Contamination or serendipity – doing the wrong thing by chance
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - August 31, 2020 Category: Biochemistry Authors: Newman, J. van Raaij, M.J. Tags: contaminants editorial Source Type: research
Detecting the nature and solving the crystal structure of a contaminant protein from an opportunistic pathogen
The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is...
Source: Acta Crystallographica Section F - August 27, 2020 Category: Biochemistry Authors: Pederzoli, R. Tarantino, D. Gourlay, L.J. Chaves-Sanjuan, A. Bolognesi, M. Tags: contaminant proteins cyanase hydratase Serratia research communications Source Type: research
The structure of the Moco carrier protein from Rippkaea orientalis
The molybdenum cofactor (Moco) is the prosthetic group of all molybdenum-dependent enzymes except for nitrogenase. The multistep biosynthesis pathway of Moco and its function in molybdenum-dependent enzymes are already well understood. The mechanisms of Moco transfer, storage and insertion, on the other hand, are not. In the cell, Moco is usually not found in its free form and remains bound to proteins because of its sensitivity to oxidation. The green alga Chlamydomonas reinhardtii harbors a Moco carrier protein (MCP) that binds and protects Moco but is devoid of enzymatic function. It has been speculated that this MCP ac...
Source: Acta Crystallographica Section F - August 27, 2020 Category: Biochemistry Authors: Krausze, J. Hercher, T.W. Archna, A. Kruse, T. Tags: molybdenum cofactor Moco carrier protein Rossmann fold molecular docking research communications Source Type: research
Crystal structures of native cytochrome c6 from Thermosynechococcus elongatus in two different space groups and implications for its oligomerization
Native cytochrome c6 was purified from an extract of strain BP-1 of the thermophilic cyanobacterium Thermosynechococcus elongatus. The protein was crystallized, and with only slight modifications of the buffer and vapour-diffusion conditions two different space groups were observed, namely H3 and C2. Both crystal structures were solved; they contained three and six molecules per asymmetric unit and were refined to 1.7 and 2.25 Å resolution, respectively. To date, the structure of native cytochrome c6 from T. elongatus has only been reported as a monomer using NMR spectroscopy, i.e. without addressing putative oligomer...
Source: Acta Crystallographica Section F - August 19, 2020 Category: Biochemistry Authors: Falke, S. Feiler, C. Chapman, H. Sarrou, I. Tags: cyanobacteria photosynthesis oligomerization mass spectrometry redox model protein research communications Source Type: research
Crystal structure of the Escherichia coli transcription termination factor Rho
During the crystal structure analysis of an ATP-binding cassette (ABC) transporter overexpressed in Escherichia coli, a contaminant protein was crystallized. The identity of the contaminant was revealed by mass spectrometry to be the Escherichia coli transcription terminator factor Rho, structures of which had been previously determined in different conformational states. Although Rho was present at only ∼ 1% of the target protein (a bacterial homolog of the eukaryotic ABC transporter of mitochondria from Novosphingobium aromaticivorans; NaAtm1), it preferentially crystallized in space group C2 as thin plates that diffra...
Source: Acta Crystallographica Section F - August 19, 2020 Category: Biochemistry Authors: Fan, C. Rees, D.C. Tags: membrane-protein purification crystallization contaminant transcription termination factor cryoEM research communications Source Type: research
Tetragonal crystal form of the cyanobacterial bicarbonate-transporter regulator SbtB from Synechocystis sp. PCC 6803
The PII-like protein SbtB has been identified as a regulator of SbtA, which is one of the key bicarbonate transporters in cyanobacteria. While SbtB from Synechocystis sp. PCC 6803 has previously been shown to be a trimer, a new crystal form is reported here which crystallizes in what is thought to be a non-native tetramer in the crystal, with the C-terminus in an extended conformation. The crystal structure shows the formation of an intermolecular disulfide bond at Cys94 between SbtB monomers, which may stabilize this conformation in the crystal. This motivates the need for future studies to investigate the potential role ...
Source: Acta Crystallographica Section F - August 18, 2020 Category: Biochemistry Authors: Bu, G. Simmons, C.R. Nielsen, D.R. Nannenga, B.L. Tags: cyanobacteria bicarbonate transport SbtB Synechocystis research communications Source Type: research
Binding of inhibitors to active-site mutants of CD1, the enigmatic catalytic domain of histone deacetylase 6
The zinc hydrolase histone deacetylase 6 (HDAC6) is unique among vertebrate deacetylases in that it contains two catalytic domains, designated CD1 and CD2. Both domains are fully functional as lysine deacetylases in vitro. However, the in vivo function of only the CD2 domain is well defined, whereas that of the CD1 domain is more enigmatic. Three X-ray crystal structures of HDAC6 CD1 – inhibitor complexes are now reported to broaden the understanding of affinity determinants in the active site. Notably, cocrystallization with inhibitors was facilitated by using active-site mutants of zebrafish HDAC6 CD1. The first mutant...
Source: Acta Crystallographica Section F - August 18, 2020 Category: Biochemistry Authors: Osko, J.D. Christianson, D.W. Tags: zinc enzymes hydrolases enzyme inhibitors drug design research communications Source Type: research
The structure of PfGH50B, an agarase from the marine bacterium Pseudoalteromonas fuliginea PS47
The recently identified marine bacterium Pseudoalteromonas fuliginea sp. PS47 possesses a polysaccharide-utilization locus dedicated to agarose degradation. In particular, it contains a gene (locus tag EU509_06755) encoding a β -agarase that belongs to glycoside hydrolase family 50 (GH50), PfGH50B. The 2.0 Å resolution X-ray crystal structure of PfGH50B reveals a rare complex multidomain fold that was found in two of the three previously determined GH50 structures. The structure comprises an N-terminal domain with a carbohydrate-binding module (CBM)-like fold fused to a C-terminal domain by a rigid linker. The CBM-li...
