Structural and biophysical studies of new l-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase
This work reports the results of random mutagenesis of the Escherichia coli class 2 l-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the l-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these muta...
Source: Acta Crystallographica Section D - June 28, 2022 Category: Biochemistry Authors: Loch, J.I. Klonecka, A. K ą dzio ł ka, K. Bonarek, P. Barciszewski, J. Imiolczyk, B. Brzezinski, K. Gilski, M. Jaskolski, M. Tags: l-asparaginases Ntn-hydrolases random mutagenesis leukemia research papers Source Type: research
Development of CryoVR, a virtual reality training system for hands-on cryoEM operations
Cryogenic electron microscopy (cryoEM) has emerged as a revolutionary method for solving high-resolution structures and studying the dynamics of macromolecular complexes and viruses in near-native states. However, the availability of the equipment, and the time and cost needed for training, severely limit the opportunities for training. To solve these problems, a virtual reality-based training system, CryoVR, has been developed to prepare trainees before operating real-world cryoEM equipment. This paper describes the design and assessment of CryoVR (available at https://www.purdue.edu/cryoVR), which helps users learn cryoE...
Source: Acta Crystallographica Section D - June 27, 2022 Category: Biochemistry Authors: Dong, J. Li, D. Ozcan, K. Wan, D. Jiang, W. Chen, Y. Tags: CryoVR cryoEM virtual reality virtual reality training human – computer interaction structural biology research papers Source Type: research
AutoPX: a new software package to process X-ray diffraction data from biomacromolecular crystals
A new software package, autoPX, for processing X-ray diffraction data from biomacromolecular crystals is reported. This processing software package is designed on the basis of novel methods such as the location of diffraction spots by an improved Canny operator, indexing by a modified Fourier transform, a novel definition of mosaicity that expresses the dispersion state of reciprocal diffraction spots, and the correction of predicted diffraction spot coordinates by homography transform. New programming of some traditional algorithms necessary for integration and scaling is also included. Several examples of crystal structu...
Source: Acta Crystallographica Section D - June 27, 2022 Category: Biochemistry Authors: Wang, L. Yun, Y. Zhu, Z. Niu, L. Tags: autoPX X-ray diffraction data processing biomacromolecular crystals Canny operator Fourier transform mosaicity homography transform research papers Source Type: research
Microsecond melting and revitrification of cryo samples: protein structure and beam-induced motion
A novel approach to time-resolved cryo-electron microscopy (cryo-EM) has recently been introduced that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in the liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time resolution of just a few microseconds, this approach is notably fast enough to study the domain motions that are typically associated with the activity of proteins but which have previously remained inaccessible. Here, crucial details are added to the characterization of the method. It is shown that single-...
Source: Acta Crystallographica Section D - June 14, 2022 Category: Biochemistry Authors: Harder, O.F. Voss, J.M. Olshin, P.K. Drabbels, M. Lorenz, U.J. Tags: microsecond time-resolved cryo-EM laser melting revitrification single-particle reconstructions beam-induced motion research papers Source Type: research
Biochemical and structural insights into an unusual, alkali-metal-independent S-adenosyl-l-homocysteine hydrolase from Synechocystis sp. PCC 6803
The mesophilic cyanobacterium Synechocystis sp. PCC 6803 encodes an S-adenosyl-l-homocysteine hydrolase (SAHase) of archaeal origin in its genome. SAHases are essential enzymes involved in the regulation of cellular S-adenosyl-l-methionine (SAM)-dependent methylation reactions. They are usually active as homotetramers or, less commonly, as homodimers. A SAHase subunit is composed of two major domains: a cofactor (NAD+)-binding domain and a substrate (S-adenosyl-l-homocysteine)-binding domain. These are connected by a hinge element that is also a coordination site for an alkali-metal cation that influences domain movement d...
Source: Acta Crystallographica Section D - June 14, 2022 Category: Biochemistry Authors: Malecki, P.H. Imiolczyk, B. Barciszewski, J. Czyrko-Horczak, J. Sliwiak, J. Gawel, M. Wozniak, K. Jaskolski, M. Brzezinski, K. Tags: cyanobacteria Synechocystis sp. PCC 6803 S-adenosyl-l-homocysteine hydrolase cellular methylation protein oligomerization metal cation-binding proteins protein dynamics research papers Source Type: research
Structural basis of ligand recognition and transport by Sfh2, a yeast phosphatidylinositol transfer protein of the Sec14 superfamily
This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2. (Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - June 14, 2022 Category: Biochemistry Authors: Chen, L. Tan, L. Im, Y.J. Tags: lipid transport yeast Sfh2 Sec14 phosphatidylinositol squalene lipid-transfer proteins research papers Source Type: research
Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate
Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and l-aspartate- β -semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidi...
