The temperature dependence of accurate structure factors of l-alanine and taurine was measured at the SPring-8 BL02B1 beamline. The quality of the structure factors is evaluated by charge density and quantum crystallographic studies. The effects of small amounts of twinning on the charge density study for taurine are also described.
Multi-temperature high-quality structure factors of l-alanine and taurine were re-measured at the SPring-8 BL02B1 beamline for method development in quantum crystallography. The quality of the data was evaluated by comparison with previous studies. In the case of taurine, we found that the data quality was highly affected by small amounts of twinning. Residual electron density around the sulfur atoms observed in a previous study [Hibbs et al. (2003). Chem. A Eur. J.9, 1075–1084] disappeared with the re-measured data. X-ray wavefunction refinements were carried out on these data. The difference electron density between the X-ray constrained wavefunction (XCW) results and the Hartree–Fock charge density showed a positive difference electron density around the nucleus and a negative difference electron density between the bonds. These features were consistent with those reported [Hupf et al. (2023). J. Chem. Phys.158, 124103]. It was found that the deformation density around the nucleus and between bonds due to electron correlations and electronic polarization could be confirmed by the XCW method using the present structure factors.
在SPring-8 BL02B1光束线上测定了l-丙氨酸和牛磺酸精确结构因子的温度依赖性。通过电荷密度和量子晶体学研究来评价结构因子的质量。本文还描述了少量孪生对牛磺酸电荷密度研究的影响。在SPring-8 BL02B1光束线上重新测量了l-丙氨酸和牛磺酸的多温度高质量结构因子，用于量子晶体学方法的发展。通过与以往研究的比较来评估数据的质量。在牛磺酸的情况下，我们发现数据质量受到少量双胞胎的高度影响。在先前的研究中观察到硫原子周围的剩余电子密度[Hibbs等人（2003）]。化学。一个欧元。J.9, 1075-1084]随重测数据消失。对这些数据进行了x射线波函数改进。x射线约束波函数（XCW）结果与Hartree-Fock电荷密度之间的电子密度差显示原子核周围的电子密度差为正，键之间的电子密度差为负。这些特征与报道[Hupf et al.(2023)]一致。j .化学。Phys.158, 124103]。发现原子核周围和键间由电子相关和电子极化引起的变形密度可以用XCW方法用现有的结构因子来确定。
{"title":"Accurate temperature dependence of structure factors of l-alanine and taurine for quantum crystallography","authors":"Mibuki Hayashi , Takashi Nishioka , Hidetaka Kasai , Eiji Nishibori","doi":"10.1107/S2052252525002647","DOIUrl":"10.1107/S2052252525002647","url":null,"abstract":"<div><div>The temperature dependence of accurate structure factors of <span>l</span>-alanine and taurine was measured at the SPring-8 BL02B1 beamline. The quality of the structure factors is evaluated by charge density and quantum crystallographic studies. The effects of small amounts of twinning on the charge density study for taurine are also described.</div></div><div><div>Multi-temperature high-quality structure factors of <span>l</span>-alanine and taurine were re-measured at the SPring-8 BL02B1 beamline for method development in quantum crystallography. The quality of the data was evaluated by comparison with previous studies. In the case of taurine, we found that the data quality was highly affected by small amounts of twinning. Residual electron density around the sulfur atoms observed in a previous study [Hibbs <em>et al.</em> (2003). <em>Chem. A Eur. J.</em><strong>9</strong>, 1075–1084] disappeared with the re-measured data. X-ray wavefunction refinements were carried out on these data. The difference electron density between the X-ray constrained wavefunction (XCW) results and the Hartree–Fock charge density showed a positive difference electron density around the nucleus and a negative difference electron density between the bonds. These features were consistent with those reported [Hupf <em>et al.</em> (2023). <em>J. Chem. Phys.</em><strong>158</strong>, 124103]. It was found that the deformation density around the nucleus and between bonds due to electron correlations and electronic polarization could be confirmed by the XCW method using the present structure factors.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 384-392"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525001484
Courtney J. Tremlett , Jack Stubbs , William S. Stuart , Patrick D. Shaw Stewart , Jonathan West , Allen M. Orville , Ivo Tews , Nicholas J. Harmer
Developments in macromolecular crystallography now allow the use of microcrystals for structural analysis through advanced beamlines and techniques such as microcrystal electron diffraction and room-temperature crystallography. This review addresses methods of matching microcrystal preparation and sample delivery. The use of microcrystals enhances the possibilities in fields such as time-resolved crystallography.
