Open-access and free articles in Acta Crystallographica Section D: Structural Biology
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Acta Crystallographica Section D: Structural Biology welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function articles that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such articles should present new insights into biology, chemistry or structure. Articles on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Articles on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability.en-gbCopyright (c) 2024 International Union of CrystallographyInternational Union of CrystallographyInternational Union of CrystallographytextActa Crystallographica Section D: Structural Biology welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function articles that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such articles should present new insights into biology, chemistry or structure. Articles on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Articles on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability.Open-access and free articles in Acta Crystallographica Section D Structural Biologyhttps://journals.iucr.orgurn:issn:0907-4449text/html2002-01-01T00:00+00:00monthly1Acta Crystallographica Section D Structural BiologyCopyright (c) 2024 International Union of Crystallographyurn:issn:0907-4449med@iucr.orgOpen-access and free articles in Acta Crystallographica Section D: Structural Biologyhttp://journals.iucr.org/logos/rss10d.gif
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Still imageAdvanced exploitation of unmerged reflection data during processing and refinement with autoPROC and BUSTER
http://scripts.iucr.org/cgi-bin/paper?qu5003
The validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) − F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.UNMERGED REFLECTION DATA; FITNESS ANALYSIS; REFLECTION AUDITING; VALIDATION AGAINST DIFFRACTION DATA; RADIATION DAMAGEtextVonrhein, C.Flensburg, C.Keller, P.Fogh, R.Sharff, A.Tickle, I.J.Bricogne, G.2024-02-27The final models for macromolecular X-ray crystallography studies are usually not only the result of refinement against some version of scaled and merged reflection data, but are often also analysed and validated purely against such merged data. Here, various examples are presented to show how the availability and use of unmerged reflection data can lead to better model analysis and improved model parametrization, as well as providing a path to better data processing and scaling.International Union of CrystallographyThe validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) − F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.Advanced exploitation of unmerged reflection data during processing and refinement with autoPROC and BUSTERurn:issn:2059-7983doi:10.1107/S2059798324001487https://creativecommons.org/licenses/by/4.0/text/htmlen2024-02-27https://creativecommons.org/licenses/by/4.0/801482059-79833med@iucr.org158March 2024Acta Crystallographica Section D: Structural Biologyresearch papers2059-7983Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii
http://scripts.iucr.org/cgi-bin/paper?jb5061
Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.MEVALONATE KINASES; GHMP ENZYME SUPERFAMILY; FEEDBACK INHIBITION; RAMAZZOTTIUS VARIEORNATUS; METHANOCOCCOIDES BURTONIItextEsquirol, L.Newman, J.Nebl, T.Scott, C.Vickers, C.Sainsbury, F.Peat, T.S.2024-02-27This work reports the purification, biochemical characterization and high-resolution structures of two novel mevalonate kinases: one from the extremotolerant tardigrade Ramazzottius varieornatus at 2 Å resolution and one from the psychrophilic archaeon Methanococcoides burtonii at 2.2 Å resolution.International Union of CrystallographyMevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtoniiurn:issn:2059-7983doi:10.1107/S2059798324001360https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983203med@iucr.org32152024-02-2780https://creativecommons.org/licenses/by/4.0/research papers2059-7983March 2024Acta Crystallographica Section D: Structural BiologyThe crystal structure of mycothiol disulfide reductase (Mtr) provides mechanistic insight into the specific low-molecular-weight thiol reductase activity of Actinobacteria
http://scripts.iucr.org/cgi-bin/paper?gi5042
Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reductases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future antituberculosis drug development.LOW-MOLECULAR-WEIGHT THIOLS; MYCOTHIOL DISULFIDE REDUCTASE; OXIDOREDUCTASES; REDOX HOMEOSTASIS; FLAVOENZYMES; X-RAY CRYSTALLOGRAPHY; DOCKING; PROTEIN STRUCTURE; ACTINOBACTERIAtextGutiérrez-Fernández, J.Hersleth, H.-P.Hammerstad, M.2024-02-19The crystal structure of mycothiol disulfide reductase (Mtr) was determined for the first time. The structure shows a highly conserved and enlarged substrate-binding pocket, providing insight into the substrate-binding mode and specificity of Mtrs.International Union of CrystallographyLow-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reductases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future antituberculosis drug development.The crystal structure of mycothiol disulfide reductase (Mtr) provides mechanistic insight into the specific low-molecular-weight thiol reductase activity of Actinobacteriaurn:issn:2059-7983doi:10.1107/S205979832400113Xhttps://creativecommons.org/licenses/by/4.0/text/htmlenMarch 2024Acta Crystallographica Section D: Structural Biology2059-7983research papers2024-02-1980https://creativecommons.org/licenses/by/4.0/med@iucr.org32059-7983181193A service-based approach to cryoEM facility processing pipelines at eBIC
http://scripts.iucr.org/cgi-bin/paper?ic5123
Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.CRYOEM FACILITIES; EBIC; PROCESSING PIPELINES; CLOUD COMPUTINGtextHorstmann, A.Riggs, S.Chaban, Y.Clare, D.K.de Freitas, G.Farmer, D.Howe, A.Morris, K.L.Hatton, D.2024-02-20The automation of cryoEM pipelines to aid data-quality analysis during acquisition poses a number of challenges, particularly at facilities where the expected data rates are high. The use of a modern service-based architecture deployed on Diamond Light Source's on-premises cloud computing cluster to tackle some of these challenges is demonstrated, focusing on the provision of an early-stage processing pipeline for electron tomography that produces first-attempt reconstructed volumes within minutes of tilt-series acquisition.International Union of CrystallographyElectron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.A service-based approach to cryoEM facility processing pipelines at eBICurn:issn:2059-7983doi:10.1107/S2059798324000986https://creativecommons.org/licenses/by/4.0/text/htmlenhttps://creativecommons.org/licenses/by/4.0/802024-02-201801742059-79833med@iucr.orgActa Crystallographica Section D: Structural BiologyMarch 2024research papers2059-7983Using cryo-EM to understand the assembly pathway of respiratory complex I
http://scripts.iucr.org/cgi-bin/paper?ih5004
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.SINGLE-PARTICLE CRYO-EM; RESPIRATORY COMPLEX I; PROTON-PUMPING NADH UBIQUINONE OXIDOREDUCTASE; COMPLEX I ASSEMBLY; ASSEMBLY FACTORStextLaube, E.Schiller, J.Zickermann, V.Vonck, J.2024-02-19Single-particle cryo-EM is a powerful technique to study the assembly process of the largest mitochondrial respiratory chain complex, NADH:ubiquinone oxidoreductase or complex I. Here, the new insights that have been gained into the molecular functions of assembly factors are reviewed.International Union of CrystallographyComplex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.Using cryo-EM to understand the assembly pathway of respiratory complex Iurn:issn:2059-7983doi:10.1107/S205979832400086Xhttps://creativecommons.org/licenses/by/4.0/text/htmlenMarch 2024Acta Crystallographica Section D: Structural Biologyresearch papers2059-79832024-02-1980https://creativecommons.org/licenses/by/4.0/2059-7983159med@iucr.org3173Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket
http://scripts.iucr.org/cgi-bin/paper?ud5050
To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.3CLPRO; SARS-COV-2; FRAGMENT SCREENING; COVALENT BINDERS; SURFACE PLASMON RESONANCE; X-RAY CRYSTALLOGRAPHYtextHuang, C.-Y.Metz, A.Lange, R.Artico, N.Potot, C.Hazemann, J.Müller, M.Dos Santos, M.Chambovey, A.Ritz, D.Eris, D.Meyer, S.Bourquin, G.Sharpe, M.Mac Sweeney, A.2024-01-30X-ray crystallographic screening of SARS-CoV-2 3CL protease resulted in 29 fragment hits, including two isatin-based reversible covalent binders, and revealed a strong influence of the crystal form used for fragment soaking on the bound conformations of three additional reference fragments.International Union of CrystallographyTo identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocketurn:issn:2059-7983doi:10.1107/S2059798324000329https://creativecommons.org/licenses/by/4.0/text/htmlen80https://creativecommons.org/licenses/by/4.0/2024-01-301362059-7983123med@iucr.org2Acta Crystallographica Section D: Structural BiologyFebruary 2024research papers2059-7983Structural flexibility of Toscana virus nucleoprotein in the presence of a single-chain camelid antibody
http://scripts.iucr.org/cgi-bin/paper?jc5062
Phenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.BUNYAVIRALES; TOSCANA VIRUS; NUCLEOPROTEIN FLEXIBILITYtextPapageorgiou, N.Baklouti, A.Lichière, J.Desmyter, A.Canard, B.Coutard, B.Ferron, F.2024-01-24Structural rearrangement of the Toscana virus nucleoprotein is induced by a single-chain camelid antibody.International Union of CrystallographyPhenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.Structural flexibility of Toscana virus nucleoprotein in the presence of a single-chain camelid antibodyurn:issn:2059-7983doi:10.1107/S2059798324000196https://creativecommons.org/licenses/by/4.0/text/htmlenFebruary 2024Acta Crystallographica Section D: Structural Biology2059-7983research papers2024-01-24https://creativecommons.org/licenses/by/4.0/802med@iucr.org1132059-7983122Investigation of how gate residues in the main channel affect the catalytic activity of Scytalidium thermophilum catalase
http://scripts.iucr.org/cgi-bin/paper?gm5099
Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.CATALASES; PHENOL OXIDASES; MAIN CHANNEL GATE RESIDUES; OXIDOREDUCTASES; SCYTALIDIUM THERMOPHILUM; CATALASE VARIANTStextYuzugullu Karakus, Y.Goc, G.Zengin Karatas, M.Balci Unver, S.Yorke, B.A.Pearson, A.R.2024-01-24Scytalidium thermophilum produces a catalase enzyme that is capable of oxidizing o-diphenolic and some p-diphenolic compounds in the absence of hydrogen peroxide. To better understand the role of the main channel in phenol oxidase activity, the gate residues Glu484 and Thr188 in the upper part of the main channel were investigated in a combined kinetic, spectroscopic and structural study.International Union of CrystallographyCatalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.Investigation of how gate residues in the main channel affect the catalytic activity of Scytalidium thermophilum catalaseurn:issn:2059-7983doi:10.1107/S2059798323011063https://creativecommons.org/licenses/by/4.0/text/htmlen1122059-7983101med@iucr.org280https://creativecommons.org/licenses/by/4.0/2024-01-24research papers2059-7983Acta Crystallographica Section D: Structural BiologyFebruary 2024From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotrons
http://scripts.iucr.org/cgi-bin/paper?qu5005
Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump–probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.TIME-RESOLVED SERIAL CRYSTALLOGRAPHY; SYNCHROTRONS; XFELS; STRUCTURAL PHOTOBIOLOGY; REACTION-INTERMEDIATE STATES; BACTERIORHODOPSIN; CRYO-TRAPPINGtextCaramello, N.Royant, A.2024-01-24This review constitutes an overview of the current status of time-resolved crystallography performed at synchrotrons and XFELs on timescales ranging from femtoseconds to minutes. Methods, potential biases, instruments and examples are presented and compared with those for the cryo-trapping of reaction-intermediate states.International Union of CrystallographyOver the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump–probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotronsurn:issn:2059-7983doi:10.1107/S2059798323011002https://creativecommons.org/licenses/by/4.0/text/htmlen79med@iucr.org22059-79836080https://creativecommons.org/licenses/by/4.0/2024-01-242059-7983feature articlesActa Crystallographica Section D: Structural BiologyFebruary 2024A web-based dashboard for RELION metadata visualization
http://scripts.iucr.org/cgi-bin/paper?ic5122
Cryo-electron microscopy (cryo-EM) has witnessed radical progress in the past decade, driven by developments in hardware and software. While current software packages include processing pipelines that simplify the image-processing workflow, they do not prioritize the in-depth analysis of crucial metadata, limiting troubleshooting for challenging data sets. The widely used RELION software package lacks a graphical native representation of the underlying metadata. Here, two web-based tools are introduced: relion_live.py, which offers real-time feedback on data collection, aiding swift decision-making during data acquisition, and relion_analyse.py, a graphical interface to represent RELION projects by plotting essential metadata including interactive data filtration and analysis. A useful script for estimating ice thickness and data quality during movie pre-processing is also presented. These tools empower researchers to analyse data efficiently and allow informed decisions during data collection and processing.CRYO-ELECTRON MICROSCOPY; RELION; ICE THICKNESS ESTIMATION; GRAPHICAL USER INTERFACE; WEB-BASED CRYO-EM TOOLStextGonzález-Rodríguez, N.Areán-Ulloa, E.Fernández-Leiro, R.2024-01-24relion_live.py and relion_analyse.py, two web-based tools to enhance cryo-EM data processing in RELION, are introduced, providing an interface for real-time feedback on data collection and simplified interpretation of metadata. Additionally, an analytical script for ice-quality estimation is provided, empowering researchers to make informed decisions to improve data quality and accessibility in cryo-EM.International Union of CrystallographyCryo-electron microscopy (cryo-EM) has witnessed radical progress in the past decade, driven by developments in hardware and software. While current software packages include processing pipelines that simplify the image-processing workflow, they do not prioritize the in-depth analysis of crucial metadata, limiting troubleshooting for challenging data sets. The widely used RELION software package lacks a graphical native representation of the underlying metadata. Here, two web-based tools are introduced: relion_live.py, which offers real-time feedback on data collection, aiding swift decision-making during data acquisition, and relion_analyse.py, a graphical interface to represent RELION projects by plotting essential metadata including interactive data filtration and analysis. A useful script for estimating ice thickness and data quality during movie pre-processing is also presented. These tools empower researchers to analyse data efficiently and allow informed decisions during data collection and processing.A web-based dashboard for RELION metadata visualizationurn:issn:2059-7983doi:10.1107/S2059798323010902https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersFebruary 2024Acta Crystallographica Section D: Structural Biology2med@iucr.org932059-79831002024-01-24https://creativecommons.org/licenses/by/4.0/80The High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX), an ancillary tool for the macromolecular crystallography beamlines at the ESRF
http://scripts.iucr.org/cgi-bin/paper?qi5004
This article describes the High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX) at the ESRF, and highlights new and complementary research opportunities that can be explored using this facility. The laboratory is dedicated to investigating interactions between macromolecules and gases in crystallo, and finds applications in many fields of research, including fundamental biology, biochemistry, and environmental and medical science. At present, the HPMX laboratory offers the use of different high-pressure cells adapted for helium, argon, krypton, xenon, nitrogen, oxygen, carbon dioxide and methane. Important scientific applications of high pressure to macromolecules at the HPMX include noble-gas derivatization of crystals to detect and map the internal architecture of proteins (pockets, tunnels and channels) that allows the storage and diffusion of ligands or substrates/products, the investigation of the catalytic mechanisms of gas-employing enzymes (using oxygen, carbon dioxide or methane as substrates) to possibly decipher intermediates, and studies of the conformational fluctuations or structure modifications that are necessary for proteins to function. Additionally, cryo-cooling protein crystals under high pressure (helium or argon at 2000 bar) enables the addition of cryo-protectant to be avoided and noble gases can be employed to produce derivatives for structure resolution. The high-pressure systems are designed to process crystals along a well defined pathway in the phase diagram (pressure–temperature) of the gas to cryo-cool the samples according to the three-step `soak-and-freeze method'. Firstly, crystals are soaked in a pressurized pure gas atmosphere (at 294 K) to introduce the gas and facilitate its interactions within the macromolecules. Samples are then flash-cooled (at 100 K) while still under pressure to cryo-trap macromolecule–gas complexation states or pressure-induced protein modifications. Finally, the samples are recovered after depressurization at cryo-temperatures. The final section of this publication presents a selection of different typical high-pressure experiments carried out at the HPMX, showing that this technique has already answered a wide range of scientific questions. It is shown that the use of different gases and pressure conditions can be used to probe various effects, such as mapping the functional internal architectures of enzymes (tunnels in the haloalkane dehalogenase DhaA) and allosteric sites on membrane-protein surfaces, the interaction of non-inert gases with proteins (oxygen in the hydrogenase ReMBH) and pressure-induced structural changes of proteins (tetramer dissociation in urate oxidase). The technique is versatile and the provision of pressure cells and their application at the HPMX is gradually being extended to address new scientific questions.HIGH PRESSURE; MACROMOLECULAR CRYSTALS; GAS DERIVATIVES; PROTEIN CHANNELS; HPMX; HIGH-PRESSURE FREEZING LABORATORY FOR MACROMOLECULAR CRYSTALLOGRAPHY; ESRFtextCarpentier, P.van der Linden, P.Mueller-Dieckmann, C.2024-01-24The High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX) at the ESRF allows the preparation of gas derivatives of macromolecular