Source: Acta Crystallographica Section F - August 18, 2020 Category: Biochemistry Authors: Pluvinage, B. Robb, C.S. Jeffries, R. Boraston, A.B. Tags: glycoside hydrolase agarase marine bacterium agarose neoagaro-oligosaccharides research communications Source Type: research
High-resolution structure of the alcohol dehydrogenase domain of the bifunctional bacterial enzyme AdhE
The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Å resolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - August 18, 2020 Category: Biochemistry Authors: Azmi, L. Bragginton, E.C. Cadby, I.T. Byron, O. Roe, A.J. Lovering, A.L. Gabrielsen, M. Tags: alcohol dehydrogenase AdhE Escherichia coli research communications Source Type: research
Characterization and structure of glyceraldehyde-3-phosphate dehydrogenase type 1 from Escherichia coli
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of d-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55 ° C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Å resolut...
Source: Acta Crystallographica Section F - August 18, 2020 Category: Biochemistry Authors: Zhang, L. Liu, M.R. Yao, Y.C. Bostrom, I.K. Wang, Y.D. Chen, A.Q. Li, J.X. Gu, S.H. Ji, C.N. Tags: glyceraldehyde-3-phosphate dehydrogenase type 1 Escherichia coli crystal structure X-ray diffraction thermostability research communications Source Type: research
Crystal structure of the extracellular domain of the receptor-like kinase TMK3 from Arabidopsis thaliana
Transmembrane kinases (TMKs) are members of the plant receptor-like kinase (RLK) family. TMKs are characterized by an extracellular leucine-rich-repeat (LRR) domain, a single transmembrane region and a cytoplasmic kinase domain. TMKs have been shown to act as critical modulators of cell expansion and cell proliferation. Here, the crystal structure of the extracellular domain of TMK3 (TMK3-ECD) was determined to a resolution of 2.06 Å , with an Rwork of 17.69% and an Rfree of 20.58%. Similar to the extracellular domain of TMK1, the TMK3-ECD structure contains two solenoids with 13 LRRs and a non-LRR region (316 – 364...
Source: Acta Crystallographica Section F - July 28, 2020 Category: Biochemistry Authors: Chen, H. Kong, Y. Chen, J. Li, L. Li, X. Yu, F. Ming, Z. Tags: transmembrane kinases extracellular domain LRR-RLKs non-LRR region TMK3 protein crystallography research communications Source Type: research
Structural characterization of three noncanonical NTF2-like superfamily proteins: implications for polyketide biosynthesis
Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp. CM020, a protein in the biosynthetic pa...
Source: Acta Crystallographica Section F - July 28, 2020 Category: Biochemistry Authors: Vuksanovic, N. Zhu, X. Serrano, D.A. Siitonen, V. Mets ä -Ketel ä , M. Melan ç on, C.E. Silvaggi, N.R. Tags: NTF2-like superfamily polyketide cyclases actinorhodin alnumycin secondary metabolism research communications Source Type: research
Structural analysis of a novel substrate-free form of the aminoglycoside 6 ′ -N-acetyltransferase from Enterococcus faecium
In this study, the crystal structure of Ef-AAC(6 ′ )-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6 ′ )-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6 ′ )-Ii. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 27, 2020 Category: Biochemistry Authors: Jang, H. Kwon, S. Jeong, C.-S. Lee, C.W. Hwang, J. Jung, K.H. Lee, J.H. Park, H.H. Tags: aminoglycoside acetyltransferases Enterococcus faecium acetyl-CoA conformational selection induced fit research communications Source Type: research
Crystal structure of the Rab-binding domain of Rab11 family-interacting protein 2
The small GTPases Rab11, Rab14 and Rab25 regulate membrane trafficking through the recruitment of Rab11 family-interacting proteins (FIPs) to endocytic compartments. FIPs are multi-domain effector proteins that have a highly conserved Rab-binding domain (RBD) at their C-termini. Several structures of complexes of Rab11 with RBDs have previously been determined, including those of Rab11 – FIP2 and Rab11 – FIP3. In addition, the structures of the Rab14 – FIP1 and Rab25 – FIP2 complexes have been determined. All of the RBD structures contain a central parallel coiled coil in the RBD that binds to the switch 1 and swit...
Source: Acta Crystallographica Section F - July 27, 2020 Category: Biochemistry Authors: Kearney, A.M. Khan, A.R. Tags: Rab11 family-interacting protein 2 ab initio phasing Rab-binding domain GTPases research communications Source Type: research
Structure of cyclin-dependent kinase 2 (CDK2) in complex with the specific and potent inhibitor CVT-313
CVT-313 is a potent CDK2 inhibitor that was identified by screening a purine-analogue library and is currently in preclinical studies. Since this molecule has the potential to be developed as a CDK2 inhibitor for cancer therapy, the potency of CVT-313 to bind and stabilize CDK2 was evaluated, together with its ability to inhibit aberrant cell proliferation. CVT-313 increased the melting temperature of CDK2 by 7 ° C in thermal stabilization studies, thus indicating its protein-stabilizing effect. CVT-313 inhibited the growth of human lung carcinoma cell line A549 in a dose-dependent manner, with an IC50 of 1.2 µ M, wh...
Source: Acta Crystallographica Section F - July 27, 2020 Category: Biochemistry Authors: Talapati, S.R. Nataraj, V. Pothuganti, M. Gore, S. Ramachandra, M. Antony, T. More, S.S. Krishnamurthy, N.R. Tags: CDK2 cyclin-dependent kinase 2 CVT-313 crystal structure X-ray crystallography cancer therapy research communications Source Type: research