Advancements in macromolecular crystallography, driven by improved sources and cryocooling techniques, have enabled the use of increasingly smaller crystals for structure determination, with microfocus beamlines now widely accessible. Initially developed for challenging samples, these techniques have culminated in advanced beamlines such as VMXm. Here, an in vacuo sample environment improves the signal-to-noise ratio in X-ray diffraction experiments, and thus enables the use of submicrometre crystals. The advancement of techniques such as microcrystal electron diffraction (MicroED) for atomic-level insights into charged states and hydrogen positions, along with room-temperature crystallography to observe physiological states via serial crystallography, has driven a resurgence in the use of microcrystals. Reproducibly preparing small crystals, especially from samples that typically yield larger crystals, requires considerable effort, as no one singular approach guarantees optimal crystals for every technique. This review discusses methods for generating such small crystals, including mechanical crushing and batch crystallization with seeding, and evaluates their compatibility with microcrystal data-collection modalities. Additionally, we examine sample-delivery methods, which are crucial for selecting appropriate crystallization strategies. Establishing reliable protocols for sample preparation and delivery opens new avenues for macromolecular crystallography, particularly in the rapidly progressing field of time-resolved crystallography.
{"title":"Small but mighty: the power of microcrystals in structural biology","authors":"Courtney J. Tremlett , Jack Stubbs , William S. Stuart , Patrick D. Shaw Stewart , Jonathan West , Allen M. Orville , Ivo Tews , Nicholas J. Harmer","doi":"10.1107/S2052252525001484","DOIUrl":"10.1107/S2052252525001484","url":null,"abstract":"<div><div>Developments in macromolecular crystallography now allow the use of microcrystals for structural analysis through advanced beamlines and techniques such as microcrystal electron diffraction and room-temperature crystallography. This review addresses methods of matching microcrystal preparation and sample delivery. The use of microcrystals enhances the possibilities in fields such as time-resolved crystallography.</div></div><div><div>Advancements in macromolecular crystallography, driven by improved sources and cryocooling techniques, have enabled the use of increasingly smaller crystals for structure determination, with microfocus beamlines now widely accessible. Initially developed for challenging samples, these techniques have culminated in advanced beamlines such as VMXm. Here, an <em>in vacuo</em> sample environment improves the signal-to-noise ratio in X-ray diffraction experiments, and thus enables the use of submicrometre crystals. The advancement of techniques such as microcrystal electron diffraction (MicroED) for atomic-level insights into charged states and hydrogen positions, along with room-temperature crystallography to observe physiological states via serial crystallography, has driven a resurgence in the use of microcrystals. Reproducibly preparing small crystals, especially from samples that typically yield larger crystals, requires considerable effort, as no one singular approach guarantees optimal crystals for every technique. This review discusses methods for generating such small crystals, including mechanical crushing and batch crystallization with seeding, and evaluates their compatibility with microcrystal data-collection modalities. Additionally, we examine sample-delivery methods, which are crucial for selecting appropriate crystallization strategies. Establishing reliable protocols for sample preparation and delivery opens new avenues for macromolecular crystallography, particularly in the rapidly progressing field of time-resolved crystallography.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 262-279"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143624589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525001447
Rahul Shukla , Emmanuel Aubert , Mariya Brezgunova , Sébastien Lebègue , Marc Fourmigué , Enrique Espinosa
<div><div>This study establishes that hydrogen-, halogen- and chalcogen-bonding intermolecular and non-covalent intramolecular interactions are driven by a face-to-face orientation of electrophilic (charge-depleted) and nucleophilic (charge-concentrated) regions, which is the origin of the specific geometries found in synthons and supramolecular motifs.</div></div><div><div>A four-membered <em>R</em><sub>2</sub><sup>2</sup>(4) supramolecular motif formed by S⋯S and S⋯I chalcogen-bonding interactions in the crystal structure of 4-iodo-1,3-dithiol-2-one (C<sub>3</sub>HIOS<sub>2</sub>, IDT) is analysed and compared with a similar <em>R</em><sub>2</sub><sup>2</sup>(4) motif (stabilized by Se⋯Se and Se⋯O chalcogen bonds) observed in the previously reported crystal structure of selenaphthalic anhydride (C<sub>8</sub>H<sub>4</sub>O<sub>2</sub>Se, SePA) through detailed charge density analysis. Our investigation reveals that the chalcogen-bonding interactions participating in the <em>R</em><sub>2</sub><sup>2</sup>(4) motifs observed in the two structures have the same characteristic orientation of local electrostatic electrophilic⋯nucleophilic interactions while involving different types of atoms. We carried out Cambridge Structural Database