crystals suitable for X-ray diffraction data collection on macromolecular crystallography beamlines. Information obtained from pressurized crystals and/or gas-derivatized structures enables the improved understanding of specific issues in structural biology, such as the internal functional architecture of proteins, the interactions and reactivity of gases with macromolecules and functional structural changes including ligand-binding processes.International Union of CrystallographyThis article describes the High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX) at the ESRF, and highlights new and complementary research opportunities that can be explored using this facility. The laboratory is dedicated to investigating interactions between macromolecules and gases in crystallo, and finds applications in many fields of research, including fundamental biology, biochemistry, and environmental and medical science. At present, the HPMX laboratory offers the use of different high-pressure cells adapted for helium, argon, krypton, xenon, nitrogen, oxygen, carbon dioxide and methane. Important scientific applications of high pressure to macromolecules at the HPMX include noble-gas derivatization of crystals to detect and map the internal architecture of proteins (pockets, tunnels and channels) that allows the storage and diffusion of ligands or substrates/products, the investigation of the catalytic mechanisms of gas-employing enzymes (using oxygen, carbon dioxide or methane as substrates) to possibly decipher intermediates, and studies of the conformational fluctuations or structure modifications that are necessary for proteins to function. Additionally, cryo-cooling protein crystals under high pressure (helium or argon at 2000 bar) enables the addition of cryo-protectant to be avoided and noble gases can be employed to produce derivatives for structure resolution. The high-pressure systems are designed to process crystals along a well defined pathway in the phase diagram (pressure–temperature) of the gas to cryo-cool the samples according to the three-step `soak-and-freeze method'. Firstly, crystals are soaked in a pressurized pure gas atmosphere (at 294 K) to introduce the gas and facilitate its interactions within the macromolecules. Samples are then flash-cooled (at 100 K) while still under pressure to cryo-trap macromolecule–gas complexation states or pressure-induced protein modifications. Finally, the samples are recovered after depressurization at cryo-temperatures. The final section of this publication presents a selection of different typical high-pressure experiments carried out at the HPMX, showing that this technique has already answered a wide range of scientific questions. It is shown that the use of different gases and pressure conditions can be used to probe various effects, such as mapping the functional internal architectures of enzymes (tunnels in the haloalkane dehalogenase DhaA) and allosteric sites on membrane-protein surfaces, the interaction of non-inert gases with proteins (oxygen in the hydrogenase ReMBH) and pressure-induced structural changes of proteins (tetramer dissociation in urate oxidase). The technique is versatile and the provision of pressure cells and their application at the HPMX is gradually being extended to address new scientific questions.The High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX), an ancillary tool for the macromolecular crystallography beamlines at the ESRFurn:issn:2059-7983doi:10.1107/S2059798323010707https://creativecommons.org/licenses/by/4.0/text/htmlenhttps://creativecommons.org/licenses/by/4.0/802024-01-24922med@iucr.org802059-7983Acta Crystallographica Section D: Structural BiologyFebruary 20242059-7983research papersDeep residual networks for crystallography trained on synthetic data
http://scripts.iucr.org/cgi-bin/paper?qi5003
The use of artificial intelligence to process diffraction images is challenged by the need to assemble large and precisely designed training data sets. To address this, a codebase called Resonet was developed for synthesizing diffraction data and training residual neural networks on these data. Here, two per-pattern capabilities of Resonet are demonstrated: (i) interpretation of crystal resolution and (ii) identification of overlapping lattices. Resonet was tested across a compilation of diffraction images from synchrotron experiments and X-ray free-electron laser experiments. Crucially, these models readily execute on graphics processing units and can thus significantly outperform conventional algorithms. While Resonet is currently utilized to provide real-time feedback for macromolecular crystallography users at the Stanford Synchrotron Radiation Lightsource, its simple Python-based interface makes it easy to embed in other processing frameworks. This work highlights the utility of physics-based simulation for training deep neural networks and lays the groundwork for the development of additional models to enhance diffraction collection and analysis.ARTIFICIAL INTELLIGENCE; SERIAL CRYSTALLOGRAPHY; ROTATION CRYSTALLOGRAPHY; SYNCHROTRONS; XFELStextMendez, D.Holton, J.M.Lyubimov, A.Y.Hollatz, S.Mathews, I.I.Cichosz, A.Martirosyan, V.Zeng, T.Stofer, R.Liu, R.Song, J.McPhillips, S.Soltis, M.Cohen, A.E.2024-01-01Artificial intelligence was used to characterize the diffraction in images from serial and rotation crystallography experiments. Forward simulations were used to train models to infer B factors, resolutions and the presence of crystal splitting from single diffraction images.International Union of CrystallographyThe use of artificial intelligence to process diffraction images is challenged by the need to assemble large and precisely designed training data sets. To address this, a codebase called Resonet was developed for synthesizing diffraction data and training residual neural networks on these data. Here, two per-pattern capabilities of Resonet are demonstrated: (i) interpretation of crystal resolution and (ii) identification of overlapping lattices. Resonet was tested across a compilation of diffraction images from synchrotron experiments and X-ray free-electron laser experiments. Crucially, these models readily execute on graphics processing units and can thus significantly outperform conventional algorithms. While Resonet is currently utilized to provide real-time feedback for macromolecular crystallography users at the Stanford Synchrotron Radiation Lightsource, its simple Python-based interface makes it easy to embed in other processing frameworks. This work highlights the utility of physics-based simulation for training deep neural networks and lays the groundwork for the development of additional models to enhance diffraction collection and analysis.Deep residual networks for crystallography trained on synthetic dataurn:issn:2059-7983doi:10.1107/S2059798323010586https://creativecommons.org/licenses/by/4.0/text/htmlenActa Crystallographica Section D: Structural BiologyJanuary 2024research papers2059-7983https://creativecommons.org/licenses/by/4.0/802024-01-0143262059-79831med@iucr.orgThe TR-icOS setup at the ESRF: time-resolved microsecond UV–Vis absorption spectroscopy on protein crystals
http://scripts.iucr.org/cgi-bin/paper?qi5005
The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump–probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump–probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm−2, providing experimental laser and delay parameters for a successful TR-MX experiment.PUMP-PROBE SPECTROSCOPY; PHOTOACTIVABLE PROTEINS; IN CRYSTALLO OPTICAL SPECTROSCOPY; BACTERIORHODOPSIN; SERIAL SYNCHROTRON CRYSTALLOGRAPHYtextEngilberge, S.Caramello, N.Bukhdruker, S.Byrdin, M.Giraud, T.Jacquet, P.Scortani, D.Biv, R.Gonzalez, H.Broquet, A.van der Linden, P.Rose, S.L.Flot, D.Balandin, T.Gordeliy, V.Lahey-Rudolph, J.M.Roessle, M.de Sanctis, D.Leonard, G.A.Mueller-Dieckmann, C.Royant, A.2024-01-01A setup to measure time-resolved UV–Vis absorption spectra from small protein crystals using a pump–probe scheme with microsecond-to-second delays is described.International Union of CrystallographyThe technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump–probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump–probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm−2, providing experimental laser and delay parameters for a successful TR-MX experiment.The TR-icOS setup at the ESRF: time-resolved microsecond UV–Vis absorption spectroscopy on protein crystalsurn:issn:2059-7983doi:10.1107/S2059798323010483https://creativecommons.org/licenses/by/4.0/text/htmlen162059-79831med@iucr.org252024-01-01https://creativecommons.org/licenses/by/4.0/80research papers2059-7983January 2024Acta Crystallographica Section D: Structural BiologyModes and model building in SHELXE
http://scripts.iucr.org/cgi-bin/paper?qu5004
Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson–Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.MODEL BUILDING; PHASING; DENSITY MODIFICATION; MRSAD; SHELXEtextUsón, I.Sheldrick, G.M.2024-01-01All alternative SHELXE modes, using single or combined sources of starting phase information, are described. Side-chain tracing now completes model building in SHELXE to enhance density modification.International Union of CrystallographyDensity modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson–Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.Modes and model building in SHELXEurn:issn:2059-7983doi:10.1107/S2059798323010082https://creativecommons.org/licenses/by/4.0/text/htmlen2024-01-0180https://creativecommons.org/licenses/by/4.0/2059-79834med@iucr.org115January 2024Acta Crystallographica Section D: Structural Biologyresearch papers2059-7983Using graphlet degree vectors to predict atomic displacement parameters in protein structures
http://scripts.iucr.org/cgi-bin/paper?di5068
In structural biology, atomic displacement parameters, commonly used in the form of B values, describe uncertainties in atomic positions. Their distribution over the structure can provide hints on local structural reliability and mobility. A spatial macromolecular model can be represented by a graph whose nodes are atoms and whose edges correspond to all interatomic contacts within a certain distance. Small connected subgraphs, called graphlets, provide information about the wiring of a particular atom. The multiple linear regression approach based on this information aims to predict a distribution of values of isotropic atomic displacement parameters (B values) within a protein structure, given the atomic coordinates and molecular packing. By modeling the dynamic component of atomic uncertainties, this method allows the B values obtained from experimental crystallographic or cryo-electron microscopy studies to be reproduced relatively well.ATOMIC DISPLACEMENT PARAMETERS; GRAPHLET DEGREE VECTORS; INTERATOMIC CONTACTS; MACROMOLECULEStextPražnikar, J.2023-11-21The components of the graphlet degree vector, which describes the complexity of the wiring of a given atom, can be used in a multiple linear regression model to predict atomic displacement parameters in protein structures.International Union of CrystallographyIn structural biology, atomic displacement parameters, commonly used in the form of B values, describe uncertainties in atomic positions. Their distribution over the structure can provide hints on local structural reliability and mobility. A spatial macromolecular model can be represented by a graph whose nodes are atoms and whose edges correspond to all interatomic contacts within a certain distance. Small connected subgraphs, called graphlets, provide information about the wiring of a particular atom. The multiple linear regression approach based on this information aims to predict a distribution of values of isotropic atomic displacement parameters (B values) within a protein structure, given the atomic coordinates and molecular packing. By modeling the dynamic component of atomic uncertainties, this method allows the B values obtained from experimental crystallographic or cryo-electron microscopy studies to be reproduced relatively well.Using graphlet degree vectors to predict atomic displacement parameters in protein structuresurn:issn:2059-7983doi:10.1107/S2059798323009142https://creativecommons.org/licenses/by/4.0/text/htmlen11092059-798312med@iucr.org11192023-11-21https://creativecommons.org/licenses/by/4.0/79research papers2059-7983December 2023Acta Crystallographica Section D: Structural BiologyImproved joint X-ray and neutron refinement procedure in Phenix
http://scripts.iucr.org/cgi-bin/paper?qo5007
Neutron diffraction is one of the three crystallographic techniques (X-ray, neutron and electron diffraction) used to determine the atomic structures of molecules. Its particular strengths derive from the fact that H (and D) atoms are strong neutron scatterers, meaning that their positions, and thus protonation states, can be derived from crystallographic maps. However, because of technical limitations and experimental obstacles, the quality of neutron diffraction data is typically much poorer (completeness, resolution and signal to noise) than that of X-ray diffraction data for the same sample. Further, refinement is more complex as it usually requires additional parameters to describe the H (and D) atoms. The increase in the number of parameters may be mitigated by using the `riding hydrogen' refinement strategy, in which the positions of H atoms without a rotational degree of freedom are inferred from their neighboring heavy atoms. However, this does not address the issues related to poor data quality. Therefore, neutron structure determination often relies on the presence of an X-ray data set for joint X-ray and neutron (XN) refinement. In this approach, the X-ray data serve to compensate for the deficiencies of the neutron diffraction data by refining one model simultaneously against the X-ray and neutron data sets. To be applicable, it is assumed that both data sets are highly isomorphous, and preferably collected from the same crystals and at the same temperature. However, the approach has a number of limitations that are discussed in this work by comparing four separately re-refined neutron models. To address the limitations, a new method for joint XN refinement is introduced that optimizes two different models against the different data sets. This approach is tested using neutron models and data deposited in the Protein Data Bank. The efficacy of refining models with H atoms as riding or as individual atoms is also investigated.MACROMOLECULAR CRYSTALLOGRAPHY; NEUTRON DIFFRACTION; JOINT XN REFINEMENTtextLiebschner, D.Afonine, P.V.Poon, B.K.Moriarty, N.W.Adams, P.D.2023-11-09The improved joint X-ray and neutron refinement procedure in Phenix optimizes two different models against the X-ray and neutron data sets. This approach is shown to reduce overfitting compared with refining the models separately.International Union of CrystallographyNeutron diffraction is one of the three crystallographic techniques (X-ray, neutron and electron diffraction) used to determine the atomic structures of molecules. Its particular strengths derive from the fact that H (and D) atoms are strong neutron scatterers, meaning that their positions, and thus protonation states, can be derived from crystallographic maps. However, because of technical limitations and experimental obstacles, the quality of neutron diffraction data is typically much poorer (completeness, resolution and signal to noise) than that of X-ray diffraction data for the same sample. Further, refinement is more complex as it usually requires additional parameters to describe the H (and D) atoms. The increase in the number of parameters may be mitigated by using the `riding hydrogen' refinement strategy, in which the positions of H atoms without a rotational degree of freedom are inferred from their neighboring heavy atoms. However, this does not address the issues related to poor data quality. Therefore, neutron structure determination often relies on the presence of an X-ray data set for joint X-ray and neutron (XN) refinement. In this approach, the X-ray data serve to compensate for the deficiencies of the neutron diffraction data by refining one model simultaneously against the X-ray and neutron data sets. To be applicable, it is assumed that both data sets are highly isomorphous, and preferably collected from the same crystals and at the same temperature. However, the approach has a number of limitations that are discussed in this work by comparing four separately re-refined neutron models. To address the limitations, a new method for joint XN refinement is introduced that optimizes two different models against the different data sets. This approach is tested using neutron models and data deposited in the Protein Data Bank. The efficacy of refining models with H atoms as riding or as individual atoms is also investigated.Improved joint X-ray and neutron refinement procedure in Phenixurn:issn:2059-7983doi:10.1107/S2059798323008914https://creativecommons.org/licenses/by/4.0/text/htmlenActa Crystallographica Section D: Structural BiologyDecember 20232059-7983research papers79https://creativecommons.org/licenses/by/4.0/2023-11-091093med@iucr.org122059-79831079The bad and the good of trends in model building and refinement for sparse-data regions: pernicious forms of overfitting versus good new tools and predictions
http://scripts.iucr.org/cgi-bin/paper?qo5006
