Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of high-quality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the air – water interface. Here, it is shown that coating cryo-EM grids with a negatively charged...
Source: Acta Crystallographica Section D - October 20, 2022 Category: Biochemistry Authors: Hreb í k, D. Gondov á , M. Valentov á , L. F ü zik, T. P ř idal, A. Nov á č ek, J. Plevka, P. Tags: cryo-EM sample preparation ssDNA coating cryo-EM grids aggregation research papers Source Type: research
A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking
Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0 – ...
Source: Acta Crystallographica Section D - October 20, 2022 Category: Biochemistry Authors: Trewhella, J. Vachette, P. Bierma, J. Blanchet, C. Brookes, E. Chakravarthy, S. Chatzimagas, L. Cleveland, T.E. Cowieson, N. Crossett, B. Duff, A.P. Franke, D. Gabel, F. Gillilan, R.E. Graewert, M. Grishaev, A. Guss, J.M. Hammel, M. Hopkins, J. Huang, Q. Tags: biomolecular small-angle scattering X-ray scattering neutron scattering standards benchmarking standards scattering-profile calculation research papers Source Type: research
Verification: model-free phasing with enhanced predicted models in ARCIMBOLDO_SHREDDER
Structure predictions have matched the accuracy of experimental structures from close homologues, providing suitable models for molecular replacement phasing. Even in predictions that present large differences due to the relative movement of domains or poorly predicted areas, very accurate regions tend to be present. These are suitable for successful fragment-based phasing as implemented in ARCIMBOLDO. The particularities of predicted models are inherently addressed in the new predicted_model mode, rendering preliminary treatment superfluous but also harmless. B-value conversion from predicted LDDT or error estimates, the ...
Source: Acta Crystallographica Section D - October 20, 2022 Category: Biochemistry Authors: Medina, A. Jim é nez, E. Caballero, I. Castellv í , A. Trivi ñ o Valls, J. Alcorlo, M. Molina, R. Hermoso, J.A. Sammito, M.D. Borges, R. Us ó n, I. Tags: phasing ARCIMBOLDO ARCIMBOLDO_SHREDDER fragment-based molecular replacement verification model bias predictions AlphaFold RoseTTAFold research papers Source Type: research
Model building and beyond
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - September 29, 2022 Category: Biochemistry Authors: Agirre, J. Joosten, R.P. Roseman, A. Tags: CCP4 Study Weekend model building integrative structural biology editorial Source Type: research
Structural basis for the transformation of the traditional medicine berberine by bacterial nitroreductase
The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Wen, H.-Y. Pan, L.-B. Ma, S.-R. Yang, X.-Y. Hu, J.-C. Zhao, H.-F. Gao, Z.-Q. Dong, Y.-H. Wang, Y. Zhang, H. Tags: berberine bacterial nitroreductases Escherichia coli NfsB flavoproteins crystal structure substrate recognition research papers Source Type: research
Structural features of chloroplast trigger factor determined at 2.6 Å resolution
The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis – trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their b...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Carius, Y. Ries, F. Gries, K. Trentmann, O. Lancaster, C.R.D. Willmund, F. Tags: molecular chaperones chaperone trigger factor chloroplasts Chlamydomonas reinhardtii co-translational folding PPIases research papers Source Type: research
MembraneDyn: simulating the dynamics of supported membrane stacks on the nanosecond timescale
The static structure factor and the undulation dynamics of a solid-supported membrane stack have previously been calculated by Romanov and Ul'yanov [Romanov& Ul'yanov (2002). Phys. Rev. E, 66, 061701]. Based on this prior work, the calculation has been extended to cover the membrane dynamics, i.e. the intermediate scattering function as a Fourier transform of the van Hove correlation function of the membrane stack. Fortran code which calculates the intermediate scattering function for a membrane stack on a solid support is presented. It allows the static and dynamic scattering functions to be calculated according to the de...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Hayward, D.W. Jaksch, S. Fomina, M. Dubey, P.S. Frielinghaus, H. Holderer, O. Monkenbusch, M. Tags: dynamical simulations materials modelling grazing-incidence diffraction neutron spin-echo spectroscopy supported membrane stacks time-dependent intermediate scattering function research papers Source Type: research
Substrate-induced dimerization of elaiophylin glycosyltransferase reveals a novel self-activating form of glycosyltransferase for symmetric glycosylation
Elaiophylin (Ela), a unique 16-membered symmetric macrodiolide antibiotic, displays broad biological activity. Two rare 2-deoxy-l-fucose moieties at the ends of Ela are critical for its activity. Previously, elaiophylin glycosyltransferase (ElaGT) was identified as the enzyme that is responsible for the symmetric glycosylation of Ela, acting as a potential enzymatic tool for enhancing the diversity and activity of Ela. However, a symmetric catalytic mechanism has never been reported for a glycosyltransferase (GT). To explore the catalytic mechanism, the structure of ElaGT was determined in four forms: the apo form and Ela-...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Xu, T. Gan, Q. Liu, Q. Chen, R. Zhen, X. Zhang, C. Liu, J. Tags: elaiophylin glycosyltransferases crystal structure dimer antibiotics symmetric glycosylation research papers Source Type: research
Dependence of crystallographic atomic displacement parameters on temperature (25 – 150 K) for complexes of horse liver alcohol dehydrogenase
Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1 – 1.3 Å resolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and st...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Plapp, B.V. Gakhar, L. Subramanian, R. Tags: B factors anisotropic displacement parameters enzyme dynamics TLS analysis hydrogen transfer alcohol dehydrogenases helium cryostat X-ray crystallography research papers Source Type: research
Crystallographic and X-ray scattering study of RdfS, a recombination directionality factor from an integrative and conjugative element
The recombination directionality factors from Mesorhizobium spp. (RdfS) are involved in regulating the excision and transfer of integrative and conjugative elements. Here, solution small-angle X-ray scattering, and crystallization and preliminary structure solution of RdfS from Mesorhizobium japonicum R7A are presented. RdfS crystallizes in space group P212121, with evidence of eightfold rotational crystallographic/noncrystallographic symmetry. Initial structure determination by molecular replacement using ab initio models yielded a partial model (three molecules), which was completed after manual inspection revealed unmod...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Verdonk, C.J. Marshall, A.C. Ramsay, J.P. Bond, C.S. Tags: RdfS recombination directionality factor Mesorhizobium noncrystallographic symmetry winged helix – turn helix domain DNA binding excisionase research papers Source Type: research
Atomic resolution studies of S1 nuclease complexes reveal details of RNA interaction with the enzyme despite multiple lattice-translocation defects
Conclusions derived from this comparison are valid for the whole S1/P1 nuclease family. For proper model building and refinement, multiple lattice-translocation defects present in the measured diffraction data needed to be solved. Two different approaches were tested and compared. Correction of the measured intensities proved to be superior to the use of the dislocation model of asymmetric units with partial occupancy of individual chains. As the crystals suffered from multiple lattice translocations, equations for their correction were derived de novo. The presented approach to the correction of multiple lattice-transloca...
Source: Acta Crystallographica Section D - September 27, 2022 Category: Biochemistry Authors: Ad á mkov á , K. Koval', T. Ø stergaard, L.H. Du š kov á , J. Mal ý , M. Š vecov á , L. Sk á lov á , T. Kolenko, P. Dohn á lek, J. Tags: S1 nuclease Aspergillus oryzae lattice-translocation defects nucleotides nucleosides complexes research papers Source Type: research
Crystal structure of a novel homodimeric d-allulose 3-epimerase from a Clostridia bacterium
d-Allulose, a low-calorie rare sugar with various physiological functions, is mainly produced through the isomerization of d-fructose by ketose 3-epimerases (KEases), which exhibit various substrate specificities. A novel KEase from a Clostridia bacterium (CDAE) was identified to be a d-allulose 3-epimerase and was further characterized as thermostable and metal-dependent. In order to explore its structure – function relationship, the crystal structure of CDAE was determined using X-ray diffraction at 2.10 Å resolution, revealing a homodimeric d-allulose 3-epimerase structure with extensive interactions formed at th...
Source: Acta Crystallographica Section D - August 30, 2022 Category: Biochemistry Authors: Xie, X. Tian, Y. Ban, X. Li, C. Yang, H. Li, Z. Tags: d-allulose ketose 3-epimerases crystal structure hydrophobic pocket Clostridia research papers Source Type: research
Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states
In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-li...
Source: Acta Crystallographica Section D - August 30, 2022 Category: Biochemistry Authors: Chen, A. Mindrebo, J.T. Davis, T.D. Kim, W.E. Katsuyama, Y. Jiang, Z. Ohnishi, Y. Noel, J.P. Burkart, M.D. Tags: ketosynthases fatty-acid synthases Escherichia coli ketosynthase FabB decarboxylative condensation reaction acyl carrier proteins protein cross-linked complex research papers Source Type: research
CCP4 Cloud for structure determination and project management in macromolecular crystallography
Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links betwe...
Source: Acta Crystallographica Section D - August 30, 2022 Category: Biochemistry Authors: Krissinel, E. Lebedev, A.A. Uski, V. Ballard, C.B. Keegan, R.M. Kovalevskiy, O. Nicholls, R.A. Pannu, N.S. Skub á k, P. Berrisford, J. Fando, M. Lohkamp, B. Wojdyr, M. Simpkin, A.J. Thomas, J.M.H. Oliver, C. Vonrhein, C. Chojnowski, G. Basle, A. Purkiss, Tags: CCP4 CCP4 Cloud macromolecular crystallography distributed computing data management project management research papers Source Type: research
Structural bases for the higher adherence to ACE2 conferred by the SARS-CoV-2 spike Q498Y substitution
A remarkable number of SARS-CoV-2 variants and other as yet unmonitored lineages harbor amino-acid substitutions with the potential to modulate the interface between the spike receptor-binding domain (RBD) and its receptor ACE2. The naturally occurring Q498Y substitution, which is present in currently circulating SARS-CoV-2 variants, has drawn the attention of several investigations. While computational predictions and in vitro binding studies suggest that Q498Y increases the binding affinity of the spike protein for ACE2, experimental in vivo models of infection have shown that a triple mutant carrying the Q498Y replaceme...
Source: Acta Crystallographica Section D - August 25, 2022 Category: Biochemistry Authors: Erausquin, E. Glaser, F. Fern á ndez-Recio, J. L ´ pez-Sagaseta, J. Tags: SARS-CoV-2 COVID-19 X-ray structure spike protein Q498Y mutation binding affinity spike protein receptor binding domain RBD – ACE2 complex ACE2 receptor research papers Source Type: research
