dependent regulation of electron transport chains.","PeriodicalId":6903,"journal":{"name":"Acta Crystallographica Section A Foundations and Advances","volume":"139 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139362119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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