Model building and refinement, and the validation of their correctness, are very effective and reliable at local resolutions better than about 2.5 Å for both crystallography and cryo-EM. However, at local resolutions worse than 2.5 Å both the procedures and their validation break down and do not ensure reliably correct models. This is because in the broad density at lower resolution, critical features such as protein backbone carbonyl O atoms are not just less accurate but are not seen at all, and so peptide orientations are frequently wrongly fitted by 90–180°. This puts both backbone and side chains into the wrong local energy minimum, and they are then worsened rather than improved by further refinement into a valid but incorrect rotamer or Ramachandran region. On the positive side, new tools are being developed to locate this type of pernicious error in PDB depositions, such as CaBLAM, EMRinger, Pperp diagnosis of ribose puckers, and peptide flips in PDB-REDO, while interactive modeling in Coot or ISOLDE can help to fix many of them. Another positive trend is that artificial intelligence predictions such as those made by AlphaFold2 contribute additional evidence from large multiple sequence alignments, and in high-confidence parts they provide quite good starting models for loops, termini or whole domains with otherwise ambiguous density.OVERFITTING AT LOW RESOLUTION; MACHINE-LEARNING PREDICTIONS; CABLAM VALIDATION; ISOLDE REBUILDING; RAMACHANDRAN REFINEMENT PROBLEMStextRichardson, J.S.Williams, C.J.Chen, V.B.Prisant, M.G.Richardson, D.C.2023-11-03The explicit refinement of Ramachandran, rotamer and clash criteria at now-prevalent lower resolutions (2.5–4 Å) has made the current, traditional model validation at the Protein Data Bank nearly useless in this range, since quite poor structures can have perfect scores. Fortunately, new criteria and programs such as ISOLDE, CaBLAM and AlphaFold are coming to the rescue, are already very useful and should be extensible into an effective new community standard.International Union of CrystallographyModel building and refinement, and the validation of their correctness, are very effective and reliable at local resolutions better than about 2.5 Å for both crystallography and cryo-EM. However, at local resolutions worse than 2.5 Å both the procedures and their validation break down and do not ensure reliably correct models. This is because in the broad density at lower resolution, critical features such as protein backbone carbonyl O atoms are not just less accurate but are not seen at all, and so peptide orientations are frequently wrongly fitted by 90–180°. This puts both backbone and side chains into the wrong local energy minimum, and they are then worsened rather than improved by further refinement into a valid but incorrect rotamer or Ramachandran region. On the positive side, new tools are being developed to locate this type of pernicious error in PDB depositions, such as CaBLAM, EMRinger, Pperp diagnosis of ribose puckers, and peptide flips in PDB-REDO, while interactive modeling in Coot or ISOLDE can help to fix many of them. Another positive trend is that artificial intelligence predictions such as those made by AlphaFold2 contribute additional evidence from large multiple sequence alignments, and in high-confidence parts they provide quite good starting models for loops, termini or whole domains with otherwise ambiguous density.The bad and the good of trends in model building and refinement for sparse-data regions: pernicious forms of overfitting versus good new tools and predictionsurn:issn:2059-7983doi:10.1107/S2059798323008847https://creativecommons.org/licenses/by/4.0/text/htmlenhttps://creativecommons.org/licenses/by/4.0/792023-11-03107812med@iucr.org10712059-7983Acta Crystallographica Section D: Structural BiologyDecember 20232059-7983research papersNeutron crystallographic refinement with REFMAC5 from the CCP4 suite
http://scripts.iucr.org/cgi-bin/paper?qe5005
Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand-recognition processes and protein–protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localization of only the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with REFMAC5 afforded models characterized by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium–low resolution.NEUTRON MACROMOLECULAR CRYSTALLOGRAPHY; CRYSTALLOGRAPHIC REFINEMENT; H ATOMS; REFMAC5; CCP4textCatapano, L.Long, F.Yamashita, K.Nicholls, R.A.Steiner, R.A.Murshudov, G.N.2023-11-03The macromolecular refinement package REFMAC5 from the CCP4 suite has been extended by the incorporation of algorithms for neutron crystallography.International Union of CrystallographyHydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand-recognition processes and protein–protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localization of only the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with REFMAC5 afforded models characterized by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium–low resolution.Neutron crystallographic refinement with REFMAC5 from the CCP4 suiteurn:issn:2059-7983doi:10.1107/S2059798323008793https://creativecommons.org/licenses/by/4.0/text/htmlen10702059-79831056med@iucr.org1279https://creativecommons.org/licenses/by/4.0/2023-11-03research papers2059-7983Acta Crystallographica Section D: Structural BiologyDecember 2023Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes
http://scripts.iucr.org/cgi-bin/paper?tz5111
In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a `sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV–Vis absorption spectroscopy.FIXED-TARGET SERIAL CRYSTALLOGRAPHY; SAMPLE SUPPORTS; SANDWICHED SILICON NITRIDE MEMBRANES; ANAEROBIC DATA COLLECTION; HEMOGLOBINtextBjelčić, M.Sigfridsson Clauss, K.G.V.Aurelius, O.Milas, M.Nan, J.Ursby, T.2023-11-01Anaerobic, room-temperature X-ray diffraction and absorption spectroscopy is performed using sandwiched silicon nitride membranes, prepared using 3D-printed tools, in an oxygen-free environment with a reducing agent. Using crystals of hemoglobin as a model system, it was demonstrated that this method ensures oxygen-free data collection.International Union of CrystallographyIn recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a `sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV–Vis absorption spectroscopy.Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranesurn:issn:2059-7983doi:10.1107/S205979832300880Xhttps://creativecommons.org/licenses/by/4.0/text/htmlenActa Crystallographica Section D: Structural BiologyNovember 20232059-7983research papershttps://creativecommons.org/licenses/by/4.0/792023-11-01102511med@iucr.org10182059-7983Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae
http://scripts.iucr.org/cgi-bin/paper?jc5061
Fucoidanases (EC 3.2.1.–) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-l-Fucp2S-(1,4)-α-l-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10−3 Uf µM−1. By crystal structure determination of Mef1 at 1.8 Å resolution, a single-domain organization comprising a (β/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.FUCOIDANASES; GH107; CRYSTAL STRUCTURE; ([BETA]/[ALPHA])8-BARREL; CA2+ SITE; MEF1; MURICAUDA ECKLONIAEtextMikkelsen, M.D.Tran, V.H.N.Meier, S.Nguyen, T.T.Holck, J.Cao, H.T.T.Van, T.T.T.Thinh, P.D.Meyer, A.S.Morth, J.P.2023-10-25The first structural determination of the α(1,4)-linkage-specific fucoidan hydrolase Mef1 (GH107) is reported, including the positioning of two calcium sites and the two main amino acids involved in the active-site hydrolytic mechanism. The structural determination also led to the discovery of a water wire leading from the exterior into the active site of the enzyme.International Union of CrystallographyFucoidanases (EC 3.2.1.–) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-l-Fucp2S-(1,4)-α-l-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10−3 Uf µM−1. By crystal structure determination of Mef1 at 1.8 Å resolution, a single-domain organization comprising a (β/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniaeurn:issn:2059-7983doi:10.1107/S2059798323008732https://creativecommons.org/licenses/by/4.0/text/htmlen104310262059-798311med@iucr.orghttps://creativecommons.org/licenses/by/4.0/792023-10-25research papers2059-7983Acta Crystallographica Section D: Structural BiologyNovember 2023Cocrystallization of ubiquitin–deubiquitinase complexes through disulfide linkage
http://scripts.iucr.org/cgi-bin/paper?nj5320
Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB–Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB–Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.DEUBIQUITINASES; UBIQUITIN; DEUBIQUITINASE-UBIQUITIN COMPLEXES; DISULFIDE-LINKED PROTEIN COMPLEXEStextNegron Teron, K.I.Das, C.2023-10-25The crystal structures of two disulfide-linked ubiquitin-bound bacterial deubiquitinases reveal the mode of ubiquitin binding. These structures show that disulfide linking is an effective strategy for capturing covalent ubiquitin–deubiquitinase complex structures.International Union of CrystallographyStructural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB–Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB–Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.Cocrystallization of ubiquitin–deubiquitinase complexes through disulfide linkageurn:issn:2059-7983doi:10.1107/S2059798323008501https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersActa Crystallographica Section D: Structural BiologyNovember 20231055med@iucr.org112059-7983104479https://creativecommons.org/licenses/by/4.0/2023-10-25Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallography
http://scripts.iucr.org/cgi-bin/paper?di5067
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.FORMATE DEHYDROGENASES; CANDIDA BOIDINII; PROTEIN ENGINEERING; X-RAY CRYSTALLOGRAPHY; STRUCTURAL BIOLOGY; STRUCTURAL DYNAMICS; TURKISH LIGHT SOURCE; TURKISH DELIGHTtextGul, M.Yuksel, B.Bulut, H.DeMirci, H.2023-10-20This study presents the atomic X-ray crystal structures of wild-type Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) and its Val120Thr mutant, revealing new hydrogen bonds and increased stability in the active site of the mutant. These findings offer valuable insights for protein engineering, potentially improving the efficiency and electron transfer of CbFDH for applications in biofuel production and industrial chemical synthesis from carbon dioxide.International Union of CrystallographyCandida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallographyurn:issn:2059-7983doi:10.1107/S2059798323008070https://creativecommons.org/licenses/by/4.0/text/htmlenresearch papers2059-7983Acta Crystallographica Section D: Structural BiologyNovember 202310172059-79831010med@iucr.org1179https://creativecommons.org/licenses/by/4.0/2023-10-20The impact of molecular variants, crystallization conditions and the space group on ligand–protein complexes: a case study on bacterial phosphotriesterase
http://scripts.iucr.org/cgi-bin/paper?nj5323
A bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38 Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P—O bonds with the exposed β-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand–protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase–organophosphate structures.CRYSTALLIZATION CONDITIONS; ZINC IONS; PHOSPHOTRIESTERASES; ORGANOPHOSPHATES; SPACE GROUPS; BIOSCAVENGERS; EXPRESSION SYSTEMStextDym, O.Aggarwal, N.Ashani, Y.Leader, H.Albeck, S.Unger, T.Hamer-Rogotner, S.Silman, I.Tawfik, D.S.Sussman, J.L.2023-10-20This study provides valuable insights into the challenges and considerations involved in the use of X-ray crystallography to study the 3D structures of ligand–protein complexes and highlights the importance of careful experimental design and rigorous data analysis in ensuring the validity of the structures obtained. A bacterial phosphotriesterase served as an experimental paradigm and novel insights were yielded into the role of the bimetal center of the enzyme in stabilizing the transition state for the hydrolysis of substrates.International Union of CrystallographyA bacterial phosphotriesterase was employed as an experimental paradigm to examine the effects of multiple factors, such as the molecular constructs, the ligands used during protein expression and purification, the crystallization conditions and the space group, on the visualization of molecular complexes of ligands with a target enzyme. In this case, the ligands used were organophosphates that are fragments of the nerve agents and insecticides on which the enzyme acts as a bioscavenger. 12 crystal structures of various phosphotriesterase constructs obtained by directed evolution were analyzed, with resolutions of up to 1.38 Å. Both apo forms and holo forms, complexed with the organophosphate ligands, were studied. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing the organophosphate complexes of the enzyme. The study revealed that the tags used for protein expression can lodge in the active site and hinder ligand binding. Furthermore, the space group in which the protein crystallizes can significantly impact the visualization of bound ligands. It was also observed that the crystallization precipitants can compete with, and even preclude, ligand binding, leading to false positives or to the incorrect identification of lead drug candidates. One of the co-crystallization conditions enabled the definition of the spaces that accommodate the substituents attached to the P atom of several products of organophosphate substrates after detachment of the leaving group. The crystal structures of the complexes of phosphotriesterase with the organophosphate products reveal similar short interaction distances of the two partially charged O atoms of the P—O bonds with the exposed β-Zn2+ ion and the buried α-Zn2+ ion. This suggests that both Zn2+ ions have a role in stabilizing the transition state for substrate hydrolysis. Overall, this study provides valuable insights into the challenges and considerations involved in studying the crystal structures of ligand–protein complexes, highlighting the importance of careful experimental design and rigorous data analysis in ensuring the accuracy and reliability of the resulting phosphotriesterase–organophosphate structures.The impact of molecular variants, crystallization conditions and the space group on ligand–protein complexes: a case study on bacterial phosphotriesteraseurn:issn:2059-7983doi:10.1107/S2059798323007672https://creativecommons.org/licenses/by/4.0/text/htmlenNovember 2023Acta Crystallographica Section D: Structural Biology2059-7983research papers2023-10-20https://creativecommons.org/licenses/by/4.0/7911med@iucr.org9922059-79831009Crystal structures of the DExH-box RNA helicase DHX9
http://scripts.iucr.org/cgi-bin/paper?ag5044
DHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.DHX9; RNA HELICASES; DEXH BOX; ADPtextLee, Y.-T.Sickmier, E.A.Grigoriu, S.Castro, J.Boriack-Sjodin, P.A.2023-10-20The first crystal structures of the human, dog and cat DHX9 proteins were determined. The structures of mammalian DHX9 proteins share a similar structural fold with the previously reported structure of the Drosophila melanogaster DHX9 orthologue MLE. The human DHX9 structure provides a useful starting point for structure-guided drug design of DHX9 inhibitors.International Union of CrystallographyDHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.Crystal structures of the DExH-box RNA helicase DHX9urn:issn:2059-7983doi:10.1107/S2059798323007611https://creativecommons.org/licenses/by/4.0/text/htmlen2023-10-20https://creativecommons.org/licenses/by/4.0/7911med@iucr.org9802059-7983991November 2023Acta Crystallographica Section D: Structural Biology2059-7983research papersDomain structure and cross-linking in a giant adhesin from the Mobiluncus mulieris bacterium
http://scripts.iucr.org/cgi-bin/paper?jb5059
Cell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.BACTERIAL ADHESINS; IG-LIKE DOMAINS; INTRAMOLECULAR CROSS-LINKS; CELL ADHESIONtextYoung, P.G.Paynter, J.M.Wardega, J.K.Middleditch, M.J.Payne, L.S.Baker, E.N.Squire, C.J.2023-10-20An adhesin from Mobiluncus mulieris, a bacterium associated with persistence in bacterial vaginosis, contains 51 repeat Ig-like domains. Each domain displays cross-linking including intramolecular ester, isopeptide, disulfide and thioester bonds. This giant 7651-residue protein, by far the largest in the bacterial proteome, is presumably retained because of its critical pathogenic role.International Union of CrystallographyCell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.Domain structure and cross-linking in a giant adhesin from the Mobiluncus mulieris bacteriumurn:issn:2059-7983doi:10.1107/S2059798323007507https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersNovember 2023Acta Crystallographica Section D: Structural Biologymed@iucr.org112059-79839719792023-10-2079https://creativecommons.org/licenses/by/4.0/Increasing the bulk of the 1TEL–target linker and retaining the 10×His tag in a 1TEL–CMG2-vWa construct improves crystal order and diffraction limits
http://scripts.iucr.org/cgi-bin/paper?di5066
TELSAM-fusion crystallization has the potential to become a revolutionary tool for the facile crystallization of proteins. TELSAM fusion can increase the crystallization rate and enable crystallization at low protein concentrations, in some cases with minimal crystal contacts [Nawarathnage et al. (2022), Open Biol. 12, 210271]. Here, requirements for the linker composition between 1TEL and a fused CMG2 vWa domain were investigated. Ala-Ala, Ala-Val, Thr-Val and Thr-Thr linkers were evaluated, comparing metrics for crystallization propensity and crystal order. The effect on crystallization of removing or retaining the purification tag was then tested. It was discovered that increasing the linker bulk and retaining the 10×His purification tag improved the diffraction resolution, likely by decreasing the number of possible vWa-domain orientations in the crystal. Additionally, it was discovered that some vWa-domain binding modes are correlated with scrambling of the 1TEL polymer orientation in crystals and an effective mitigation strategy for this pathology is presented.POLYMER-MEDIATED PROTEIN CRYSTALLIZATION; BEST PRACTICES; 1TEL; TELSAM; CMG2; ANTXR2; VWA DOMAINS; POLYMER FLIPPINGtextGajjar, P.L.Pedroza Romo, M.J.Litchfield, C.M.Callahan, M.Redd, N.Nawarathnage, S.Soleimani, S.Averett, J.Wilson, E.Lewis, A.Stewart, C.Tseng, Y.-J.Doukov, T.Lebedev, A.Moody, J.D.2023-09-25Using a 1TEL–CMG2-vWa construct, evidence is provided to support limiting the flexibility of linkers between TELSAM and proteins of interest and considering retaining polyhistidine purification tags in TELSAM-fusion constructs. The phenomenon of TELSAM-polymer flipping is also identified and a correction strategy is developed.International Union of CrystallographyTELSAM-fusion crystallization has the potential to become a revolutionary tool for the facile crystallization of proteins. TELSAM fusion can increase the crystallization rate and enable crystallization at low protein concentrations, in some cases with minimal crystal contacts [Nawarathnage et al. (2022), Open Biol. 12, 210271]. Here, requirements for the linker composition between 1TEL and a fused CMG2 vWa domain were investigated. Ala-Ala, Ala-Val, Thr-Val and Thr-Thr linkers were evaluated, comparing metrics for crystallization propensity and crystal order. The effect on crystallization of removing or retaining the purification tag was then tested. It was discovered that increasing the linker bulk and retaining the 10×His purification tag improved the diffraction resolution, likely by decreasing the number of possible vWa-domain orientations in the crystal. Additionally, it was discovered that some vWa-domain binding modes are correlated with scrambling of the 1TEL polymer orientation in crystals and an effective mitigation strategy for this pathology is presented.Increasing the bulk of the 1TEL–target linker and retaining the 10×His tag in a 1TEL–CMG2-vWa construct improves crystal order and diffraction limitsurn:issn:2059-7983doi:10.1107/S2059798323007246https://creativecommons.org/licenses/by/4.0/text/htmlenresearch papers2059-7983October 2023Acta Crystallographica Section D: Structural Biology2059-7983925med@iucr.org109432023-09-2579https://creativecommons.org/licenses/by/4.0/Elucidating polymorphs of crystal structures by intensity-based hierarchical clustering analysis of multiple diffraction data sets
