Data to knowledge: how to get meaning from your result Berman, Helen M., Gabanyi, Margaret J. In: 2014. @article{noKey,
title = {Data to knowledge: how to get meaning from your result},
author = {Berman, Helen M., Gabanyi, Margaret J.},
url = {https://journals.iucr.org/m/issues/2015/01/00/dc5004/index.html},
doi = {https://doi.org/10.1107/S2052252514023306},
year = {2014},
date = {2014-01-01},
abstract = {Structural and functional studies require the development of sophisticated �Big Data� technologies and software to increase the knowledge derived and ensure reproducibility of the data. This paper presents summaries of the Structural Biology Knowledge Base, the VIPERdb Virus Structure Database, evaluation of homology modeling by the Protein Model Portal, the ProSMART tool for conformation-independent structure comparison, the LabDB �super� laboratory information management system and the Cambridge Structural Database. These techniques and technologies represent important tools for the transformation of crystallographic data into knowledge and information, in an effort to address the problem of non-reproducibility of experimental results.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Structural and functional studies require the development of sophisticated �Big Data� technologies and software to increase the knowledge derived and ensure reproducibility of the data. This paper presents summaries of the Structural Biology Knowledge Base, the VIPERdb Virus Structure Database, evaluation of homology modeling by the Protein Model Portal, the ProSMART tool for conformation-independent structure comparison, the LabDB �super� laboratory information management system and the Cambridge Structural Database. These techniques and technologies represent important tools for the transformation of crystallographic data into knowledge and information, in an effort to address the problem of non-reproducibility of experimental results. |
Software developments in automated structure solution and crystallographic studies of the Sso10a2 and human C1 inhibitor protein Waterreus, W.J. In: 2013. @article{noKey,
title = {Software developments in automated structure solution and crystallographic studies of the Sso10a2 and human C1 inhibitor protein},
author = {Waterreus, W.J.},
url = {https://hdl.handle.net/1887/22715},
doi = {undefined},
year = {2013},
date = {2013-01-01},
abstract = {CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process. The new MULTICOMB program, discussed in chapter 3, provides a new phase combination algorithm for the density modification step of the structure solution process. MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction (SAD) data directly, accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment. Testing of these new algorithms with over 100 real data sets showed a dramatic improvement over state-of-the-art methods. These novel methods were also applied in solving the new structure of the DNA-binding protein Sso10a2 from Sulfolobus solfataricus reported in chapter 4. This structure provides insight to the observed differences in behaviour between Sso10a2 and its close homolog Sso10a. The last chapter of this work describes the crystallization conditions for a recombinant, fully glycosylated form of the human C1 inhibitor protein, which is involved in hereditary angioedema, a potentially life threatening condition.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process. The new MULTICOMB program, discussed in chapter 3, provides a new phase combination algorithm for the density modification step of the structure solution process. MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction (SAD) data directly, accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment. Testing of these new algorithms with over 100 real data sets showed a dramatic improvement over state-of-the-art methods. These novel methods were also applied in solving the new structure of the DNA-binding protein Sso10a2 from Sulfolobus solfataricus reported in chapter 4. This structure provides insight to the observed differences in behaviour between Sso10a2 and its close homolog Sso10a. The last chapter of this work describes the crystallization conditions for a recombinant, fully glycosylated form of the human C1 inhibitor protein, which is involved in hereditary angioedema, a potentially life threatening condition. |
Structural and functional characterization of ScsC, a periplasmic thioredoxin-like protein from Salmonella enterica serovar Typhimurium Shepherd, Mark, Heras, Begoña In: 2013. @article{noKey,
title = {Structural and functional characterization of ScsC, a periplasmic thioredoxin-like protein from Salmonella enterica serovar Typhimurium},
author = {Shepherd, Mark, Heras, Begoña},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797457/},
doi = {https://doi.org/10.1089/ars.2012.4939},
year = {2013},
date = {2013-01-01},
abstract = {Aims: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses. Results: Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of �-sheets and a-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress. Innovation and Conclusion: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity. Antioxid. Redox Signal. 19, 1494�1506.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Aims: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses. Results: Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of �-sheets and a-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress. Innovation and Conclusion: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity. Antioxid. Redox Signal. 19, 1494�1506. |
Biochemical characterization of the Nup62� 58� 54 nucleoporin complex and mutational analysis of the exportin CRM1 Chug, Hema In: 2013. @article{noKey,
title = {Biochemical characterization of the Nup62� 58� 54 nucleoporin complex and mutational analysis of the exportin CRM1},
author = {Chug, Hema},
url = {https://d-nb.info/1059570157/34},
doi = {undefined},
year = {2013},
date = {2013-01-01},
abstract = {Nuclear pore complexes (NPCs) are giant molecular machines embedded in the double
membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the
NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins
(Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer.