searches for synthons and supramolecular motifs involving chalcogen-, halogen- and hydrogen-bonding (ChB, XB and HB) interactions. Geometrical characterizations and topological analyses of the electron density ρ(<strong>r</strong>) and its negative Laplacian function [<em>L</em>(<strong>r</strong>) = −∇<sup>2</sup>ρ(<strong>r</strong>)] indicate that all the bonding interactions forming the motifs are driven by local electrophilic⋯nucleophilic interactions between complementary charge concentration (CC) and charge depletion (CD) sites present in the valence shells of the atoms, regardless of the atoms and functional groups involved. The graph-set assignment <em>G</em><sub><em>d</em></sub><sup><em>a</em></sup>(<em>n</em>) (<em>G</em> = <em>C</em>, <em>R</em>, <em>D</em> or <em>S</em>), formerly developed by Etter [<em>Acc. Chem. Res.</em> (1990), <strong>23</strong>, 120–126] for HB interactions, is a convenient way to describe the connectivity in supramolecular motifs based on electrophilic⋯nucleophilic interactions (such as ChB, XB and HB interactions), exchanging the number of atomic acceptors (<em>a</em>) and donors (<em>d</em>) with the number of nucleophilic (<em>n</em>: CC) and electrophilic (<em>e</em>: CD) sites, and the number of atoms building the motif <em>n</em> by <em>m</em>, leading to the new graph-set assignment <em>G</em><sub><em>e</em></sub><sup><em>n</em></sup>(<em>m</em>) (<em>G</em> = <em>C</em>, <em>R</em>, <em>D</em> or <em>S</em>). Geometrical preferences in the molecular assembly of synthons and other supramolecular motifs are governed by the relative positions of CC and CD sites through CC⋯CD interactions that, in most cases, align with the internuclear directions within a <15° range de
{"title":"The origin of synthons and supramolecular motifs: beyond atoms and functional groups","authors":"Rahul Shukla , Emmanuel Aubert , Mariya Brezgunova , Sébastien Lebègue , Marc Fourmigué , Enrique Espinosa","doi":"10.1107/S2052252525001447","DOIUrl":"10.1107/S2052252525001447","url":null,"abstract":"<div><div>This study establishes that hydrogen-, halogen- and chalcogen-bonding intermolecular and non-covalent intramolecular interactions are driven by a face-to-face orientation of electrophilic (charge-depleted) and nucleophilic (charge-concentrated) regions, which is the origin of the specific geometries found in synthons and supramolecular motifs.</div></div><div><div>A four-membered <em>R</em><sub>2</sub><sup>2</sup>(4) supramolecular motif formed by S⋯S and S⋯I chalcogen-bonding interactions in the crystal structure of 4-iodo-1,3-dithiol-2-one (C<sub>3</sub>HIOS<sub>2</sub>, IDT) is analysed and compared with a similar <em>R</em><sub>2</sub><sup>2</sup>(4) motif (stabilized by Se⋯Se and Se⋯O chalcogen bonds) observed in the previously reported crystal structure of selenaphthalic anhydride (C<sub>8</sub>H<sub>4</sub>O<sub>2</sub>Se, SePA) through detailed charge density analysis. Our investigation reveals that the chalcogen-bonding interactions participating in the <em>R</em><sub>2</sub><sup>2</sup>(4) motifs observed in the two structures have the same characteristic orientation of local electrostatic electrophilic⋯nucleophilic interactions while involving different types of atoms. We carried out Cambridge Structural Database searches for synthons and supramolecular motifs involving chalcogen-, halogen- and hydrogen-bonding (ChB, XB and HB) interactions. Geometrical characterizations and topological analyses of the electron density ρ(<strong>r</strong>) and its negative Laplacian function [<em>L</em>(<strong>r</strong>) = −∇<sup>2</sup>ρ(<strong>r</strong>)] indicate that all the bonding interactions forming the motifs are driven by local electrophilic⋯nucleophilic interactions between complementary charge concentration (CC) and charge depletion (CD) sites present in the valence shells of the atoms, regardless of the atoms and functional groups involved. The graph-set assignment <em>G</em><sub><em>d</em></sub><sup><em>a</em></sup>(<em>n</em>) (<em>G</em> = <em>C</em>, <em>R</em>, <em>D</em> or <em>S</em>), formerly developed by Etter [<em>Acc. Chem. Res.</em> (1990), <strong>23</strong>, 120–126] for HB interactions, is a convenient way to describe the connectivity in supramolecular motifs based on electrophilic⋯nucleophilic interactions (such as ChB, XB and HB interactions), exchanging the number of atomic acceptors (<em>a</em>) and donors (<em>d</em>) with the number of nucleophilic (<em>n</em>: CC) and electrophilic (<em>e</em>: CD) sites, and the number of atoms building the motif <em>n</em> by <em>m</em>, leading to the new graph-set assignment <em>G</em><sub><em>e</em></sub><sup><em>n</em></sup>(<em>m</em>) (<em>G</em> = <em>C</em>, <em>R</em>, <em>D</em> or <em>S</em>). Geometrical preferences in the molecular assembly of synthons and other supramolecular motifs are governed by the relative positions of CC and CD sites through CC⋯CD interactions that, in most cases, align with the internuclear directions within a <15° range de","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 334-357"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525001721
Bruno Landeros-Rivera , Julia Contreras-García , Ángel Martín Pendás
The development of quantum crystallography depends on the availability of reliable theoretical electron densities. This work demonstrates a non-negligible code dependence of these densities and warns against their blind use.