http://scripts.iucr.org/cgi-bin/paper?wa5143
In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.POLYMORPH ANALYSIS; HIGH-DATA-RATE MACROMOLECULAR CRYSTALLOGRAPHY; HIERARCHICAL CLUSTERING ANALYSIS; AUTOMATED STRUCTURE ANALYSIStextMatsuura, H.Sakai, N.Toma-Fukai, S.Muraki, N.Hayama, K.Kamikubo, H.Aono, S.Kawano, Y.Yamamoto, M.Hirata, K.2023-10-25Single-step intensity-based hierarchical clustering is demonstrated to allow the detection of structural polymorphs in diffraction data sets obtained from multiple crystals. By splitting data sets collected using a continuous helical scheme into several chunks, both inter-crystal and intra-crystal polymorphs can successfully be analyzed.International Union of CrystallographyIn macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.Elucidating polymorphs of crystal structures by intensity-based hierarchical clustering analysis of multiple diffraction data setsurn:issn:2059-7983doi:10.1107/S2059798323007039https://creativecommons.org/licenses/by/4.0/text/htmlen10med@iucr.org9092059-79839242023-10-25https://creativecommons.org/licenses/by/4.0/792059-7983research papersOctober 2023Acta Crystallographica Section D: Structural BiologyA user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography
http://scripts.iucr.org/cgi-bin/paper?wa5145
Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831–4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.X-RAY CRYSTALLOGRAPHY; SYNCHROTRONS; XFELS; SAMPLE DELIVERY; FIXED TARGETS; CYCLIC OLEFIN COPOLYMER; MICROFLUIDICS; SERIAL CRYSTALLOGRAPHY; ROOM-TEMPERATURE SFXtextLiu, Z.Gu, K.K.Shelby, M.L.Gilbile, D.Lyubimov, A.Y.Russi, S.Cohen, A.E.Narayanasamy, S.R.Botha, S.Kupitz, C.Sierra, R.G.Poitevin, F.Gilardi, A.Lisova, S.Coleman, M.A.Frank, M.Kuhl, T.L.2023-09-25An improved design of an all-polymer microfluidic `chip' for fixed-target serial crystallography is presented that can easily be fabricated in-house, is inexpensive and is highly modifiable to meet broad user needs for room-temperature serial crystallography at both synchrotron and XFEL light sources.International Union of CrystallographyOver the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831–4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallographyurn:issn:2059-7983doi:10.1107/S2059798323007027https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983944med@iucr.org109522023-09-2579https://creativecommons.org/licenses/by/4.0/research papers2059-7983October 2023Acta Crystallographica Section D: Structural BiologyAtypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum
http://scripts.iucr.org/cgi-bin/paper?jc5060
Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon–halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate–histidine–aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.HALOALKANE DEHALOGENASES; MYCOBACTERIUM MARINUM; DMMARA; HOMODIMERIZATION; SURFACE LOOPS; ENANTIOSELECTIVITY; X-RAY CRYSTALLOGRAPHY; SAXStextSnajdarova, K.Marques, S.M.Damborsky, J.Bednar, D.Marek, M.2023-10-20Crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures reveal a previously unobserved mode of homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions.International Union of CrystallographyHaloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon–halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate–histidine–aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.Atypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinumurn:issn:2059-7983doi:10.1107/S2059798323006642https://creativecommons.org/licenses/by/4.0/text/htmlen9709562059-798311med@iucr.orghttps://creativecommons.org/licenses/by/4.0/792023-10-20research papers2059-7983Acta Crystallographica Section D: Structural BiologyNovember 2023Announcing the launch of Protein Data Bank China as an Associate Member of the Worldwide Protein Data Bank Partnership
http://scripts.iucr.org/cgi-bin/paper?rr5235
The Protein Data Bank (PDB) is the single global archive of atomic-level, three-dimensional structures of biological macromolecules experimentally determined by macromolecular crystallography, nuclear magnetic resonance spectroscopy or three-dimensional cryo-electron microscopy. The PDB is growing continuously, with a recent rapid increase in new structure depositions from Asia. In 2022, the Worldwide Protein Data Bank (wwPDB; https://www.wwpdb.org/) partners welcomed Protein Data Bank China (PDBc; https://www.pdbc.org.cn) to the organization as an Associate Member. PDBc is based in the National Facility for Protein Science in Shanghai which is associated with the Shanghai Advanced Research Institute of Chinese Academy of Sciences, the Shanghai Institute for Advanced Immunochemical Studies and the iHuman Institute of ShanghaiTech University. This letter describes the history of the wwPDB, recently established mechanisms for adding new wwPDB data centers and the processes developed to bring PDBc into the partnership.MACROMOLECULAR CRYSTALLOGRAPHY; NUCLEAR MAGNETIC RESONANCE; THREE-DIMENSIONAL CRYO-ELECTRON MICROSCOPY; PROTEIN DATA BANK; BIOLOGICAL MAGNETIC RESONANCE BANK; ELECTRON MICROSCOPY DATA BANK; WORLDWIDE PROTEIN DATA BANKtextXu, W.Velankar, S.Patwardhan, A.Hoch, J.C.Burley, S.K.Kurisu, G.2023-08-10The formal launch of Protein Data Bank China (PDBc) is announced, and the history of the wwPDB, recently established mechanisms for adding new wwPDB data centers and the processes developed to bring PDBc into the partnership are described.International Union of CrystallographyThe Protein Data Bank (PDB) is the single global archive of atomic-level, three-dimensional structures of biological macromolecules experimentally determined by macromolecular crystallography, nuclear magnetic resonance spectroscopy or three-dimensional cryo-electron microscopy. The PDB is growing continuously, with a recent rapid increase in new structure depositions from Asia. In 2022, the Worldwide Protein Data Bank (wwPDB; https://www.wwpdb.org/) partners welcomed Protein Data Bank China (PDBc; https://www.pdbc.org.cn) to the organization as an Associate Member. PDBc is based in the National Facility for Protein Science in Shanghai which is associated with the Shanghai Advanced Research Institute of Chinese Academy of Sciences, the Shanghai Institute for Advanced Immunochemical Studies and the iHuman Institute of ShanghaiTech University. This letter describes the history of the wwPDB, recently established mechanisms for adding new wwPDB data centers and the processes developed to bring PDBc into the partnership.Announcing the launch of Protein Data Bank China as an Associate Member of the Worldwide Protein Data Bank Partnershipurn:issn:2059-7983doi:10.1107/S2059798323006381https://creativecommons.org/licenses/by/4.0/text/htmlenletters to the editor2059-7983September 2023Acta Crystallographica Section D: Structural Biology2059-7983792med@iucr.org97952023-08-1079https://creativecommons.org/licenses/by/4.0/Predicted models and CCP4
http://scripts.iucr.org/cgi-bin/paper?qo5005
In late 2020, the results of CASP14, the 14th event in a series of competitions to assess the latest developments in computational protein structure-prediction methodology, revealed the giant leap forward that had been made by Google's Deepmind in tackling the prediction problem. The level of accuracy in their predictions was the first instance of a competitor achieving a global distance test score of better than 90 across all categories of difficulty. This achievement represents both a challenge and an opportunity for the field of experimental structural biology. For structure determination by macromolecular X-ray crystallography, access to highly accurate structure predictions is of great benefit, particularly when it comes to solving the phase problem. Here, details of new utilities and enhanced applications in the CCP4 suite, designed to allow users to exploit predicted models in determining macromolecular structures from X-ray diffraction data, are presented. The focus is mainly on applications that can be used to solve the phase problem through molecular replacement.CCP4; PREDICTED MODELS; MOLECULAR REPLACEMENT; MACROMOLECULAR CRYSTALLOGRAPHY; STRUCTURE DETERMINATIONtextSimpkin, A.J.Caballero, I.McNicholas, S.Stevenson, K.Jiménez, E.Sánchez Rodríguez, F.Fando, M.Uski, V.Ballard, C.Chojnowski, G.Lebedev, A.Krissinel, E.Usón, I.Rigden, D.J.Keegan, R.M.2023-08-17The use of predicted models for macromolecular structure determination in CCP4 is discussed.International Union of CrystallographyIn late 2020, the results of CASP14, the 14th event in a series of competitions to assess the latest developments in computational protein structure-prediction methodology, revealed the giant leap forward that had been made by Google's Deepmind in tackling the prediction problem. The level of accuracy in their predictions was the first instance of a competitor achieving a global distance test score of better than 90 across all categories of difficulty. This achievement represents both a challenge and an opportunity for the field of experimental structural biology. For structure determination by macromolecular X-ray crystallography, access to highly accurate structure predictions is of great benefit, particularly when it comes to solving the phase problem. Here, details of new utilities and enhanced applications in the CCP4 suite, designed to allow users to exploit predicted models in determining macromolecular structures from X-ray diffraction data, are presented. The focus is mainly on applications that can be used to solve the phase problem through molecular replacement.Predicted models and CCP4urn:issn:2059-7983doi:10.1107/S2059798323006289https://creativecommons.org/licenses/by/4.0/text/htmlenresearch papers2059-7983September 2023Acta Crystallographica Section D: Structural Biology2059-7983806med@iucr.org98192023-08-1779https://creativecommons.org/licenses/by/4.0/A Python package based on robust statistical analysis for serial crystallography data processing
http://scripts.iucr.org/cgi-bin/paper?qi5001
The term robustness in statistics refers to methods that are generally insensitive to deviations from model assumptions. In other words, robust methods are able to preserve their accuracy even when the data do not perfectly fit the statistical models. Robust statistical analyses are particularly effective when analysing mixtures of probability distributions. Therefore, these methods enable the discretization of X-ray serial crystallography data into two probability distributions: a group comprising true data points (for example the background intensities) and another group comprising outliers (for example Bragg peaks or bad pixels on an X-ray detector). These characteristics of robust statistical analysis are beneficial for the ever-increasing volume of serial crystallography (SX) data sets produced at synchrotron and X-ray free-electron laser (XFEL) sources. The key advantage of the use of robust statistics for some applications in SX data analysis is that it requires minimal parameter tuning because of its insensitivity to the input parameters. In this paper, a software package called Robust Gaussian Fitting library (RGFlib) is introduced that is based on the concept of robust statistics. Two methods are presented based on the concept of robust statistics and RGFlib for two SX data-analysis tasks: (i) a robust peak-finding algorithm and (ii) an automated robust method to detect bad pixels on X-ray pixel detectors.RGFLIB; ROBUST STATISTICS; SERIAL CRYSTALLOGRAPHY; ROBUST PEAK-FINDING; ROBUST BAD PIXEL MASK MAKINGtextHadian-Jazi, M.Sadri, A.2023-08-16This article introduces RGFlib, a Python package for robust statistical analysis. The package is a useful tool for a variety of tasks in X-ray crystallography data analysis, such as peak-finding, bad pixel mask making and other outlier-detection tasks.International Union of CrystallographyThe term robustness in statistics refers to methods that are generally insensitive to deviations from model assumptions. In other words, robust methods are able to preserve their accuracy even when the data do not perfectly fit the statistical models. Robust statistical analyses are particularly effective when analysing mixtures of probability distributions. Therefore, these methods enable the discretization of X-ray serial crystallography data into two probability distributions: a group comprising true data points (for example the background intensities) and another group comprising outliers (for example Bragg peaks or bad pixels on an X-ray detector). These characteristics of robust statistical analysis are beneficial for the ever-increasing volume of serial crystallography (SX) data sets produced at synchrotron and X-ray free-electron laser (XFEL) sources. The key advantage of the use of robust statistics for some applications in SX data analysis is that it requires minimal parameter tuning because of its insensitivity to the input parameters. In this paper, a software package called Robust Gaussian Fitting library (RGFlib) is introduced that is based on the concept of robust statistics. Two methods are presented based on the concept of robust statistics and RGFlib for two SX data-analysis tasks: (i) a robust peak-finding algorithm and (ii) an automated robust method to detect bad pixels on X-ray pixel detectors.A Python package based on robust statistical analysis for serial crystallography data processingurn:issn:2059-7983doi:10.1107/S2059798323005855https://creativecommons.org/licenses/by/4.0/text/htmlenSeptember 2023Acta Crystallographica Section D: Structural Biology2059-7983research papers2023-08-16https://creativecommons.org/licenses/by/4.0/799med@iucr.org8202059-7983829LifeSoaks: a tool for analyzing solvent channels in protein crystals and obstacles for soaking experiments
http://scripts.iucr.org/cgi-bin/paper?nz5014
Due to the structural complexity of proteins, their corresponding crystal arrangements generally contain a significant amount of solvent-occupied space. These areas allow a certain degree of intracrystalline protein flexibility and mobility of solutes. Therefore, knowledge of the geometry of solvent-filled channels and cavities is essential whenever the dynamics inside a crystal are of interest. Especially in soaking experiments for structure-based drug design, ligands must be able to traverse the crystal solvent channels and reach the corresponding binding pockets. Unsuccessful screenings are sometimes attributed to the geometry of the crystal packing, but the underlying causes are often difficult to understand. This work presents LifeSoaks, a novel tool for analyzing and visualizing solvent channels in protein crystals. LifeSoaks uses a Voronoi diagram-based periodic channel representation which can be efficiently computed. The size and location of channel bottlenecks, which might hinder molecular diffusion, can be directly derived from this representation. This work presents the calculated bottleneck radii for all crystal structures in the PDB and the analysis of a new, hand-curated data set of structures obtained by soaking experiments. The results indicate that the consideration of bottleneck radii and the visual inspection of channels are beneficial for planning soaking experiments.SOLVENT CHANNELS; SOAKING; LIFESOAKS; VORONOI DIAGRAM; STRUCTURE-BASED DRUG DESIGNtextPletzer-Zelgert, J.Ehrt, C.Fender, I.Griewel, A.Flachsenberg, F.Klebe, G.Rarey, M.2023-08-10A novel tool is presented for the calculation of solvent channels and their bottlenecks, with a special emphasis on small-molecule soaking. Bottleneck radii for all crystal structures in the PDB as well as a hand-curated data set of successfully soaked structures are presented.International Union of CrystallographyDue to the structural complexity of proteins, their corresponding crystal arrangements generally contain a significant amount of solvent-occupied space. These areas allow a certain degree of intracrystalline protein flexibility and mobility of solutes. Therefore, knowledge of the geometry of solvent-filled channels and cavities is essential whenever the dynamics inside a crystal are of interest. Especially in soaking experiments for structure-based drug design, ligands must be able to traverse the crystal solvent channels and reach the corresponding binding pockets. Unsuccessful screenings are sometimes attributed to the geometry of the crystal packing, but the underlying causes are often difficult to understand. This work presents LifeSoaks, a novel tool for analyzing and visualizing solvent channels in protein crystals. LifeSoaks uses a Voronoi diagram-based periodic channel representation which can be efficiently computed. The size and location of channel bottlenecks, which might hinder molecular diffusion, can be directly derived from this representation. This work presents the calculated bottleneck radii for all crystal structures in the PDB and the analysis of a new, hand-curated data set of structures obtained by soaking experiments. The results indicate that the consideration of bottleneck radii and the visual inspection of channels are beneficial for planning soaking experiments.LifeSoaks: a tool for analyzing solvent channels in protein crystals and obstacles for soaking experimentsurn:issn:2059-7983doi:10.1107/S205979832300582Xhttps://creativecommons.org/licenses/by/4.0/text/htmlen79https://creativecommons.org/licenses/by/4.0/2023-08-10856med@iucr.org92059-7983837Acta Crystallographica Section D: Structural BiologySeptember 20232059-7983research papersCorrecting systematic errors in diffraction data with modern scaling algorithms
http://scripts.iucr.org/cgi-bin/paper?qi5002