Source: Acta Crystallographica Section D - June 8, 2022 Category: Biochemistry Authors: Chooback, L. Thomas, L.N. Blythe, N. Karsten, W. Tags: crystallography dihydrodipicolinate synthase (S)-2-bromopropionate lysine biosynthesis enzyme structure enzyme inhibition research papers Source Type: research
Structural analysis of LpqY, a substrate-binding protein from the SugABC transporter of Mycobacterium tuberculosis, provides insights into its trehalose specificity
The LpqY-SugABC transporter of Mycobacterium tuberculosis (Mtb) salvages residual trehalose across the cell membrane, which is otherwise lost during the formation of cell-wall glycoconjugates in the periplasm. LpqY, a substrate-binding protein from the SugABC transporter, acts as the primary receptor for the recognition of trehalose, leading to its transport across the cell membrane. Since trehalose is crucial for the survival and virulence of Mtb, trehalose receptors should serve as important targets for novel drug design against tuberculosis. In order to comprehend the detailed architecture and substrate specificity, the...
Source: Acta Crystallographica Section D - June 7, 2022 Category: Biochemistry Authors: Sharma, D. Singh, M. Kaur, P. Das, U. Tags: LpqY SugABC substrate-binding proteins Mycobacterium tuberculosis research papers Source Type: research
Crystal structures of the molecular class A β -lactamase TEM-171 and its complexes with tazobactam
The resistance of bacteria to β -lactam antibiotics is primarily caused by the production of β -lactamases. Here, novel crystal structures of the native β -lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical β -lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex ...
Source: Acta Crystallographica Section D - June 7, 2022 Category: Biochemistry Authors: Grigorenko, V.G. Petrova, T.E. Carolan, C. Rubtsova, M.Y. Uporov, I.V. Pereira, J. Chojnowski, G. Samygina, V.R. Lamzin, V.S. Egorov, A.M. Tags: β -lactamase TEM-171 tazobactam intermediate antibiotic resistance enzyme inhibition crystal structure UV spectroscopy research papers Source Type: research
High-resolution structure determination using high-throughput electron cryo-tomography
Tomographic reconstruction of frozen-hydrated specimens followed by extraction and averaging of sub-tomograms has successfully been used to determine the structure of macromolecules in their native environment at resolutions that are high enough to reveal molecular level interactions. The low throughput characteristic of tomographic data acquisition combined with the complex data-analysis pipeline that is required to obtain high-resolution maps, however, has limited the applicability of this technique to favorable samples or to resolutions that are too low to provide useful mechanistic information. Recently, beam image-shi...
Source: Acta Crystallographica Section D - June 7, 2022 Category: Biochemistry Authors: Liu, H.-F. Zhou, Y. Bartesaghi, A. Tags: electron cryo-tomography sub-volume averaging in situ structure determination beam-image shift tomography constrained single-particle tomography research papers Source Type: research
Sequence-assignment validation in cryo-EM models with checkMySequence
The availability of new artificial intelligence-based protein-structure-prediction tools has radically changed the way that cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register shifts remain one of the most difficult to identify and correct. Here, checkMySequence, a fast, fully automated and parameter-free method for detecting register shifts in protein models built into cryo-EM maps, is introduced. It is shown tha...
Source: Acta Crystallographica Section D - June 7, 2022 Category: Biochemistry Authors: Chojnowski, G. Tags: cryo-EM register shifts sequence assignment model validation checkMySequence research papers Source Type: research
Pentameric assembly of the Kv2.1 tetramerization domain
In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER – plasma membrane contacts. (Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - May 30, 2022 Category: Biochemistry Authors: Xu, Z. Khan, S. Schnicker, N.J. Baker, S. Tags: voltage-gated potassium channels Kv2.1 tetramerization domain crystal structure SAXS pentamer research papers Source Type: research
Mamannamana Vijayan (1941 – 2022)
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - May 25, 2022 Category: Biochemistry Authors: Baker, E.N. Tags: Vijayan obituaries Source Type: research
Structural visualization of transient interactions between the cis-acting acyltransferase and acyl carrier protein of the salinomycin modular polyketide synthase
Transient protein – protein interactions between cis-acting acyltransferase (AT) and acyl carrier protein (ACP) domains are critical for the catalysis and processivity of modular polyketide synthases (mPKSs), but are challenging for structural characterization due to the intrinsically weak binding affinity. Here, a stable complex of cis-acting AT and ACP domains from the ninth module of the salinomycin mPKS was obtained using a maleimide cross-linker and the structure of the complex was determined at 2.6 Å resolution. The crystal structure shows that the AT in combination with the ketosynthase (KS)-to-AT linker form...
Source: Acta Crystallographica Section D - May 25, 2022 Category: Biochemistry Authors: Feng, Y. Zhang, F. Huang, S. Deng, Z. Bai, L. Zheng, J. Tags: salinomycin modular polyketide synthases polyketides protein – protein interactions acyltransferases acyl carrier proteins cross-linking research papers Source Type: research
Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre
Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Å resolution when exposed to XFEL radiation, which is an improvement of 0.15 Å over previous micr...
Source: Acta Crystallographica Section D - May 25, 2022 Category: Biochemistry Authors: B å th, P. Banacore, A. B ö rjesson, P. Bosman, R. Wickstrand, C. Safari, C. Dods, R. Ghosh, S. Dahl, P. Ortolani, G. Bj ö rg Ulfarsdottir, T. Hammarin, G. Garc í a Bonete, M.-J. Vallejos, A. Ostoji ć , L. Edlund, P. Linse, J.-B. Andersson, R. Nango, Tags: membrane-protein crystallization photosynthetic reaction centre Blastochloris viridis serial crystallography lipidic cubic phase microcrystals research papers Source Type: research