The use of electronic structure methods in crystallographic data analysis, the now well known field of quantum crystallography, aids in the solution of several problems in X-ray diffraction refinement, as well as opening new avenues to access a whole new set of experimentally available observables. A key ingredient in quantum crystallography is the theoretically derived electron density, ρ, obtained from standard electronic structure codes. Here, we introduce a factor that has not been carefully considered until now. As we demonstrate, theoretically derived ρ values depend not only on the set of computational conditions used to obtain them but also on the particular computational code selected for this task. We recommend that all quantum crystallographers carefully check the convergence of ρ before undertaking any serious study.
{"title":"Code dependence of calculated crystalline electron densities. Possible lessons for quantum crystallography","authors":"Bruno Landeros-Rivera , Julia Contreras-García , Ángel Martín Pendás","doi":"10.1107/S2052252525001721","DOIUrl":"10.1107/S2052252525001721","url":null,"abstract":"<div><div>The development of quantum crystallography depends on the availability of reliable theoretical electron densities. This work demonstrates a non-negligible code dependence of these densities and warns against their blind use.</div></div><div><div>The use of electronic structure methods in crystallographic data analysis, the now well known field of quantum crystallography, aids in the solution of several problems in X-ray diffraction refinement, as well as opening new avenues to access a whole new set of experimentally available observables. A key ingredient in quantum crystallography is the theoretically derived electron density, ρ, obtained from standard electronic structure codes. Here, we introduce a factor that has not been carefully considered until now. As we demonstrate, theoretically derived ρ values depend not only on the set of computational conditions used to obtain them but also on the particular computational code selected for this task. We recommend that all quantum crystallographers carefully check the convergence of ρ before undertaking any serious study.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 295-306"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525002040
Michael Patzer , Christian W. Lehmann
This work presents a new iterative refinement method, comparable to Hirshfeld atom refinement, using the Hansen–Coppens multipole model charge density description to obtain accurate atomic coordinates and atomic displacements based on CRYSTAL17 periodic boundary calculations. The refinement, performed using the Python code ReCrystal, allows the user to explore the full periodic charge density in the crystalline solid state for charge density analysis of weak interactions.
A quantum crystallographic refinement methodology has been developed using theoretical multipole parameters generated directly from solid-state calculations using the CRYSTAL17 program. This refinement method is comparable to other transferable form factor approaches, such as the Invariom model, but in contrast to the Hirshfeld atom refinement, it uses theoretical multipole parameters to describe the electron density from a solid-state calculation performed with CRYSTAL17 in an iterative refinement procedure. For this purpose, a Python3 code named ReCrystal has been developed. To start ReCrystal, a CIF, a Gaussian basis set, a DFT functional and the number of CPUs must be defined. The Pack–Monkhorst and Gilat shrinking factors, which define a lattice in the first Brillouin zone, must also be specified. After k-point sampling, CRYSTAL17 calculates structure factors directly from the static electron density. Multipole parameters are generated from these structure factors using the XD program and are fixed during least-squares refinement. The refinement of the xylitol molecular crystal has shown that the hydrogen atom positions can be determined with reasonable agreement to those obtained in the neutron diffraction experiment. This indicates that the periodic boundary condition in ReCrystal is an improvement over gas phase refinement with HAR. The multipole parameters obtained from ReCrystal can be used for further charge density studies especially if weak interactions are the focus. In this work, we demonstrate the performance of ReCrystal on molecular crystals of the small molecules d/l-serine and xylitol with weak hydrogen-bonding motifs using multipole refinement. The advantage of this approach is that multipole parameters can be obtained from high-resolution calculated diffraction data, no database is required, and errors due to the model and errors resulting from the experiment are clearly separated.