X-ray diffraction enables the routine determination of the atomic structure of materials. Key to its success are data-processing algorithms that allow experimenters to determine the electron density of a sample from its diffraction pattern. Scaling, the estimation and correction of systematic errors in diffraction intensities, is an essential step in this process. These errors arise from sample heterogeneity, radiation damage, instrument limitations and other aspects of the experiment. New X-ray sources and sample-delivery methods, along with new experiments focused on changes in structure as a function of perturbations, have led to new demands on scaling algorithms. Classically, scaling algorithms use least-squares optimization to fit a model of common error sources to the observed diffraction intensities to force these intensities onto the same empirical scale. Recently, an alternative approach has been demonstrated which uses a Bayesian optimization method, variational inference, to simultaneously infer merged data along with corrections, or scale factors, for the systematic errors. Owing to its flexibility, this approach proves to be advantageous in certain scenarios. This perspective briefly reviews the history of scaling algorithms and contrasts them with variational inference. Finally, appropriate use cases are identified for the first such algorithm, Careless, guidance is offered on its use and some speculations are made about future variational scaling methods.X-RAY CRYSTALLOGRAPHY; SCALING; VARIATIONAL INFERENCE; DEEP LEARNINGtextAldama, L.A.Dalton, K.M.Hekstra, D.R.2023-08-16Emerging algorithms based on machine learning offer promise in processing new diffraction experiments.International Union of CrystallographyX-ray diffraction enables the routine determination of the atomic structure of materials. Key to its success are data-processing algorithms that allow experimenters to determine the electron density of a sample from its diffraction pattern. Scaling, the estimation and correction of systematic errors in diffraction intensities, is an essential step in this process. These errors arise from sample heterogeneity, radiation damage, instrument limitations and other aspects of the experiment. New X-ray sources and sample-delivery methods, along with new experiments focused on changes in structure as a function of perturbations, have led to new demands on scaling algorithms. Classically, scaling algorithms use least-squares optimization to fit a model of common error sources to the observed diffraction intensities to force these intensities onto the same empirical scale. Recently, an alternative approach has been demonstrated which uses a Bayesian optimization method, variational inference, to simultaneously infer merged data along with corrections, or scale factors, for the systematic errors. Owing to its flexibility, this approach proves to be advantageous in certain scenarios. This perspective briefly reviews the history of scaling algorithms and contrasts them with variational inference. Finally, appropriate use cases are identified for the first such algorithm, Careless, guidance is offered on its use and some speculations are made about future variational scaling methods.Correcting systematic errors in diffraction data with modern scaling algorithmsurn:issn:2059-7983doi:10.1107/S2059798323005776https://creativecommons.org/licenses/by/4.0/text/htmlen9med@iucr.org7962059-79838052023-08-16https://creativecommons.org/licenses/by/4.0/792059-7983topical reviewsSeptember 2023Acta Crystallographica Section D: Structural BiologyStructural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin
http://scripts.iucr.org/cgi-bin/paper?ag5043
Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS–PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS–PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein–protein interactions supports a predicted molecular basis for enzyme–carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.NATURAL PRODUCT BIOSYNTHESIS; AMIDASES; COLIBACTIN; CLBL; MICROBIOMEtextTripathi, P.Mousa, J.J.Guntaka, N.S.Bruner, S.D.2023-08-10Insight into biosynthetic pathways in the human microbiome can provide details of host–microbe interactions and beneficial symbiosis. In this report, the structure and function of a key bond-forming enzyme in the biosynthesis of the human genotoxin colibactin is presented.International Union of CrystallographyColibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS–PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS–PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein–protein interactions supports a predicted molecular basis for enzyme–carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactinurn:issn:2059-7983doi:10.1107/S2059798323005703https://creativecommons.org/licenses/by/4.0/text/htmlen79https://creativecommons.org/licenses/by/4.0/2023-08-10836med@iucr.org92059-7983830Acta Crystallographica Section D: Structural BiologySeptember 20232059-7983research papersA standard descriptor for fixed-target serial crystallography
http://scripts.iucr.org/cgi-bin/paper?gm5097
Fixed-target crystallography has become a widely used approach for serial crystallography at both synchrotron and X-ray free-electron laser (XFEL) sources. A plethora of fixed targets have been developed at different facilities and by various manufacturers, with different characteristics and dimensions and with little or no emphasis on standardization. These many fixed targets have good reasons for their design, shapes, fabrication materials and the presence or absence of apertures and fiducials, reflecting the diversity of serial experiments. Given this, it would be a Sisyphean task to design and manufacture a new standard fixed target that would satisfy all possible experimental configurations. Therefore, a simple standardized descriptor to fully describe fixed targets is proposed rather than a standardized device. This descriptor is a dictionary that could be read by fixed-target beamline software and straightforwardly allow data collection from fixed targets new to that beamline. The descriptor would therefore allow a much easier exchange of fixed targets between sources and facilitate the uptake of new fixed targets, benefiting beamlines, users and manufacturers. This descriptor was first presented at, and was developed following, a meeting of representatives from multiple synchrotron and XFEL sources in Hamburg in January 2023.FIXED TARGETS; SERIAL CRYSTALLOGRAPHY; STANDARDIZATIONtextOwen, R.L.de Sanctis, D.Pearson, A.R.Beale, J.H.2023-07-18A descriptor with a standard format and parameters is introduced for the large-area fixed targets used in serial crystallography. It is proposed that any fixed target used and developed by different facilities or groups could have its own version of this descriptor, facilitating their wider use and exchange.International Union of CrystallographyFixed-target crystallography has become a widely used approach for serial crystallography at both synchrotron and X-ray free-electron laser (XFEL) sources. A plethora of fixed targets have been developed at different facilities and by various manufacturers, with different characteristics and dimensions and with little or no emphasis on standardization. These many fixed targets have good reasons for their design, shapes, fabrication materials and the presence or absence of apertures and fiducials, reflecting the diversity of serial experiments. Given this, it would be a Sisyphean task to design and manufacture a new standard fixed target that would satisfy all possible experimental configurations. Therefore, a simple standardized descriptor to fully describe fixed targets is proposed rather than a standardized device. This descriptor is a dictionary that could be read by fixed-target beamline software and straightforwardly allow data collection from fixed targets new to that beamline. The descriptor would therefore allow a much easier exchange of fixed targets between sources and facilitate the uptake of new fixed targets, benefiting beamlines, users and manufacturers. This descriptor was first presented at, and was developed following, a meeting of representatives from multiple synchrotron and XFEL sources in Hamburg in January 2023.A standard descriptor for fixed-target serial crystallographyurn:issn:2059-7983doi:10.1107/S2059798323005429https://creativecommons.org/licenses/by/4.0/text/htmlenAugust 2023Acta Crystallographica Section D: Structural Biology2059-7983letters to the editor2023-07-1879https://creativecommons.org/licenses/by/4.0/med@iucr.org82059-7983668672Novel starting points for fragment-based drug design against mycobacterial thioredoxin reductase identified using crystallographic fragment screening
http://scripts.iucr.org/cgi-bin/paper?jb5056
The increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR.CRYSTALLOGRAPHIC FRAGMENT SCREENING; MYCOBACTERIUM TUBERCULOSIS; MYCOBACTERIUM SMEGMATIS; THIOREDOXIN REDUCTASE; ANTITUBERCULOTIC DRUGStextFüsser, F.T.Wollenhaupt, J.Weiss, M.S.Kümmel, D.Koch, O.2023-08-14Crystallographic fragment screening of a mycobacterial thioredoxin reductase revealed 56 starting points for the development of new antituberculotic drugs, with 42 fragments bound to 11 different binding sites.International Union of CrystallographyThe increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR.Novel starting points for fragment-based drug design against mycobacterial thioredoxin reductase identified using crystallographic fragment screeningurn:issn:2059-7983doi:10.1107/S2059798323005223https://creativecommons.org/licenses/by/4.0/text/htmlenActa Crystallographica Section D: Structural BiologySeptember 2023research papers2059-798379https://creativecommons.org/licenses/by/4.0/2023-08-148652059-7983857med@iucr.org9Overall protein structure quality assessment using hydrogen-bonding parameters
http://scripts.iucr.org/cgi-bin/paper?qo5003
Atomic model refinement at low resolution is often a challenging task. This is mostly because the experimental data are not sufficiently detailed to be described by atomic models. To make refinement practical and ensure that a refined atomic model is geometrically meaningful, additional information needs to be used such as restraints on Ramachandran plot distributions or residue side-chain rotameric states. However, using Ramachandran plots or rotameric states as refinement targets diminishes the validating power of these tools. Therefore, finding additional model-validation criteria that are not used or are difficult to use as refinement goals is desirable. Hydrogen bonds are one of the important noncovalent interactions that shape and maintain protein structure. These interactions can be characterized by a specific geometry of hydrogen donor and acceptor atoms. Systematic analysis of these geometries performed for quality-filtered high-resolution models of proteins from the Protein Data Bank shows that they have a distinct and a conserved distribution. Here, it is demonstrated how this information can be used for atomic model validation.ATOMIC MODEL REFINEMENT; HYDROGEN BONDS; CRYSTALLOGRAPHY; CRYO-EM; VALIDATIONtextAfonine, P.V.Sobolev, O.V.Moriarty, N.W.Terwilliger, T.C.Adams, P.D.2023-07-11A new protein structure validation method using hydrogen-bonding parameters is described.International Union of CrystallographyAtomic model refinement at low resolution is often a challenging task. This is mostly because the experimental data are not sufficiently detailed to be described by atomic models. To make refinement practical and ensure that a refined atomic model is geometrically meaningful, additional information needs to be used such as restraints on Ramachandran plot distributions or residue side-chain rotameric states. However, using Ramachandran plots or rotameric states as refinement targets diminishes the validating power of these tools. Therefore, finding additional model-validation criteria that are not used or are difficult to use as refinement goals is desirable. Hydrogen bonds are one of the important noncovalent interactions that shape and maintain protein structure. These interactions can be characterized by a specific geometry of hydrogen donor and acceptor atoms. Systematic analysis of these geometries performed for quality-filtered high-resolution models of proteins from the Protein Data Bank shows that they have a distinct and a conserved distribution. Here, it is demonstrated how this information can be used for atomic model validation.Overall protein structure quality assessment using hydrogen-bonding parametersurn:issn:2059-7983doi:10.1107/S2059798323005077https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersActa Crystallographica Section D: Structural BiologyAugust 20236938med@iucr.org6842059-7983https://creativecommons.org/licenses/by/4.0/792023-07-11Module walking using an SH3-like cell-wall-binding domain leads to a new GH184 family of muramidases
http://scripts.iucr.org/cgi-bin/paper?rr5233
Muramidases (also known as lysozymes) hydrolyse the peptidoglycan component of the bacterial cell wall and are found in many glycoside hydrolase (GH) families. Similar to other glycoside hydrolases, muramidases sometimes have noncatalytic domains that facilitate their interaction with the substrate. Here, the identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is first described, in which an SH3-like cell-wall-binding domain (CWBD) was identified by structure comparison in addition to its catalytic domain. Further, a complex between a triglycine peptide and the CWBD from T. saccata is presented that shows a possible anchor point of the peptidoglycan on the CWBD. A `domain-walking' approach, searching for other sequences with a domain of unknown function appended to the CWBD, was then used to identify a group of fungal muramidases that also contain homologous SH3-like cell-wall-binding modules, the catalytic domains of which define a new GH family. The properties of some representative members of this family are described as well as X-ray structures of the independent catalytic and SH3-like domains of the Kionochaeta sp., Thermothielavioides terrestris and Penicillium virgatum enzymes. This work confirms the power of the module-walking approach, extends the library of known GH families and adds a new noncatalytic module to the muramidase arsenal.GH184 FAMILY; LYSOZYMES; LYSINS; PEPTIDOGLYCAN CLEAVAGE; SH3-LIKE DOMAINS; MURAMIDASES; GLYCOSIDE HYDROLASE FAMILY 24; TRICHOPHAEA SACCATA; MODULE WALKINGtextMoroz, O.V.Blagova, E.Lebedev, A.A.Skov, L.K.Pache, R.A.Schnorr, K.M.Kiemer, L.Friis, E.P.Nymand-Grarup, S.Ming, L.Ye, L.Klausen, M.Cohn, M.T.Schmidt, E.G.W.Davies, G.J.Wilson, K.S.2023-07-10The identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is described in which an SH3-like cell-wall-binding domain was identified by structure comparisons in addition to its catalytic domain. A domain-walking approach was then used to identify a group of fungal muramidases that belong to a new GH family containing homologous SH3-like cell-wall-binding modules, and X-ray structures of the independent catalytic and SH3-like domains of three of them are reported.International Union of CrystallographyMuramidases (also known as lysozymes) hydrolyse the peptidoglycan component of the bacterial cell wall and are found in many glycoside hydrolase (GH) families. Similar to other glycoside hydrolases, muramidases sometimes have noncatalytic domains that facilitate their interaction with the substrate. Here, the identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is first described, in which an SH3-like cell-wall-binding domain (CWBD) was identified by structure comparison in addition to its catalytic domain. Further, a complex between a triglycine peptide and the CWBD from T. saccata is presented that shows a possible anchor point of the peptidoglycan on the CWBD. A `domain-walking' approach, searching for other sequences with a domain of unknown function appended to the CWBD, was then used to identify a group of fungal muramidases that also contain homologous SH3-like cell-wall-binding modules, the catalytic domains of which define a new GH family. The properties of some representative members of this family are described as well as X-ray structures of the independent catalytic and SH3-like domains of the Kionochaeta sp., Thermothielavioides terrestris and Penicillium virgatum enzymes. This work confirms the power of the module-walking approach, extends the library of known GH families and adds a new noncatalytic module to the muramidase arsenal.Module walking using an SH3-like cell-wall-binding domain leads to a new GH184 family of muramidasesurn:issn:2059-7983doi:10.1107/S2059798323005004https://creativecommons.org/licenses/by/4.0/text/htmlen79https://creativecommons.org/licenses/by/4.0/2023-07-107202059-7983706med@iucr.org8Acta Crystallographica Section D: Structural BiologyAugust 2023research papers2059-7983Raynals, an online tool for the analysis of dynamic light scattering
http://scripts.iucr.org/cgi-bin/paper?vo5014
Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size-distribution profile of samples containing microscopic particles in suspension or solubilized polymers. In this work, Raynals, user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov–Phillips regularization, is introduced. Its performance is evaluated on simulated and experimental data generated by different DLS instruments for several proteins and gold nanoparticles. DLS data can easily be misinterpreted and the simulation tools available in Raynals allow the limitations of the measurement and its resolution to be understood. It was designed as a tool to address the quality control of biological samples during sample preparation and optimization and it helps in the detection of aggregates, showing the influence of large particles. Lastly, Raynals provides flexibility in the way that the data are presented, allows the export of publication-quality figures, is free for academic use and can be accessed online on the eSPC data-analysis platform at https://spc.embl-hamburg.de/.RAYNALS; DYNAMIC LIGHT SCATTERING; MOLECULAR BIOPHYSICS; ONLINE DATA ANALYSIS; PROTEIN QUALITY CONTROL; HYDRODYNAMIC RADIUS; SIZE DISTRIBUTION; SOFTWAREtextBurastero, O.Draper-Barr, G.Raynal, B.Chevreuil, M.England, P.Garcia Alai, M.2023-07-10Raynals is an online, user-friendly, free (for academia) and advanced tool for the analysis of single-angle dynamic light-scattering data. Estimation of the size distribution is performed through the Tikhonov–Phillips regularization.International Union of CrystallographyDynamic light scattering (DLS) is routinely employed to assess the homogeneity and size-distribution profile of samples containing microscopic particles in suspension or solubilized polymers. In this work, Raynals, user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov–Phillips regularization, is introduced. Its performance is evaluated on simulated and experimental data generated by different DLS instruments for several proteins and gold nanoparticles. DLS data can easily be misinterpreted and the simulation tools available in Raynals allow the limitations of the measurement and its resolution to be understood. It was designed as a tool to address the quality control of biological samples during sample preparation and optimization and it helps in the detection of aggregates, showing the influence of large particles. Lastly, Raynals provides flexibility in the way that the data are presented, allows the export of publication-quality figures, is free for academic use and can be accessed online on the eSPC data-analysis platform at https://spc.embl-hamburg.de/.Raynals, an online tool for the analysis of dynamic light scatteringurn:issn:2059-7983doi:10.1107/S2059798323004862https://creativecommons.org/licenses/by/4.0/text/htmlenresearch papers2059-7983August 2023Acta Crystallographica Section D: Structural Biology6732059-79838med@iucr.org6832023-07-10https://creativecommons.org/licenses/by/4.0/79Bulk-solvent and overall scaling revisited: faster calculations, improved results. Corrigendum.