Nup62�58�54 subcomplex is a nucleoporin subcomplex in the NPC�s central channel, the
molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all
subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeatmotifs. The C-terminal half is structured into coiled-coil domains that engage in tight proteinprotein interactions to hold the complex together and to anchor it to the NPC scaffold. In this
project, I aimed to elucidate the molecular structure of the X.laevis ?FG-Nup62�58�54 complex
by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as
Nup54, in bacterial expression has been a challenge towards obtaining large amounts of
nucleoporins required for crystallographic analysis. We established a system where all three
subunits of xl?FGNup62�58�54 were co-expressed together in E.coli. The soluble amount of
Nup54 was greatly enhanced, when co-expressed together with its interaction partners.
Biochemical analysis of the recombinant ?FG-Nup62�58�54 complex revealed a monomeric, nonspherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also,
in contrast to previously published reports, the ?FG-Nup62�58�54 complex did not show any
tendency to form protein-concentration dependent higher-order oligomers. However, extensive
crystallization trials failed to crystallize ?FG-Nup62�58�54 complex, probably due to inherent
flexibility in its structure. Therefore, we generated and screened a series of single-domain
antibodies (nanobodies, Nbs) against ?FG-Nup62�58�54 complex and identified a few that
stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric
interactions. Crystallization trials of ?FG-xlNup62�58�54 complex conjugated to one such
nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction
potential to ~7.5�. Thus, nanobody conjugation of the ?FG-xlNup62�58�54 complex aided in its
crystallization and can be used as a general approach to stabilize NPC subcomplexes.
Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62�58�54 complex
is thought to recruit the Nup62�58�54 complex to the NPCs. This interaction in our experiments,
however, turned out to be surprisingly weak. This now predicts additional interaction sites for a
high avidity anchorage of the Nup62�58�54 complex to the NPC scaffold.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Nuclear pore complexes (NPCs) are giant molecular machines embedded in the double
membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the
NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins
(Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer.
Nup62�58�54 subcomplex is a nucleoporin subcomplex in the NPC�s central channel, the
molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all
subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeatmotifs. The C-terminal half is structured into coiled-coil domains that engage in tight proteinprotein interactions to hold the complex together and to anchor it to the NPC scaffold. In this
project, I aimed to elucidate the molecular structure of the X.laevis ?FG-Nup62�58�54 complex
by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as
Nup54, in bacterial expression has been a challenge towards obtaining large amounts of
nucleoporins required for crystallographic analysis. We established a system where all three
subunits of xl?FGNup62�58�54 were co-expressed together in E.coli. The soluble amount of
Nup54 was greatly enhanced, when co-expressed together with its interaction partners.
Biochemical analysis of the recombinant ?FG-Nup62�58�54 complex revealed a monomeric, nonspherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also,
in contrast to previously published reports, the ?FG-Nup62�58�54 complex did not show any
tendency to form protein-concentration dependent higher-order oligomers. However, extensive
crystallization trials failed to crystallize ?FG-Nup62�58�54 complex, probably due to inherent
flexibility in its structure. Therefore, we generated and screened a series of single-domain
antibodies (nanobodies, Nbs) against ?FG-Nup62�58�54 complex and identified a few that
stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric
interactions. Crystallization trials of ?FG-xlNup62�58�54 complex conjugated to one such
nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction
potential to ~7.5�. Thus, nanobody conjugation of the ?FG-xlNup62�58�54 complex aided in its
crystallization and can be used as a general approach to stabilize NPC subcomplexes.
Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62�58�54 complex
is thought to recruit the Nup62�58�54 complex to the NPCs. This interaction in our experiments,
however, turned out to be surprisingly weak. This now predicts additional interaction sites for a
high avidity anchorage of the Nup62�58�54 complex to the NPC scaffold. |
Insights into membrane binding of PROPPINs Busse, R. In: 2013. @article{noKey,
title = {Insights into membrane binding of PROPPINs},
author = {Busse, R.},
url = {https://pdfs.semanticscholar.org/accf/a5aaed1062f6e1be8466643e30c126673867.pdf},
doi = {undefined},
year = {2013},
date = {2013-01-01},
abstract = {Autophagy is a degradation pathway conserved in eukaryotes. Upon induction of autophagy a double layered membrane is formed de novo and engulfs the cytosolic content. After fusion of the membrane, an autophagosome vesicle is formed, which then fuses with the vacuole (or lysosome) where its content is degraded. PROPPINs, �-propeller proteins that bind polyphosphoinositides, play a role in autophagy and phosphoinositide binding depends on a conserved FRRG motif. The three yeast PROPPINs Atg18, Atg21 and Hsv2 are involved in different subtypes of autophagy.
In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co-flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide binding of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding stoichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic environment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions.
Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively.
A in vivo reconstitution system for the two human ubiquitin-like conjugation systems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Autophagy is a degradation pathway conserved in eukaryotes. Upon induction of autophagy a double layered membrane is formed de novo and engulfs the cytosolic content. After fusion of the membrane, an autophagosome vesicle is formed, which then fuses with the vacuole (or lysosome) where its content is degraded. PROPPINs, �-propeller proteins that bind polyphosphoinositides, play a role in autophagy and phosphoinositide binding depends on a conserved FRRG motif. The three yeast PROPPINs Atg18, Atg21 and Hsv2 are involved in different subtypes of autophagy.
In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co-flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide binding of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding stoichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic environment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions.
Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively.
A in vivo reconstitution system for the two human ubiquitin-like conjugation systems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields |
A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals Nederlof, I., Genderen, E. van In: 2013. @article{noKey,
title = {A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals},
author = {Nederlof, I., Genderen, E. van},
url = {http://scripts.iucr.org/cgi-bin/paper?S0907444913009700},
doi = {https://dx.doi.org/10.1107/S0907444913009700},
year = {2013},
date = {2013-01-01},
abstract = {When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e- �-2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ~100 nm thick protein crystal. A highly parallel 200 keV electron beam ([lambda] = 0.025 �) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4�. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e- �-2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ~100 nm thick protein crystal. A highly parallel 200 keV electron beam ([lambda] = 0.025 �) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4�. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins |
Crystallization and preliminary X-ray diffraction analysis of a DING protein from Pseudomonas aeruginosa PA14 Djeghader, Ahmed, Gotthard, Guillaume In: 2013. @article{noKey,
title = {Crystallization and preliminary X-ray diffraction analysis of a DING protein from Pseudomonas aeruginosa PA14},
author = {Djeghader, Ahmed, Gotthard, Guillaume},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614170/},
doi = {https://doi.org/10.1107/S1744309113005356},
year = {2013},
date = {2013-01-01},