{"title":"Solid-state calculations for iterative refinement in quantum crystallography using the multipole model","authors":"Michael Patzer , Christian W. Lehmann","doi":"10.1107/S2052252525002040","DOIUrl":"10.1107/S2052252525002040","url":null,"abstract":"<div><div>This work presents a new iterative refinement method, comparable to Hirshfeld atom refinement, using the Hansen–Coppens multipole model charge density description to obtain accurate atomic coordinates and atomic displacements based on <em>CRYSTAL17</em> periodic boundary calculations. The refinement, performed using the Python code <em>ReCrystal</em>, allows the user to explore the full periodic charge density in the crystalline solid state for charge density analysis of weak interactions.</div></div><div><div>A quantum crystallographic refinement methodology has been developed using theoretical multipole parameters generated directly from solid-state calculations using the <em>CRYSTAL17</em> program. This refinement method is comparable to other transferable form factor approaches, such as the Invariom model, but in contrast to the Hirshfeld atom refinement, it uses theoretical multipole parameters to describe the electron density from a solid-state calculation performed with <em>CRYSTAL17</em> in an iterative refinement procedure. For this purpose, a Python3 code named <em>ReCrystal</em> has been developed. To start <em>ReCrystal</em>, a CIF, a Gaussian basis set, a DFT functional and the number of CPUs must be defined. The Pack–Monkhorst and Gilat shrinking factors, which define a lattice in the first Brillouin zone, must also be specified. After <em>k</em>-point sampling, <em>CRYSTAL17</em> calculates structure factors directly from the static electron density. Multipole parameters are generated from these structure factors using the <em>XD</em> program and are fixed during least-squares refinement. The refinement of the xylitol molecular crystal has shown that the hydrogen atom positions can be determined with reasonable agreement to those obtained in the neutron diffraction experiment. This indicates that the periodic boundary condition in <em>ReCrystal</em> is an improvement over gas phase refinement with HAR. The multipole parameters obtained from <em>ReCrystal</em> can be used for further charge density studies especially if weak interactions are the focus. In this work, we demonstrate the performance of <em>ReCrystal</em> on molecular crystals of the small molecules <span>d</span>/<span>l</span>-serine and xylitol with weak hydrogen-bonding motifs using multipole refinement. The advantage of this approach is that multipole parameters can be obtained from high-resolution calculated diffraction data, no database is required, and errors due to the model and errors resulting from the experiment are clearly separated.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 322-333"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525003021
Xin Lu , Ming Yan , Yang Cai , Xi Song , Huan Chen , Mengtan Du , Zhenyi Wang , Jia’an Li , Liwen Niu , Fuxing Zeng , Quan Hao , Hongmin Zhang
This study introduces a modular scaffold strategy utilizing designed ankyrin-repeat proteins (DARPins) and a symmetric apoferritin base to overcome size limitations in single-particle cryo-EM, enabling near-atomic-resolution structural determination of medium-sized proteins like GFP and MBP. The high-symmetry, near-spherical scaffold not only resolves the common preferred-orientation challenges in single-particle cryo-EM but also reduces data-processing demands, offering a versatile platform for structural analysis of diverse proteins.
Single-particle cryo-electron microscopy (cryo-EM) has emerged as an indispensable technique in structural biology that is pivotal for deciphering protein architectures. However, the medium-sized proteins (30–40 kDa) that are prevalent in both eukaryotic and prokaryotic organisms often elude the resolving capabilities of contemporary cryo-EM methods. To address this challenge, we engineered a scaffold strategy that securely anchors proteins of interest to a robust, symmetric base via a selective adapter. Our most efficacious constructs, namely models 4 and 6c, feature a designed ankyrin-repeat protein (DARPin) rigidly linked to an octahedral human apoferritin via a helical linker. By utilizing these large, highly symmetric scaffolds (∼1 MDa), we achieved near-atomic-resolution cryo-EM structures of green fluorescent protein (GFP) and maltose-binding protein (MBP), revealing nearly all side-chain densities of GFP and the distinct structural features of MBP. The modular design of our scaffold allows the adaptation of new DARPins through minor amino-acid-sequence modifications, enabling the binding and visualization of a diverse array of proteins. The high symmetry and near-spherical shape of the scaffold not only mitigates the prevalent challenge of preferred particle orientation in cryo-EM but also significantly reduces the demands of image collection and data processing. This approach presents a versatile solution, breaking through the size constraints that have traditionally limited single-particle cryo-EM.