http://scripts.iucr.org/cgi-bin/paper?rr5234
Equations in Sections 2.3 and 2.4 of the article by Afonine et al. [Acta Cryst. (2013). D69, 625–634] are corrected.PHENIX; ANISOTROPY; BULK SOLVENT; SCALINGtextAfonine, P.V.Grosse-Kunstleve, R.W.Adams, P.D.Urzhumtsev, A.2023-06-20The article by Afonine et al. [Acta Cryst. (2013). D69, 625–634] is corrected.International Union of CrystallographyEquations in Sections 2.3 and 2.4 of the article by Afonine et al. [Acta Cryst. (2013). D69, 625–634] are corrected.Bulk-solvent and overall scaling revisited: faster calculations, improved results. Corrigendum.urn:issn:2059-7983doi:10.1107/S2059798323004825https://creativecommons.org/licenses/by/4.0/text/htmlen79https://creativecommons.org/licenses/by/4.0/2023-06-20667med@iucr.org72059-7983666Acta Crystallographica Section D: Structural BiologyJuly 20232059-7983addenda and errataCrystal structure of dihydrofolate reductase from the emerging pathogenic fungus Candida auris
http://scripts.iucr.org/cgi-bin/paper?ji5031
Candida auris has emerged as a global health problem with a dramatic spread by nosocomial transmission and a high mortality rate. Antifungal therapy for C. auris infections is currently limited due to widespread resistance to fluconazole and amphotericin B and increasing resistance to the front-line drug echinocandin. Therefore, new treatments are urgently required to combat this pathogen. Dihydrofolate reductase (DHFR) has been validated as a potential drug target for Candida species, although no structure of the C. auris enzyme (CauDHFR) has been reported. Here, crystal structures of CauDHFR are reported as an apoenzyme, as a holoenzyme and in two ternary complexes with pyrimethamine and cycloguanil, which are common antifolates, at near-atomic resolution. Preliminary biochemical and biophysical assays and antifungal susceptibility testing with a variety of classical antifolates were also performed, highlighting the enzyme-inhibition rates and the inhibition of yeast growth. These structural and functional data might provide the basis for a novel drug-discovery campaign against this global threat.CANDIDA AURIS; DIHYDROFOLATE REDUCTASE; CRYSTALLOGRAPHY; ANTIFOLATEStextKirkman, T.Sketcher, A.de Morais Barroso, V.Ishida, K.Tosin, M.Dias, M.V.B2023-07-10The first structures of Candida auris dihydrofolate reductase at near-atomic resolution are described, including apo and holo forms and complexes with two antifolate drugs: pyrimethamine and cycloguanil. C. auris is a highly significant globally emerging and resistant fungal pathogen and there is a great demand for research and progress towards novel therapeutics.International Union of CrystallographyCandida auris has emerged as a global health problem with a dramatic spread by nosocomial transmission and a high mortality rate. Antifungal therapy for C. auris infections is currently limited due to widespread resistance to fluconazole and amphotericin B and increasing resistance to the front-line drug echinocandin. Therefore, new treatments are urgently required to combat this pathogen. Dihydrofolate reductase (DHFR) has been validated as a potential drug target for Candida species, although no structure of the C. auris enzyme (CauDHFR) has been reported. Here, crystal structures of CauDHFR are reported as an apoenzyme, as a holoenzyme and in two ternary complexes with pyrimethamine and cycloguanil, which are common antifolates, at near-atomic resolution. Preliminary biochemical and biophysical assays and antifungal susceptibility testing with a variety of classical antifolates were also performed, highlighting the enzyme-inhibition rates and the inhibition of yeast growth. These structural and functional data might provide the basis for a novel drug-discovery campaign against this global threat.Crystal structure of dihydrofolate reductase from the emerging pathogenic fungus Candida aurisurn:issn:2059-7983doi:10.1107/S2059798323004709https://creativecommons.org/licenses/by/4.0/text/htmlen7458med@iucr.org7352059-7983https://creativecommons.org/licenses/by/4.0/792023-07-102059-7983research papersActa Crystallographica Section D: Structural BiologyAugust 2023Conformation-based refinement of 18-mer DNA structures
http://scripts.iucr.org/cgi-bin/paper?rr5230
Nine new crystal structures of CG-rich DNA 18-mers with the sequence 5′-GGTGGGGGC-XZ-GCCCCACC-3′, which are related to the bacterial repetitive extragenic palindromes, are reported. 18-mer oligonucleotides with the central XZ dinucleotide systematically mutated to all 16 sequences show complex behavior in solution, but all ten so far successfully crystallized 18-mers crystallized as A-form duplexes. The refinement protocol benefited from the recurrent use of geometries of the dinucleotide conformer (NtC) classes as refinement restraints in regions of poor electron density. The restraints are automatically generated at the dnatco.datmos.org web service and are available for download. This NtC-driven protocol significantly helped to stabilize the structure refinement. The NtC-driven refinement protocol can be adapted to other low-resolution data such as cryo-EM maps. To test the quality of the final structural models, a novel validation method based on comparison of the electron density and conformational similarity to the NtC classes was employed.DNA STRUCTURE; DNATCO.DATMOS.ORG; STRUCTURE VALIDATION; STRUCTURE REFINEMENT; BASE PAIRINGtextSvoboda, J.Berdár, D.Kolenko, P.Černý, J.Nováková, Z.Pavlíček, J.Schneider, B.2023-06-20The refinement and validation of nine A-form DNA 18-mer crystal structures containing both canonical and noncanonical base pairs benefits from the use of dinucleotide conformer classes (NtCs).International Union of CrystallographyNine new crystal structures of CG-rich DNA 18-mers with the sequence 5′-GGTGGGGGC-XZ-GCCCCACC-3′, which are related to the bacterial repetitive extragenic palindromes, are reported. 18-mer oligonucleotides with the central XZ dinucleotide systematically mutated to all 16 sequences show complex behavior in solution, but all ten so far successfully crystallized 18-mers crystallized as A-form duplexes. The refinement protocol benefited from the recurrent use of geometries of the dinucleotide conformer (NtC) classes as refinement restraints in regions of poor electron density. The restraints are automatically generated at the dnatco.datmos.org web service and are available for download. This NtC-driven protocol significantly helped to stabilize the structure refinement. The NtC-driven refinement protocol can be adapted to other low-resolution data such as cryo-EM maps. To test the quality of the final structural models, a novel validation method based on comparison of the electron density and conformational similarity to the NtC classes was employed.Conformation-based refinement of 18-mer DNA structuresurn:issn:2059-7983doi:10.1107/S2059798323004679https://creativecommons.org/licenses/by/4.0/text/htmlen2023-06-2079https://creativecommons.org/licenses/by/4.0/med@iucr.org72059-7983655665July 2023Acta Crystallographica Section D: Structural Biology2059-7983research papersScipion Flexibility Hub: an integrative framework for advanced analysis of conformational heterogeneity in cryoEM
http://scripts.iucr.org/cgi-bin/paper?ic5121
Understanding how structure and function meet to drive biological processes is progressively shifting the cryoEM field towards a more advanced analysis of macromolecular flexibility. Thanks to techniques such as single-particle analysis and electron tomography, it is possible to image a macromolecule in different states, information that can subsequently be extracted through advanced image-processing methods to build a richer approximation of a conformational landscape. However, the interoperability of all of these algorithms remains a challenging task that is left to users, preventing them from defining a single flexible workflow in which conformational information can be addressed by different algorithms. Therefore, in this work, a new framework integrated into Scipion is proposed called the Flexibility Hub. This framework automatically handles intercommunication between different heterogeneity software, simplifying the task of combining the software into workflows in which the quality and the amount of information extracted from flexibility analysis is maximized.SINGLE-PARTICLE ANALYSIS; IMAGE PROCESSING; CRYO-ELECTRON MICROSCOPY; PROTEIN DYNAMICS; CONTINUOUS CONFORMATIONAL VARIABILITY; SCIPION FLEXIBILITY HUBtextHerreros, D.Krieger, J.M.Fonseca, Y.Conesa, P.Harastani, M.Vuillemot, R.Hamitouche, I.Serrano Gutiérrez, R.Gragera, M.Melero, R.Jonic, S.Carazo, J.M.Sorzano, C.O.S.2023-06-16An integrative framework called Flexibility Hub is integrated into Scipion that simplifies the interoperability of different heterogeneity algorithms.International Union of CrystallographyUnderstanding how structure and function meet to drive biological processes is progressively shifting the cryoEM field towards a more advanced analysis of macromolecular flexibility. Thanks to techniques such as single-particle analysis and electron tomography, it is possible to image a macromolecule in different states, information that can subsequently be extracted through advanced image-processing methods to build a richer approximation of a conformational landscape. However, the interoperability of all of these algorithms remains a challenging task that is left to users, preventing them from defining a single flexible workflow in which conformational information can be addressed by different algorithms. Therefore, in this work, a new framework integrated into Scipion is proposed called the Flexibility Hub. This framework automatically handles intercommunication between different heterogeneity software, simplifying the task of combining the software into workflows in which the quality and the amount of information extracted from flexibility analysis is maximized.Scipion Flexibility Hub: an integrative framework for advanced analysis of conformational heterogeneity in cryoEMurn:issn:2059-7983doi:10.1107/S2059798323004497https://creativecommons.org/licenses/by/4.0/text/htmlen5845692059-79837med@iucr.orghttps://creativecommons.org/licenses/by/4.0/792023-06-16research papers2059-7983Acta Crystallographica Section D: Structural BiologyJuly 2023Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius
http://scripts.iucr.org/cgi-bin/paper?rr5232
Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.THERMOPHILIC PROTEINS; SIDEROPHORE BINDING; STRUCTURE; BIOPHYSICAL CHARACTERIZATION; CEUE; GEOBACILLUS STEAROTHERMOPHILUS; PARAGEOBACILLUS THERMOGLUCOSIDASIUStextBlagova, E.V.Miller, A.H.Bennett, M.Booth, R.L.Dodson, E.J.Duhme-Klair, A.-K.Wilson, K.S.2023-07-10The expression, characterization and structures of CeuE homologues from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, are described together with their ligand binding. The proteins show enhanced thermostability and resistance to organic chemicals; consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.International Union of CrystallographySiderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasiusurn:issn:2059-7983doi:10.1107/S2059798323004473https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersActa Crystallographica Section D: Structural BiologyAugust 20237058med@iucr.org6942059-7983https://creativecommons.org/licenses/by/4.0/792023-07-10Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF
http://scripts.iucr.org/cgi-bin/paper?ag5042
The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.AETOKTHONOTOXIN; BIOCATALYSIS; BROMINATION; ENZYMES; ROSSMANN FOLD; SITE SELECTIVITY; AETF; SUBSTRATE BINDINGtextGäfe, S.Niemann, H.H.2023-06-14The single-component flavin-dependent tryptophan halogenase AetF converts tryptophan to 5,7-dibromotryptophan during the biosynthesis of the neurotoxin aetokthonotoxin. Crystal structures of AetF with the substrates tryptophan and 5-bromotryptophan show that a flip of the indole moiety of tryptophan positions first C5 and then C7 in the same location in the active site to facilitate two successive bromination reactions.International Union of CrystallographyThe flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetFurn:issn:2059-7983doi:10.1107/S2059798323004254https://creativecommons.org/licenses/by/4.0/text/htmlen609med@iucr.org72059-798359679https://creativecommons.org/licenses/by/4.0/2023-06-142059-7983research papersActa Crystallographica Section D: Structural BiologyJuly 2023Crystal structure of the monocupin ring-cleaving dioxygenase 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp.