abstract = {DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life. They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate. In eukaryotes, DING proteins have been isolated by virtue of their implication in several diseases and biological processes. Some of them are potent inhibitors of HIV-1 replication/transcription, raising the question of their potential involvement in the human defence system. Recently, a protein from Pseudomonas aeruginosa strain PA14, named PA14DING or LapC, belonging to the DING family has been identified. The structure of PA14DING, combined with detailed biochemical characterization and comparative analysis with available DING protein structures, will be helpful in understanding the structural determinants implicated in the inhibition of HIV-�1 by DING proteins. Here, the expression, purification and crystallization of PA14DING and the collection of X-ray data to 1.9 � resolution are reported.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life. They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate. In eukaryotes, DING proteins have been isolated by virtue of their implication in several diseases and biological processes. Some of them are potent inhibitors of HIV-1 replication/transcription, raising the question of their potential involvement in the human defence system. Recently, a protein from Pseudomonas aeruginosa strain PA14, named PA14DING or LapC, belonging to the DING family has been identified. The structure of PA14DING, combined with detailed biochemical characterization and comparative analysis with available DING protein structures, will be helpful in understanding the structural determinants implicated in the inhibition of HIV-�1 by DING proteins. Here, the expression, purification and crystallization of PA14DING and the collection of X-ray data to 1.9 � resolution are reported. |
Imaging protein three-dimensional nanocrystals with cryo-EM Nederlof, Igor, Li, Yao Wang In: 2013. @article{noKey,
title = {Imaging protein three-dimensional nanocrystals with cryo-EM},
author = {Nederlof, Igor, Li, Yao Wang},
url = {http://scripts.iucr.org/cgi-bin/paper?S0907444913002734},
doi = {https://doi.org/10.1107/S0907444913002734},
year = {2013},
date = {2013-01-01},
abstract = {Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of 100 nm were imaged by 300 kV cryo-EM on a Falcon direct electron detector. The images were taken close to focus and to the eye appeared devoid of contrast. Fourier transforms of the images revealed the reciprocal lattice up to 3 � resolution in favourable cases and up to 4 � resolution for about half the crystals. The reciprocal-lattice spots showed structure, indicating that the ordering of the crystals was not uniform. Data processing revealed details at higher than 2 � resolution and indicated the presence of multiple mosaic blocks within the crystal which could be separately processed. The prospects for full three-dimensional structure determination by electron imaging of protein three-dimensional nanocrystals are discussed.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of 100 nm were imaged by 300 kV cryo-EM on a Falcon direct electron detector. The images were taken close to focus and to the eye appeared devoid of contrast. Fourier transforms of the images revealed the reciprocal lattice up to 3 � resolution in favourable cases and up to 4 � resolution for about half the crystals. The reciprocal-lattice spots showed structure, indicating that the ordering of the crystals was not uniform. Data processing revealed details at higher than 2 � resolution and indicated the presence of multiple mosaic blocks within the crystal which could be separately processed. The prospects for full three-dimensional structure determination by electron imaging of protein three-dimensional nanocrystals are discussed. |
The structure of LsrB from Yersinia pestis complexed with autoinducer-2 Kavanaugh, Jeffrey S., Gakhar, Lokesh In: 2011. @article{noKey,
title = {The structure of LsrB from Yersinia pestis complexed with autoinducer-2},
author = {Kavanaugh, Jeffrey S., Gakhar, Lokesh},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3232125/},
doi = {https://doi.org/10.1107/S1744309111042953},
year = {2011},
date = {2011-01-01},
abstract = {The crystal structure of LsrB from Yersinia pestis complexed with autoinducer-2 (AI-2; space group P212121, unit-cell parameters a = 40.61, b = 61.03, c = 125.23 �) has been solved by molecular replacement using the structure of LsrB from Salmonella typhimurium (PDB entry 1tjy) and refined to R = 0.180 (R free = 0.213) at 1.75 � resolution. The electron density for bound AI-2 and the stereochemistry of the AI-2-binding site are consistent with bound AI-2 adopting the (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran conformation, just as has been observed in the crystal structures of the Salmonella typhimurium and Sinorhizobium meliloti LsrB�AI-2 complexes.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
The crystal structure of LsrB from Yersinia pestis complexed with autoinducer-2 (AI-2; space group P212121, unit-cell parameters a = 40.61, b = 61.03, c = 125.23 �) has been solved by molecular replacement using the structure of LsrB from Salmonella typhimurium (PDB entry 1tjy) and refined to R = 0.180 (R free = 0.213) at 1.75 � resolution. The electron density for bound AI-2 and the stereochemistry of the AI-2-binding site are consistent with bound AI-2 adopting the (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran conformation, just as has been observed in the crystal structures of the Salmonella typhimurium and Sinorhizobium meliloti LsrB�AI-2 complexes. |
Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium Jarrott, R., Shouldice, S. R. In: 2010. @article{noKey,
title = {Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium},
author = {Jarrott, R., Shouldice, S. R.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864702/},
doi = {https://doi.org/10.1107/S1744309110011942},
year = {2010},
date = {2010-01-01},
abstract = {Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-�12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 � and belonged to space groups P21, P21212 and C2, respectively.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-�12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 � and belonged to space groups P21, P21212 and C2, respectively. |
Structural insights into mechanisms of the small RNA methyltransferase HEN1 Huang, Ying, Ji, Lijuan In: 2009. @article{noKey,
title = {Structural insights into mechanisms of the small RNA methyltransferase HEN1},
author = {Huang, Ying, Ji, Lijuan},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125239/},
doi = {https://doi.org/10.1038/nature08433},
year = {2009},
date = {2009-01-01},
abstract = {RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ~20�30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide3�6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3�5,7,8. Here we report the 3.1 � crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2'-O-methylation mechanism.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ~20�30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide3�6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3�5,7,8. Here we report the 3.1 � crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2'-O-methylation mechanism. |
The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins Gräslund, Susanne, Sagemark, Johanna In: 2007. @article{noKey,
title = {The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins},
author = {Gräslund, Susanne, Sagemark, Johanna},
url = {https://www.sciencedirect.com/science/article/abs/pii/S1046592807002896?via%3Dihub},
doi = {https://doi.org/10.1016/j.pep.2007.11.008},
year = {2007},
date = {2007-01-01},
abstract = {Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials. |
A program for automated optimization of initial crystallization conditions Abdellatif, T. Ben Haj, Kozielski, F. In: 2006. @article{noKey,
title = {A program for automated optimization of initial crystallization conditions},
author = {Abdellatif, T. Ben Haj, Kozielski, F.},
url = {http://scripts.iucr.org/cgi-bin/paper?S0021889806007072},
doi = {https://doi.org/10.1107/S0021889806007072},
year = {2006},
date = {2006-01-01},
abstract = {Protein crystallization is a difficult and time-consuming task, because to obtain a
crystal, optimization steps are required almost systematically. A tool that
simplifies the optimization of crystallization conditions, and that can be used by
any crystallographer to design a crystallization plate and to visualize its content,
has become a paramount necessity. A free and open-source application has been
developed to automate this task. It is based on a graphical user interface (GUI)
that allows a personalized crystallization plate to be designed. All data used and
generated are saved in XML documents, which allow reuse of the information.
The steps involved in preparing a crystallization plate and the functions of the
GUI designed to perform these steps are described. Plans for future
development are presented. The program was written in Java. The application
and its documentation are available under CeCILL license, which is a Free
Software license agreement.},
keywords = {ROCKMAKER},
pubstate = {published},
tppubtype = {article}
}
Protein crystallization is a difficult and time-consuming task, because to obtain a
crystal, optimization steps are required almost systematically. A tool that
simplifies the optimization of crystallization conditions, and that can be used by
any crystallographer to design a crystallization plate and to visualize its content,
has become a paramount necessity. A free and open-source application has been
developed to automate this task. It is based on a graphical user interface (GUI)
that allows a personalized crystallization plate to be designed. All data used and
generated are saved in XML documents, which allow reuse of the information.
The steps involved in preparing a crystallization plate and the functions of the
GUI designed to perform these steps are described. Plans for future
development are presented. The program was written in Java. The application
and its documentation are available under CeCILL license, which is a Free
Software license agreement. |