{"title":"A large, general and modular DARPin–apoferritin scaffold enables the visualization of small proteins by cryo-EM","authors":"Xin Lu , Ming Yan , Yang Cai , Xi Song , Huan Chen , Mengtan Du , Zhenyi Wang , Jia’an Li , Liwen Niu , Fuxing Zeng , Quan Hao , Hongmin Zhang","doi":"10.1107/S2052252525003021","DOIUrl":"10.1107/S2052252525003021","url":null,"abstract":"<div><div>This study introduces a modular scaffold strategy utilizing designed ankyrin-repeat proteins (DARPins) and a symmetric apoferritin base to overcome size limitations in single-particle cryo-EM, enabling near-atomic-resolution structural determination of medium-sized proteins like GFP and MBP. The high-symmetry, near-spherical scaffold not only resolves the common preferred-orientation challenges in single-particle cryo-EM but also reduces data-processing demands, offering a versatile platform for structural analysis of diverse proteins.</div></div><div><div>Single-particle cryo-electron microscopy (cryo-EM) has emerged as an indispensable technique in structural biology that is pivotal for deciphering protein architectures. However, the medium-sized proteins (30–40 kDa) that are prevalent in both eukaryotic and prokaryotic organisms often elude the resolving capabilities of contemporary cryo-EM methods. To address this challenge, we engineered a scaffold strategy that securely anchors proteins of interest to a robust, symmetric base via a selective adapter. Our most efficacious constructs, namely models 4 and 6c, feature a designed ankyrin-repeat protein (DARPin) rigidly linked to an octahedral human apoferritin via a helical linker. By utilizing these large, highly symmetric scaffolds (∼1 MDa), we achieved near-atomic-resolution cryo-EM structures of green fluorescent protein (GFP) and maltose-binding protein (MBP), revealing nearly all side-chain densities of GFP and the distinct structural features of MBP. The modular design of our scaffold allows the adaptation of new DARPins through minor amino-acid-sequence modifications, enabling the binding and visualization of a diverse array of proteins. The high symmetry and near-spherical shape of the scaffold not only mitigates the prevalent challenge of preferred particle orientation in cryo-EM but also significantly reduces the demands of image collection and data processing. This approach presents a versatile solution, breaking through the size constraints that have traditionally limited single-particle cryo-EM.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 393-402"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902094","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525002581
Roman Gajda , Michał Chodkiewicz , Dongzhou Zhang , Phuong Nguyen , Vitali Prakapenka , Krzysztof Wozniak
The crystal structure of cubo-ice (ice VII) has been established by single-crystal X-ray diffraction using both synchrotron and laboratory data collected at high pressure. X-ray diffraction data in both cases were refined with Hirshfeld atom refinement. Various structural models including those with ‘split’ positions of atoms were refined.
In the refinement of the crystal structures of ice, the best results obtained so far have been with neutron diffraction because the most troublemaking aspects are the hydrogen atoms. In nine out of twenty ice structures, the hydrogen atoms are disordered, which makes proper refinement more difficult. In our previous paper describing the structure of ice VI we proved that, using Hirshfeld atom refinement (HAR) based on synchrotron X-ray data, it is possible to obtain results comparable with those from neutron experiments. In this work, we investigate another structure of high-pressure disordered ice, cubo-ice (ice VII). Single crystals of cubo-ice were grown under pressure in diamond anvil cells. X-ray diffraction measurements were conducted at a synchrotron source facility (APS, University of Chicago, USA) as well as on our regular in-house laboratory diffractometer with Ag radiation. The data collected were further refined with HAR. Comparison of the structural parameters obtained with those derived from neutron diffraction showed very good agreement in terms of bond lengths and fairly good agreement in terms of hydrogen atom ADPs. We were also able to perform unconstrained refinements with various split-atom models.
{"title":"Structure of ice VII with Hirshfeld atom refinement","authors":"Roman Gajda , Michał Chodkiewicz , Dongzhou Zhang , Phuong Nguyen , Vitali Prakapenka , Krzysztof Wozniak","doi":"10.1107/S2052252525002581","DOIUrl":"10.1107/S2052252525002581","url":null,"abstract":"<div><div>The crystal structure of cubo-ice (ice VII) has been established by single-crystal X-ray diffraction using both synchrotron and laboratory data collected at high pressure. X-ray diffraction data in both cases were refined with Hirshfeld atom refinement. Various structural models including those with ‘split’ positions of atoms were refined.</div></div><div><div>In the refinement of the crystal structures of ice, the best results obtained so far have been with neutron diffraction because the most troublemaking aspects are the hydrogen atoms. In nine out of twenty ice structures, the hydrogen atoms are disordered, which makes proper refinement more difficult. In our previous paper describing the structure of ice VI we proved that, using Hirshfeld atom refinement (HAR) based on synchrotron X-ray data, it is possible to obtain results comparable with those from neutron experiments. In this work, we investigate another structure of high-pressure disordered ice, cubo-ice (ice VII). Single crystals of cubo-ice were grown under pressure in diamond anvil cells. X-ray diffraction measurements were conducted at a synchrotron source facility (APS, University of Chicago, USA) as well as on our regular in-house laboratory diffractometer with Ag radiation. The data collected were further refined with HAR. Comparison of the structural parameters obtained with those derived from neutron diffraction showed very good agreement in terms of bond lengths and fairly good agreement in terms of hydrogen atom ADPs. We were also able to perform unconstrained refinements with various split-atom models.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 288-294"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525001526
Yuriy Chushkin , Federico Zontone
A review of plane-wave coherent X-ray diffraction imaging in small-angle X-ray scattering geometry is presented, together with a discussion of the new opportunities offered by fourth-generation synchrotron sources.