http://scripts.iucr.org/cgi-bin/paper?nz5013
5-Nitrosalicylate 1,2-dioxygenase (5NSDO) is an iron(II)-dependent dioxygenase involved in the aerobic degradation of 5-nitroanthranilic acid by the bacterium Bradyrhizobium sp. It catalyzes the opening of the 5-nitrosalicylate aromatic ring, a key step in the degradation pathway. Besides 5-nitrosalicylate, the enzyme is also active towards 5-chlorosalicylate. The X-ray crystallographic structure of the enzyme was solved at 2.1 Å resolution by molecular replacement using a model from the AI program AlphaFold. The enzyme crystallized in the monoclinic space group P21, with unit-cell parameters a = 50.42, b = 143.17, c = 60.07 Å, β = 107.3°. 5NSDO belongs to the third class of ring-cleaving dioxygenases. Members of this family convert para-diols or hydroxylated aromatic carboxylic acids and belong to the cupin superfamily, which is one of the most functionally diverse protein classes and is named on the basis of a conserved β-barrel fold. 5NSDO is a tetramer composed of four identical subunits, each folded as a monocupin domain. The iron(II) ion in the enzyme active site is coordinated by His96, His98 and His136 and three water molecules with a distorted octahedral geometry. The residues in the active site are poorly conserved compared with other dioxygenases of the third class, such as gentisate 1,2-dioxygenase and salicylate 1,2-dioxygenase. Comparison with these other representatives of the same class and docking of the substrate into the active site of 5NSDO allowed the identification of residues which are crucial for the catalytic mechanism and enzyme selectivity.DIOXYGENASES; 5-NITROSALICYLATE 1,2-DIOXYGENASE; BRADYRHIZOBIUM SP JS329; X-RAY CRYSTALLOGRAPHY; CUPINS; SUBSTRATE SPECIFICITY; AROMATIC CATABOLISMtextEppinger, E.Stolz, A.Ferraroni, M.2023-06-16The crystal structure of the monocupin 5-nitrosalicylate 1,2-dioxygenase, an iron(II)-dependent ring-cleaving dioxygenase, from Bradyrhizobium sp. was determined by molecular replacement using a theoretical model obtained by AlphaFold2. Comparison with structures of other members of the same class and docking of the substrate allowed identification of the residues responsible for its very unusual enzyme selectivity.International Union of Crystallography5-Nitrosalicylate 1,2-dioxygenase (5NSDO) is an iron(II)-dependent dioxygenase involved in the aerobic degradation of 5-nitroanthranilic acid by the bacterium Bradyrhizobium sp. It catalyzes the opening of the 5-nitrosalicylate aromatic ring, a key step in the degradation pathway. Besides 5-nitrosalicylate, the enzyme is also active towards 5-chlorosalicylate. The X-ray crystallographic structure of the enzyme was solved at 2.1 Å resolution by molecular replacement using a model from the AI program AlphaFold. The enzyme crystallized in the monoclinic space group P21, with unit-cell parameters a = 50.42, b = 143.17, c = 60.07 Å, β = 107.3°. 5NSDO belongs to the third class of ring-cleaving dioxygenases. Members of this family convert para-diols or hydroxylated aromatic carboxylic acids and belong to the cupin superfamily, which is one of the most functionally diverse protein classes and is named on the basis of a conserved β-barrel fold. 5NSDO is a tetramer composed of four identical subunits, each folded as a monocupin domain. The iron(II) ion in the enzyme active site is coordinated by His96, His98 and His136 and three water molecules with a distorted octahedral geometry. The residues in the active site are poorly conserved compared with other dioxygenases of the third class, such as gentisate 1,2-dioxygenase and salicylate 1,2-dioxygenase. Comparison with these other representatives of the same class and docking of the substrate into the active site of 5NSDO allowed the identification of residues which are crucial for the catalytic mechanism and enzyme selectivity.Crystal structure of the monocupin ring-cleaving dioxygenase 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp.urn:issn:2059-7983doi:10.1107/S2059798323004199https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersJuly 2023Acta Crystallographica Section D: Structural Biologymed@iucr.org72059-79836326402023-06-1679https://creativecommons.org/licenses/by/4.0/Structure–function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role
http://scripts.iucr.org/cgi-bin/paper?jc5058
Multicopper oxidases are promiscuous biocatalysts with great potential for the production of industrial compounds. This study is focused on the elucidation of the structure–function determinants of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila (TtLMCO1), which is capable of oxidizing both ascorbic acid and phenolic compounds and thus is functionally categorized between the ascorbate oxidases and fungal ascomycete laccases (asco-laccases). The crystal structure of TtLMCO1, determined using an AlphaFold2 model due to a lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations showed that the ability of TtLMCO1 to oxidize ortho-substituted phenols stems from the movement of two polar amino acids at the hydrophilic side of the substrate-binding region, providing structural evidence for the promiscuity of this enzyme.LACCASE-LIKE MULTICOPPER OXIDASES; LMCOS; BIOCATALYSTS; CRYSTAL STRUCTURE; THERMOTHELOMYCES THERMOPHILA; MOLECULAR DOCKINGtextKosinas, C.Zerva, A.Topakas, E.Dimarogona, M.2023-06-16The crystal structure of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila was refined at 1.9 Å resolution. Ligand-docking simulations reveal conformational changes that influence substrate specificity.International Union of CrystallographyMulticopper oxidases are promiscuous biocatalysts with great potential for the production of industrial compounds. This study is focused on the elucidation of the structure–function determinants of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila (TtLMCO1), which is capable of oxidizing both ascorbic acid and phenolic compounds and thus is functionally categorized between the ascorbate oxidases and fungal ascomycete laccases (asco-laccases). The crystal structure of TtLMCO1, determined using an AlphaFold2 model due to a lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations showed that the ability of TtLMCO1 to oxidize ortho-substituted phenols stems from the movement of two polar amino acids at the hydrophilic side of the substrate-binding region, providing structural evidence for the promiscuity of this enzyme.Structure–function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological roleurn:issn:2059-7983doi:10.1107/S2059798323004175https://creativecommons.org/licenses/by/4.0/text/htmlen2023-06-1679https://creativecommons.org/licenses/by/4.0/med@iucr.org72059-7983641654July 2023Acta Crystallographica Section D: Structural Biology2059-7983research papersProtein–macrocycle polymorphism: crystal form IV of the Ralstonia solanacearum lectin–sulfonato-calix[8]arene complex
http://scripts.iucr.org/cgi-bin/paper?gm5094
Controlled protein assembly and crystallization is necessary as a means of generating diffraction-quality crystals as well as providing a basis for new types of biomaterials. Water-soluble calixarenes are useful mediators of protein crystallization. Recently, it was demonstrated that Ralstonia solanacearum lectin (RSL) co-crystallizes with anionic sulfonato-calix[8]arene (sclx8) in three space groups. Two of these co-crystals only grow at pH ≤ 4 where the protein is cationic, and the crystal packing is dominated by the calixarene. This paper describes a fourth RSL–sclx8 co-crystal, which was discovered while working with a cation-enriched mutant. Crystal form IV grows at high ionic strength in the pH range 5–6. While possessing some features in common with the previous forms, the new structure reveals alternative calixarene binding modes. The occurrence of C2-symmetric assemblies, with the calixarene at special positions, appears to be an important result for framework fabrication. Questions arise regarding crystal screening and exhaustive searching for polymorphs.[BETA]-PROPELLERS; CRYSTAL ENGINEERING LANDSCAPE; LECTINS; PROTEIN ASSEMBLY; SULFONATO-CALIX[8]ARENE FORM IV; RALSTONIA SOLANACEARUMtextMockler, N.M.Ramberg, K.O.Crowley, P.B.2023-06-14Calixarene-mediated protein assembly provides a basis for the development of new types of biomaterials. A fourth structure of the Ralstonia solanacearum lectin–sulfonato-calix[8]arene complex expands the crystal-engineering landscape and suggests an alternative pH trigger of assembly.International Union of CrystallographyControlled protein assembly and crystallization is necessary as a means of generating diffraction-quality crystals as well as providing a basis for new types of biomaterials. Water-soluble calixarenes are useful mediators of protein crystallization. Recently, it was demonstrated that Ralstonia solanacearum lectin (RSL) co-crystallizes with anionic sulfonato-calix[8]arene (sclx8) in three space groups. Two of these co-crystals only grow at pH ≤ 4 where the protein is cationic, and the crystal packing is dominated by the calixarene. This paper describes a fourth RSL–sclx8 co-crystal, which was discovered while working with a cation-enriched mutant. Crystal form IV grows at high ionic strength in the pH range 5–6. While possessing some features in common with the previous forms, the new structure reveals alternative calixarene binding modes. The occurrence of C2-symmetric assemblies, with the calixarene at special positions, appears to be an important result for framework fabrication. Questions arise regarding crystal screening and exhaustive searching for polymorphs.Protein–macrocycle polymorphism: crystal form IV of the Ralstonia solanacearum lectin–sulfonato-calix[8]arene complexurn:issn:2059-7983doi:10.1107/S2059798323003832https://creativecommons.org/licenses/by/4.0/text/htmlen631med@iucr.org72059-798362479https://creativecommons.org/licenses/by/4.0/2023-06-142059-7983research papersActa Crystallographica Section D: Structural BiologyJuly 2023Sequence-assignment validation in protein crystal structure models with checkMySequence
http://scripts.iucr.org/cgi-bin/paper?qo5004
Sequence-register shifts remain one of the most elusive errors in experimental macromolecular models. They may affect model interpretation and propagate to newly built models from older structures. In a recent publication, it was shown that register shifts in cryo-EM models of proteins can be detected using a systematic reassignment of short model fragments to the target sequence. Here, it is shown that the same approach can be used to detect register shifts in crystal structure models using standard, model-bias-corrected electron-density maps (2mFo − DFc). Five register-shift errors in models deposited in the PDB detected using this method are described in detail.MACROMOLECULAR CRYSTALLOGRAPHY; REGISTER SHIFTS; FINDMYSEQUENCE; MODEL VALIDATION; CHECKMYSEQUENCE; SEQUENCE VALIDATIONtextChojnowski, G.2023-06-14It is shown that checkMySequence, an automated method for validating sequence assignment in cryo-EM structures of proteins, can be used to validate crystal structure models.International Union of CrystallographySequence-register shifts remain one of the most elusive errors in experimental macromolecular models. They may affect model interpretation and propagate to newly built models from older structures. In a recent publication, it was shown that register shifts in cryo-EM models of proteins can be detected using a systematic reassignment of short model fragments to the target sequence. Here, it is shown that the same approach can be used to detect register shifts in crystal structure models using standard, model-bias-corrected electron-density maps (2mFo − DFc). Five register-shift errors in models deposited in the PDB detected using this method are described in detail.Sequence-assignment validation in protein crystal structure models with checkMySequenceurn:issn:2059-7983doi:10.1107/S2059798323003765https://creativecommons.org/licenses/by/4.0/text/htmlen5687med@iucr.org5592059-7983https://creativecommons.org/licenses/by/4.0/792023-06-142059-7983research papersActa Crystallographica Section D: Structural BiologyJuly 2023Cysteine synthase: multiple structures of a key enzyme in cysteine synthesis and a potential drug target for Chagas disease and leishmaniasis
http://scripts.iucr.org/cgi-bin/paper?di5064
Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-l-serine into l-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein–protein inhibitors.AMINO-ACID METABOLISM; CYSTEINE SYNTHASE; PLP-DEPENDENT ENZYMES; X-RAY CRYSTALLOGRAPHY; CHAGAS DISEASE; LEISHMANIASIS; TRYPANOSOMA THEILERI; TRYPANOSOMA CRUZI; LEISHMANIA INFANTUMtextSowerby, K.Freitag-Pohl, S.Murillo, A.M.Silber, A.M.Pohl, E.2023-05-19Biochemical and structural analyses of cysteine synthase, the key enzyme in cysteine biosynthesis, from the protozoan pathogens Trypanosoma cruzi, T. theileri and Leishmania infantum are presented. This enzyme is a potential drug target for neglected tropical diseases such as Chagas disease and leishmaniasis.International Union of CrystallographyChagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-l-serine into l-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein–protein inhibitors.Cysteine synthase: multiple structures of a key enzyme in cysteine synthesis and a potential drug target for Chagas disease and leishmaniasisurn:issn:2059-7983doi:10.1107/S2059798323003613https://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983research papersActa Crystallographica Section D: Structural BiologyJune 20235306med@iucr.org5182059-7983https://creativecommons.org/licenses/by/4.0/792023-05-19The CCP4 suite: integrative software for macromolecular crystallography
http://scripts.iucr.org/cgi-bin/paper?ai5011
The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.COLLABORATIVE COMPUTATIONAL PROJECT NO. 4; CCP4; CRYSTALLOGRAPHY SOFTWARE; MACROMOLECULAR CRYSTALLOGRAPHYtextAgirre, J.Atanasova, M.Bagdonas, H.Ballard, C.B.Baslé, A.Beilstein-Edmands, J.Borges, R.J.Brown, D.G.Burgos-Mármol, J.J.Berrisford, J.M.Bond, P.S.Caballero, I.Catapano, L.Chojnowski, G.Cook, A.G.Cowtan, K.D.Croll, T.I.Debreczeni, J.É.Devenish, N.E.Dodson, E.J.Drevon, T.R.Emsley, P.Evans, G.Evans, P.R.Fando, M.Foadi, J.Fuentes-Montero, L.Garman, E.F.Gerstel, M.Gildea, R.J.Hatti, K.Hekkelman, M.L.Heuser, P.Hoh, S.W.Hough, M.A.Jenkins, H.T.Jiménez, E.Joosten, R.P.Keegan, R.M.Keep, N.Krissinel, E.B.Kolenko, P.Kovalevskiy, O.Lamzin, V.S.Lawson, D.M.Lebedev, A.A.Leslie, A.G.W.Lohkamp, B.Long, F.Malý, M.McCoy, A.J.McNicholas, S.J.Medina, A.Millán, C.Murray, J.W.Murshudov, G.N.Nicholls, R.A.Noble, M.E.M.Oeffner, R.Pannu, N.S.Parkhurst, J.M.Pearce, N.Pereira, J.Perrakis, A.Powell, H.R.Read, R.J.Rigden, D.J.Rochira, W.Sammito, M.Sánchez Rodríguez, F.Sheldrick, G.M.Shelley, K.L.Simkovic, F.Simpkin, A.J.Skubak, P.Sobolev, E.Steiner, R.A.Stevenson, K.Tews, I.Thomas, J.M.H.Thorn, A.Valls, J.T.Uski, V.Usón, I.Vagin, A.Velankar, S.Vollmar, M.Walden, H.Waterman, D.Wilson, K.S.Winn, M.D.Winter, G.Wojdyr, M.Yamashita, K.2023-05-30This is the Collaborative Computational Project No. 4 (CCP4) general citation article. Please reference it alongside any CCP4 software used in published research.International Union of CrystallographyThe Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.The CCP4 suite: integrative software for macromolecular crystallographyurn:issn:2059-7983doi:10.1107/S2059798323003595https://creativecommons.org/licenses/by/4.0/text/htmlenhttps://creativecommons.org/licenses/by/4.0/792023-05-304616med@iucr.org4492059-7983Acta Crystallographica Section D: Structural BiologyJune 20232059-7983research papersAnalysis and validation of overall N-glycan conformation in Privateer
http://scripts.iucr.org/cgi-bin/paper?qe5003
The oligosaccharides in N-glycosylation provide key structural and functional contributions to a glycoprotein. These contributions are dependent on the composition and overall conformation of the glycans. The Privateer software allows structural biologists to evaluate and improve the atomic structures of carbohydrates, including N-glycans; this software has recently been extended to check glycan composition through the use of glycomics data. Here, a broadening of the scope of the software to analyse and validate the overall conformation of N-glycans is presented, focusing on a newly compiled set of glycosidic linkage torsional preferences harvested from a curated set of glycoprotein models.GLYCOBIOLOGY; VALIDATION; PRIVATEER; N-GLYCANS; TORSION ANGLEStextDialpuri, J.S.Bagdonas, H.Atanasova, M.Schofield, L.C.Hekkelman, M.L.Joosten, R.P.Agirre, J.2023-05-23The Privateer software allows structural biologists to evaluate and improve the atomic structures of carbohydrates, including N-glycans. This software has recently been extended to check glycan composition through the use of glycomics data, and the broadening of its scope is presented in this article.International Union of CrystallographyThe oligosaccharides in N-glycosylation provide key structural and functional contributions to a glycoprotein. These contributions are dependent on the composition and overall conformation of the glycans. The Privateer software allows structural biologists to evaluate and improve the atomic structures of carbohydrates, including N-glycans; this software has recently been extended to check glycan composition through the use of glycomics data. Here, a broadening of the scope of the software to analyse and validate the overall conformation of N-glycans is presented, focusing on a newly compiled set of glycosidic linkage torsional preferences harvested from a curated set of glycoprotein models.Analysis and validation of overall N-glycan conformation in Privateerurn:issn:2059-7983doi:10.1107/S2059798323003510https://creativecommons.org/licenses/by/4.0/text/htmlen4724622059-79836med@iucr.orghttps://creativecommons.org/licenses/by/4.0/792023-05-23research papers2059-7983Acta Crystallographica Section D: Structural BiologyJune 2023Near-atomic resolution reconstructions from in situ revitrified cryo samples
http://scripts.iucr.org/cgi-bin/paper?id5012
A microsecond time-resolved version of cryo-electron microscopy (cryo-EM) has recently been introduced to enable observation of the fast conformational motions of proteins. The technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in the liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. Two alternative implementations of the technique have previously been described, using either an optical microscope or performing revitrification experiments in situ. Here, it is shown that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within the spatial resolution. Interestingly, it is observed that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.MICROSECOND MELTING AND REVITRIFICATION; MICROSECOND TIME-RESOLVED CRYO-EM; PROTEIN DYNAMICS; TIME-RESOLVED ELECTRON MICROSCOPY; PREFERENTIAL ORIENTATIONtextBongiovanni, G.Harder, O.F.Voss, J.M.Drabbels, M.Lorenz, U.J.2023-05-23Near-atomic resolution reconstructions can be obtained from in situ melted and revitrified cryo samples. Revitrification results in a more homogeneous angular distribution.International Union of CrystallographyA microsecond time-resolved version of cryo-electron microscopy (cryo-EM) has recently been introduced to enable observation of the fast conformational motions of proteins. The technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in the liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. Two alternative implementations of the technique have previously been described, using either an optical microscope or performing revitrification experiments in situ. Here, it is shown that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within the spatial resolution. Interestingly, it is observed that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.Near-atomic resolution reconstructions from in situ revitrified cryo samplesurn:issn:2059-7983doi:10.1107/S2059798323003431https://creativecommons.org/licenses/by/4.0/text/htmlenhttps://creativecommons.org/licenses/by/4.0/792023-05-234784732059-79836med@iucr.orgActa Crystallographica Section D: Structural BiologyJune 2023research papers2059-7983The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctX
http://scripts.iucr.org/cgi-bin/paper?gi5041
Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX–SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.HELIX BENDING; PROTEIN-PROTEIN INTERACTIONS; TYPE III SECRETION SYSTEMS; YSCX; YSCY; ASCX; AEROMONAS HYDROPHILA; YERSINIA ENTEROCOLITICA; TYPE III SECRETION CHAPERONE SCTYtextGilzer, D.Kowal, J.L.Flottmann, F.Niemann, H.H.2023-05-19SctX proteins are secreted substrates and are essential components of type III secretion systems. The dedicated chaperone SctY escorts SctX proteins to the secretion machinery, where the hydrophobic helix at the C-terminus of SctX mediates recognition by the export apparatus. New crystal structures suggest that in the absence of the export gate this hydrophobic helix kinks and folds back onto the chaperone, presumably to shield it from the solvent.International Union of CrystallographyGram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX–SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctXurn:issn:2059-7983doi:10.1107/S2059798323003248https://creativecommons.org/licenses/by/4.0/text/htmlen6med@iucr.org5082059-79835172023-05-19https://creativecommons.org/licenses/by/4.0/792059-7983research papersJune 2023Acta Crystallographica Section D: Structural BiologyStructural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans
http://scripts.iucr.org/cgi-bin/paper?jc5055
In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.LIGNOCELLULOSE DEGRADATION; GLUCURONYL ESTERASES; HEMICELLULOSE; [ALPHA]/[BETA] HYDROLASES; BIOMASS CONVERSION; RARE XYLANS; LENTITHECIUM FLUVIATILEtextMazurkewich, S.Scholzen, K.C.Brusch, R.H.Poulsen, J.-C.N.Theibich, Y.Hüttner, S.Olsson, L.Larsbrink, J.Lo Leggio, L.2023-05-25Structural investigation of a presumed fungal glucuronoyl esterase reveals a classical serine hydrolase active site but an unusual ligand-binding site. Functional analysis showed a lack of activity on a wide array of substrates commonly utilized by this family of enzymes. It is hypothesized that this enzyme needs complex plant cell-wall substructures for activity.International Union of CrystallographyIn plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylansurn:issn:2059-7983doi:10.1107/S205979832300325Xhttps://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983545med@iucr.org65552023-05-2579https://creativecommons.org/licenses/by/4.0/research papers2059-7983June 2023Acta Crystallographica Section D: Structural BiologyStructural insight into an anti-BRIL Fab as a G-protein-coupled receptor crystallization chaperone
http://scripts.iucr.org/cgi-bin/paper?nj5317
Structure determination of G-protein-coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti-BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL-fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high-resolution crystal structure of the BRIL–SRP2070Fab complex. The structure of the BRIL–SRP2070Fab complex was determined at 2.1 Å resolution. This high-resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL–SRP2070Fab co-crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL-fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure-based drug design of membrane-protein drug targets.CRYSTAL STRUCTURE; CRYSTAL PACKING; APOCYTOCHROME B562; GPCRS; CRYSTALLIZATION CHAPERONES; ANTI-BRIL FABStextMiyagi, H.Suzuki, M.Yasunaga, M.Asada, H.Iwata, S.Saito, J.2023-04-26The structure of a complex between BRIL and an anti-BRIL antibody (SRP2070Fab) has been determined at high resolution. This work presents a detailed elucidation of the interaction between BRIL and SRP2070Fab, which may help to improve the function of SRP2070Fab as a crystallization chaperone for membrane proteins.International Union of CrystallographyStructure determination of G-protein-coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti-BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL-fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high-resolution crystal structure of the BRIL–SRP2070Fab complex. The structure of the BRIL–SRP2070Fab complex was determined at 2.1 Å resolution. This high-resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL–SRP2070Fab co-crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL-fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure-based drug design of membrane-protein drug targets.Structural insight into an anti-BRIL Fab as a G-protein-coupled receptor crystallization chaperoneurn:issn:2059-7983doi:10.1107/S205979832300311Xhttps://creativecommons.org/licenses/by/4.0/text/htmlen2059-7983435med@iucr.org54412023-04-2679https://creativecommons.org/licenses/by/4.0/research papers2059-7983May 2023Acta Crystallographica Section D: Structural BiologyThe X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report. Corrigendum
http://scripts.iucr.org/cgi-bin/paper?jc9047
A figure in the article by Barbarin-Bocahu & Graille [(2022), Acta Cryst. D78, 517–531] is corrected.STRUCTURAL BIOLOGY; PHASE PROBLEM; ALPHAFOLD; MOLECULAR REPLACEMENT; MACHINE-LEARNING 3D MODELS; CORRIGENDUMtextBarbarin-Bocahu, I.Graille, M.2023-03-30The article by Barbarin-Bocahu & Graille [(2022), Acta Cryst. D78, 517–531] is corrected.International Union of CrystallographyA figure in the article by Barbarin-Bocahu & Graille [(2022), Acta Cryst. D78, 517–531] is corrected.The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report. Corrigendumurn:issn:2059-7983doi:10.1107/S2059798323002826text/htmlen2059-7983addenda and errataActa Crystallographica Section D: Structural BiologyApril 20233534med@iucr.org3532059-7983792023-03-30Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain
http://scripts.iucr.org/cgi-bin/paper?rr5231
Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a β-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the β-bulge loop is required for supercoiling activity. It has previously been shown that the β-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β-bulge loop as a minimal latch is reported. It is shown that the β-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.REVERSE GYRASES; HELICASES; TOPOISOMERASES; LATCH; POSITIVE DNA SUPERCOILING; EXTREMOPHILES; INTER-DOMAIN COMMUNICATIONtextMhaindarkar, V.P.Rasche, R.Kümmel, D.Rudolph, M.G.Klostermeier, D.2023-05-19The latch domain in reverse gyrases mediates the cooperation of the helicase and topoisomerase domains. In Thermotoga maritima reverse gyrase this latch can be designed into a minimal β-bulge loop that has natural precedence in Thermosipho africanus reverse gyrase. The properties and functionalities of different latch domains across reverse gyrases are discussed.International Union of CrystallographyReverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a β-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the β-bulge loop is required for supercoiling activity. It has previously been shown that the β-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β-bulge loop as a minimal latch is reported. It is shown that the β-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domainurn:issn:2059-7983doi:10.1107/S2059798323002565https://creativecommons.org/licenses/by/4.0/text/htmlen5072059-7983498med@iucr.org679https://creativecommons.org/licenses/by/4.0/2023-05-19research papers2059-7983Acta Crystallographica Section D: Structural BiologyJune 2023GEMMI and Servalcat restrain REFMAC5
http://scripts.iucr.org/cgi-bin/paper?qe5004
Macromolecular refinement uses experimental data together with prior chemical knowledge (usually digested into geometrical restraints) to optimally fit an atomic structural model into experimental data, while ensuring that the model is chemically plausible. In the CCP4 suite this chemical knowledge is stored in a Monomer Library, which comprises a set of restraint dictionaries. To use restraints in refinement, the model is analysed and template restraints from the dictionary are used to infer (i) restraints between concrete atoms and (ii) the positions of riding hydrogen atoms. Recently, this mundane process has been overhauled. This was also an opportunity to enhance the Monomer Library with new features, resulting in a small improvement in REFMAC5 refinement. Importantly, the overhaul of this part of CCP4 has increased flexibility and eased experimentation, opening up new possibilities.REFMAC5; GEMMI; SERVALCAT; MODEL REFINEMENTtextYamashita, K.Wojdyr, M.Long, F.Nicholls, R.A.Murshudov, G.N.2023-05-04The restraint-generation part of the macromolecular atomic model-refinement program REFMAC5 has been delegated to GEMMI. A controller program was implemented in Servalcat to distribute tasks between GEMMI and REFMAC5.International Union of CrystallographyMacromolecular refinement uses experimental data together with prior chemical knowledge (usually digested into geometrical restraints) to optimally fit an atomic structural model into experimental data, while ensuring that the model is chemically plausible. In the CCP4 suite this chemical knowledge is stored in a Monomer Library, which comprises a set of restraint dictionaries. To use restraints in refinement, the model is analysed and template restraints from the dictionary are used to infer (i) restraints between concrete atoms and (ii) the positions of riding hydrogen atoms. Recently, this mundane process has been overhauled. This was also an opportunity to enhance the Monomer Library with new features, resulting in a small improvement in REFMAC5 refinement. Importantly, the overhaul of this part of CCP4 has increased flexibility and eased experimentation, opening up new possibilities.GEMMI and Servalcat restrain REFMAC5urn:issn:2059-7983doi:10.1107/S2059798323002413https://creativecommons.org/licenses/by/4.0/text/htmlenresearch papers2059-7983Acta Crystallographica Section D: Structural BiologyMay 20233733682059-79835med@iucr.orghttps://creativecommons.org/licenses/by/4.0/792023-05-04FLEXR: automated multi-conformer model building using electron-density map sampling
http://scripts.iucr.org/cgi-bin/paper?qe5002
Protein conformational dynamics that may inform biology often lie dormant in high-resolution electron-density maps. While an estimated ∼18% of side chains in high-resolution models contain alternative conformations, these are underrepresented in current PDB models due to difficulties in manually detecting, building and inspecting alternative conformers. To overcome this challenge, we developed an automated multi-conformer modeling program, FLEXR. Using Ringer-based electron-density sampling, FLEXR builds explicit multi-conformer models for refinement. Thereby, it bridges the gap of detecting hidden alternate states in electron-density maps and including them in structural models for refinement, inspection and deposition. Using a series of high-quality crystal structures (0.8–1.85 Å resolution), we show that the multi-conformer models produced by FLEXR uncover new insights that are missing in models built either manually or using current tools. Specifically, FLEXR models revealed hidden side chains and backbone conformations in ligand-binding sites that may redefine protein–ligand binding mechanisms. Ultimately, the tool facilitates crystallographers with opportunities to include explicit multi-conformer states in their high-resolution crystallographic models. One key advantage is that such models may better reflect interesting higher energy features in electron-density maps that are rarely consulted by the community at large, which can then be productively used for ligand discovery downstream. FLEXR is open source and publicly available on GitHub at https://github.com/TheFischerLab/FLEXR.CRYSTALLOGRAPHIC REFINEMENT; MODEL BUILDING; MULTI-CONFORMER; OCCUPANCY; CONFORMATIONAL FLEXIBILITY; FLEXRtextStachowski, T.R.Fischer, M.2023-04-18Alternative conformations are underrepresented in current PDB models due to difficulties in manually detecting, building and inspecting multiple conformers. To overcome this shortcoming, an automated multi-conformer modeling program, FLEXR, has been developed that uses Ringer-based electron-density sampling to explicitly build multi-conformer models for refinement.International Union of CrystallographyProtein conformational dynamics that may inform biology often lie dormant in high-resolution electron-density maps. While an estimated ∼18% of side chains in high-resolution models contain alternative conformations, these are underrepresented in current PDB models due to difficulties in manually detecting, building and inspecting alternative conformers. To overcome this challenge, we developed an automated multi-conformer modeling program, FLEXR. Using Ringer-based electron-density sampling, FLEXR builds explicit multi-conformer models for refinement. Thereby, it bridges the gap of detecting hidden alternate states in electron-density maps and including them in structural models for refinement, inspection and deposition. Using a series of high-quality crystal structures (0.8–1.85 Å resolution), we show that the multi-conformer models produced by FLEXR uncover new insights that are missing in models built either manually or using current tools. Specifically, FLEXR models revealed hidden side chains and backbone conformations in ligand-binding sites that may redefine protein–ligand binding mechanisms. Ultimately, the tool facilitates crystallographers with opportunities to include explicit multi-conformer states in their high-resolution crystallographic models. One key advantage is that such models may better reflect interesting higher energy features in electron-density maps that are rarely consulted by the community at large, which can then be productively used for ligand discovery downstream. FLEXR is open source and publicly available on GitHub at https://github.com/TheFischerLab/FLEXR.FLEXR: automated multi-conformer model building using electron-density map samplingurn:issn:2059-7983doi:10.1107/S2059798323002498https://creativecommons.org/licenses/by/4.0/text/htmlen2023-04-18https://creativecommons.org/licenses/by/4.0/793542059-79835med@iucr.org367May 2023Acta Crystallographica Section D: Structural Biologyresearch papers2059-7983Conformational transition of the Ixodes ricinus salivary serpin Iripin-4
http://scripts.iucr.org/cgi-bin/paper?jv5014
Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Å and the structure of the cleaved conformation was solved at 2.0 Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.SERPINS; IRIPIN-4; X-RAY STRUCTURE; NATIVE CONFORMATION; CLEAVED CONFORMATION; IXODES RICINUStextKascakova, B.Kotal, J.Havlickova, P.Vopatkova, V.Prudnikova, T.Grinkevich, P.Kuty, M.Chmelar, J.Kuta Smatanova, I.2023-04-24The crystal structures of two important conformations of a new serpin from I. ricinus, namely the partially stressed native and cleaved conformations, were solved at 2.3 and 2.0 Å resolution, respectively. The importance of the reactive-centre loop in protease inhibition was also confirmed.International Union of CrystallographyIripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Å and the structure of the cleaved conformation was solved at 2.0 Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.Conformational transition of the Ixodes ricinus salivary serpin Iripin-4urn:issn:2059-7983doi:10.1107/S2059798323002322https://creativecommons.org/licenses/by/4.0/text/htmlen419med@iucr.org52059-798340979https://creativecommons.org/licenses/by/4.0/2023-04-242059-7983research papersActa Crystallographica Section D: Structural BiologyMay 2023The structure of the complex between the arsenite oxidase from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c552
http://scripts.iucr.org/cgi-bin/paper?cb5144
The arsenite oxidase (AioAB) from Pseudorhizobium banfieldiae sp. strain NT-26 catalyzes the oxidation of arsenite to arsenate and transfers electrons to its cognate electron acceptor cytochrome c552 (cytc552). This activity underpins the ability of this organism to respire using arsenite present in contaminated environments. The crystal structure of the AioAB/cytc552 electron transfer complex reveals two A2B2/(cytc552)2 assemblies per asymmetric unit. Three of the four cytc552 molecules in the asymmetric unit dock to AioAB in a cleft at the interface between the AioA and AioB subunits, with an edge-to-edge distance of 7.5 Å between the heme of cytc552 and the [2Fe–2S] Rieske cluster in the AioB subunit. The interface between the AioAB and cytc552 proteins features electrostatic and nonpolar interactions and is stabilized by two salt bridges. A modest number of hydrogen bonds, salt bridges and relatively small, buried surface areas between protein partners are typical features of transient electron transfer complexes. Interestingly, the fourth cytc552 molecule is positioned differently between two AioAB heterodimers, with distances between its heme and the AioAB redox active cofactors that are outside the acceptable range for fast electron transfer. This unique cytc552 molecule appears to be positioned to facilitate crystal packing rather than reflecting a functional complex.ELECTRON TRANSFER COMPLEXES; X-RAY CRYSTALLOGRAPHY; ARSENITE; MOLYBDENUM ENZYMES; PSEUDORHIZOBIUM BANFIELDIAE SP. STRAIN NT-26; CYTOCHROME C552textPoddar, N.Santini, J.M.Maher, M.J.2023-03-30The crystal structure of the electron transfer complex between arsenite oxidase (AioAB) from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c552 (cytc552) is presented. Cytc552 docks within a cleft at the interface of the AioA and AioB subunits, which allows a close association between redox cofactors.; this close association presumably facilitates fast electron transfer and underpins the ability of this organism to respire in arsenic contaminated environments.International Union of CrystallographyThe arsenite oxidase (AioAB) from Pseudorhizobium banfieldiae sp. strain NT-26 catalyzes the oxidation of arsenite to arsenate and transfers electrons to its cognate electron acceptor cytochrome c552 (cytc552). This activity underpins the ability of this organism to respire using arsenite present in contaminated environments. The crystal structure of the AioAB/cytc552 electron transfer complex reveals two A2B2/(cytc552)2 assemblies per asymmetric unit. Three of the four cytc552 molecules in the asymmetric unit dock to AioAB in a cleft at the interface between the AioA and AioB subunits, with an edge-to-edge distance of 7.5 Å between the heme of cytc552 and the [2Fe–2S] Rieske cluster in the AioB subunit. The interface between the AioAB and cytc552 proteins features electrostatic and nonpolar interactions and is stabilized by two salt bridges. A modest number of hydrogen bonds, salt bridges and relatively small, buried surface areas between protein partners are typical features of transient electron transfer complexes. Interestingly, the fourth cytc552 molecule is positioned differently between two AioAB heterodimers, with distances between its heme and the AioAB redox active cofactors that are outside the acceptable range for fast electron transfer. This unique cytc552 molecule appears to be positioned to facilitate crystal packing rather than reflecting a functional complex.The structure of the complex between the arsenite oxidase from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c552urn:issn:2059-7983doi:10.1107/S2059798323002103https://creativecommons.org/licenses/by/4.0/text/htmlenApril 2023Acta Crystallographica Section D: Structural Biology2059-7983research papers2023-03-30https://creativecommons.org/licenses/by/4.0/794med@iucr.org3452059-7983352Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experiment
http://scripts.iucr.org/cgi-bin/paper?nz5010
To determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F−) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.SUBSTRUCTURE DETERMINATION; EXPERIMENTAL PHASING; MULTIVARIATE STATISTICS; DIRECT METHODS; SINGLE-WAVELENGTH ANOMALOUS DIFFRACTION; AFROtextPannu, N.S.Skubák, P.2023-03-28A new equation for the calculation of substructure-factor amplitudes for substructure detection from a single-wavelength anomalous diffraction experiment produces better results compared with the currently used estimates in test cases.International Union of CrystallographyTo determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F−) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experimenturn:issn:2059-7983doi:10.1107/S2059798323001997https://creativecommons.org/licenses/by/4.0/text/htmlenActa Crystallographica Section D: Structural BiologyApril 20232059-7983research papers79https://creativecommons.org/licenses/by/4.0/2023-03-28344med@iucr.org42059-7983339