Coherent X-ray diffraction imaging is a lens-less microscopy technique that emerged with the advent of third-generation synchrotrons, modern detectors and computers. It can image isolated micrometre-sized objects with a spatial resolution of a few nanometres. The method is based on the inversion of the speckle pattern in the far field produced by the scattering from the object under coherent illumination. The retrieval of the missing phase is performed using an iterative algorithm that numerically phases the amplitudes from the intensities of speckles measured with sufficient oversampling. Two- and three-dimensional imaging is obtained by simple inverse Fourier transform. This lens-less imaging technique has been applied to various specimens for their structural characterization on the nanoscale. Here, we review the theoretical and experimental elements of the technique, its achievements, and its limitations at third-generation synchrotrons. We also discuss the new opportunities offered by modern fourth-generation synchrotrons and outline the developments necessary to maximize the potential of the technique.
{"title":"Prospects for coherent X-ray diffraction imaging at fourth-generation synchrotron sources","authors":"Yuriy Chushkin , Federico Zontone","doi":"10.1107/S2052252525001526","DOIUrl":"10.1107/S2052252525001526","url":null,"abstract":"<div><div>A review of plane-wave coherent X-ray diffraction imaging in small-angle X-ray scattering geometry is presented, together with a discussion of the new opportunities offered by fourth-generation synchrotron sources.</div></div><div><div>Coherent X-ray diffraction imaging is a lens-less microscopy technique that emerged with the advent of third-generation synchrotrons, modern detectors and computers. It can image isolated micrometre-sized objects with a spatial resolution of a few nanometres. The method is based on the inversion of the speckle pattern in the far field produced by the scattering from the object under coherent illumination. The retrieval of the missing phase is performed using an iterative algorithm that numerically phases the amplitudes from the intensities of speckles measured with sufficient oversampling. Two- and three-dimensional imaging is obtained by simple inverse Fourier transform. This lens-less imaging technique has been applied to various specimens for their structural characterization on the nanoscale. Here, we review the theoretical and experimental elements of the technique, its achievements, and its limitations at third-generation synchrotrons. We also discuss the new opportunities offered by modern fourth-generation synchrotrons and outline the developments necessary to maximize the potential of the technique.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 280-287"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143624569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525003148
Alexander S. Novikov
The design of crystalline solids relies on understanding and controlling intermolecular and intramolecular interactions. Through theoretical charge density analysis and database mining, Shukla et al. [(2025). IUCrJ, 12, 334–357] have found a new way of viewing supramolecular assembly through the lens of electrostatic complementarity.
{"title":"Electrostatic landscapes in crystal engineering: a new perspective on synthons","authors":"Alexander S. Novikov","doi":"10.1107/S2052252525003148","DOIUrl":"10.1107/S2052252525003148","url":null,"abstract":"<div><div>The design of crystalline solids relies on understanding and controlling intermolecular and intramolecular interactions. Through theoretical charge density analysis and database mining, Shukla <em>et al.</em> [(2025). <em>IUCrJ</em>, <strong>12</strong>, 334–357] have found a new way of viewing supramolecular assembly through the lens of electrostatic complementarity.</div></div>","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 255-256"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-01DOI: 10.1107/S2052252525002106
Santosh Panjikar , Manfred S. Weiss
<div><div>High-resolution crystal structures reveal that peptide bonds in α-helices exhibit a slightly more pronounced enol-like character than those in β-strands. This can go as far as peptide oxygen atoms in protein structures being protonated.</div></div><div><div>Understanding the structural and chemical properties of peptide bonds within protein secondary structures is vital for elucidating their roles in protein folding, stability and function. This study examines the distinct characteristics of peptide bonds in α-helices and β-strands using a nonredundant data set comprising 1024 high-resolution protein crystal structures from the Protein Data Bank (PDB). The analysis reveals surprising and intriguing insights into bond lengths, angles, dihedral angles, electron-density distributions and hydrogen bonding within α-helices and β-strands. While the respective bond lengths (CN and CO) do not differ much between helices and strands, the bond angles (∠CNC<sub>α</sub> and ∠OCN) are significantly larger in strands compared with helices. Furthermore, the peptide dihedral angle (ω) in helices clusters around 180° and follows a sharp Gaussian distribution with a standard deviation of 4.1°. In contrast, the distribution of dihedral angles in strands spans a much wider range, with a more flattened Gaussian peak around 180°. This distinct difference in the distribution of dihedral angles reflects the unique structural characteristics of helices and strands, highlighting their respective conformational preferences. Additionally, if the ratio of the electron-density values (2<em>mF</em><sub>o</sub> − <em>DF</em><sub>c</sub>) at the midpoint of the CO bond and of the CN bond is calculated, a skewed distribution is observed, with the ratio being lower for helices than for strands. Moreover, higher normalized mean atomic displacement parameters (ADPs) for peptide atoms in helices relative to strands suggest increased flexibility or a more dynamic structure within helical regions. Analysis of hydrogen-bond distances between O and N atoms of the main chain reveals larger distances in helices compared with strands, indicative of distinct hydrogen-bonding patterns associated with different secondary structures. All of these observations taken together led us to conclude that peptide bonds in α-helices are different from peptide bonds in β-strands. Overall, α-helical peptide bonds seem to display a more enol-like character. This suggests that peptide oxygen atoms in helices are more likely to be protonated. These findings have several important implications for refining protein structures, particularly in regions susceptible to enol-like transitions or protonation. By recognizing the distinct bond-angle and bond-length variations associated with protonated carbonyl oxygen atoms, current refinement protocols can be adapted to apply more flexible restraints in these regions. This could improve the accuracy of modelling local geometries, where protonation or enol fo
{"title":"Peptide bonds revisited","authors":"Santosh Panjikar , Manfred S. Weiss","doi":"10.1107/S2052252525002106","DOIUrl":"10.1107/S2052252525002106","url":null,"abstract":"<div><div>High-resolution crystal structures reveal that peptide bonds in α-helices exhibit a slightly more pronounced enol-like character than those in β-strands. This can go as far as peptide oxygen atoms in protein structures being protonated.</div></div><div><div>Understanding the structural and chemical properties of peptide bonds within protein secondary structures is vital for elucidating their roles in protein folding, stability and function. This study examines the distinct characteristics of peptide bonds in α-helices and β-strands using a nonredundant data set comprising 1024 high-resolution protein crystal structures from the Protein Data Bank (PDB). The analysis reveals surprising and intriguing insights into bond lengths, angles, dihedral angles, electron-density distributions and hydrogen bonding within α-helices and β-strands. While the respective bond lengths (CN and CO) do not differ much between helices and strands, the bond angles (∠CNC<sub>α</sub> and ∠OCN) are significantly larger in strands compared with helices. Furthermore, the peptide dihedral angle (ω) in helices clusters around 180° and follows a sharp Gaussian distribution with a standard deviation of 4.1°. In contrast, the distribution of dihedral angles in strands spans a much wider range, with a more flattened Gaussian peak around 180°. This distinct difference in the distribution of dihedral angles reflects the unique structural characteristics of helices and strands, highlighting their respective conformational preferences. Additionally, if the ratio of the electron-density values (2<em>mF</em><sub>o</sub> − <em>DF</em><sub>c</sub>) at the midpoint of the CO bond and of the CN bond is calculated, a skewed distribution is observed, with the ratio being lower for helices than for strands. Moreover, higher normalized mean atomic displacement parameters (ADPs) for peptide atoms in helices relative to strands suggest increased flexibility or a more dynamic structure within helical regions. Analysis of hydrogen-bond distances between O and N atoms of the main chain reveals larger distances in helices compared with strands, indicative of distinct hydrogen-bonding patterns associated with different secondary structures. All of these observations taken together led us to conclude that peptide bonds in α-helices are different from peptide bonds in β-strands. Overall, α-helical peptide bonds seem to display a more enol-like character. This suggests that peptide oxygen atoms in helices are more likely to be protonated. These findings have several important implications for refining protein structures, particularly in regions susceptible to enol-like transitions or protonation. By recognizing the distinct bond-angle and bond-length variations associated with protonated carbonyl oxygen atoms, current refinement protocols can be adapted to apply more flexible restraints in these regions. This could improve the accuracy of modelling local geometries, where protonation or enol fo","PeriodicalId":14775,"journal":{"name":"IUCrJ","volume":"12 3","pages":"Pages 307-321"},"PeriodicalIF":2.9,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143752970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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