Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to spee...Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.展开更多
In recent years,short peptide self-assembled materials,prepared under the control of the thermolysin catalyst,have been investigated extensively and shown to acquire various morphologies and functions as building bloc...In recent years,short peptide self-assembled materials,prepared under the control of the thermolysin catalyst,have been investigated extensively and shown to acquire various morphologies and functions as building blocks for a wide range of biomaterials and device applications.However,the role played by thermolysin in this enzymatically triggered peptide self-assembly is still ambiguous.Herein,we designed a series of Fmoc-dipeptide amphiphiles to explore the catalytic role of thermolysin.展开更多
High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and easily. Experimental phasing is an essential technique in determining the three-dimensional protein s...High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and easily. Experimental phasing is an essential technique in determining the three-dimensional protein structures using single-crystal X-ray diffraction. In macromolecular crystallography, the phases are derived either by Molecular Replacement (MR) method using the atomic coordinates of a structurally similar protein or by locating the positions of heavy atoms that are intrinsic to the protein or that have been added (MIR, MIRAS, SIR, SIRAS, MAD and SAD). Availability of in-house lab data collection sources (Cu Kα and Cr Kα radiation), cryo-crystallography and improved software for heavy atom location and density modification have increased the ability to solve protein structures using SAD. SAD phasing using intrinsic anomalous scatterers like sulfur, chlorine, calcium, manganese and zinc, which are already present in the protein becomes increasingly attractive owing to the advanced phasing methods. An analysis of successful SAD phasing on three proteins, lysozyme, glucose isomerase and thermolysin based on the signal of weak anomalous scatterers such as sulfur atom and chloride ion have been carried out. This analysis also proves that even the anomalous signal provided or present naturally in a macromolecule is good enough to solve crystal structures successfully using lab source chromium-generated X-ray radiation.展开更多
Obtaining phase information for the solution of macromolecular structures is a bottleneck in X-ray crystallography. Anomalous dispersion was recognized as a powerful tool for phasing macromolecular structures. It was ...Obtaining phase information for the solution of macromolecular structures is a bottleneck in X-ray crystallography. Anomalous dispersion was recognized as a powerful tool for phasing macromolecular structures. It was used mainly to supplement the isomorphous replacement or to locate the anomalous scatterer itself. The first step in solving macromolecular structures by SAD (single-wavelength anomalous diffraction) is the location of the anomalous scatterers. The SAD method for experimental phasing has evolved substantially in the recent years. A phasing tool, 5-amino-2,4,6- triiodoisophthalic acid (I3C—magic triangle), was incorporated into three proteins, lysozyme, glucose isomerase and thermolysin using quick-soaking and co-crystallization method in order to understand the binding of metal ion with proteins. The high quality of the diffraction data, the use of chromium anode X-ray radiation and the required amount of anomalous signal enabled way for successful structure determination and automated model building. An analysis and/or comparison of the sulfur and iodine anomalous signals at the Cr Kα wavelength are discussed.展开更多
文摘Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.
基金support from the National Natural Science Foundation of China(project no.21774132)The computation was performed at ECNU Public Platform for Innovation(001).
文摘In recent years,short peptide self-assembled materials,prepared under the control of the thermolysin catalyst,have been investigated extensively and shown to acquire various morphologies and functions as building blocks for a wide range of biomaterials and device applications.However,the role played by thermolysin in this enzymatically triggered peptide self-assembly is still ambiguous.Herein,we designed a series of Fmoc-dipeptide amphiphiles to explore the catalytic role of thermolysin.
文摘High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and easily. Experimental phasing is an essential technique in determining the three-dimensional protein structures using single-crystal X-ray diffraction. In macromolecular crystallography, the phases are derived either by Molecular Replacement (MR) method using the atomic coordinates of a structurally similar protein or by locating the positions of heavy atoms that are intrinsic to the protein or that have been added (MIR, MIRAS, SIR, SIRAS, MAD and SAD). Availability of in-house lab data collection sources (Cu Kα and Cr Kα radiation), cryo-crystallography and improved software for heavy atom location and density modification have increased the ability to solve protein structures using SAD. SAD phasing using intrinsic anomalous scatterers like sulfur, chlorine, calcium, manganese and zinc, which are already present in the protein becomes increasingly attractive owing to the advanced phasing methods. An analysis of successful SAD phasing on three proteins, lysozyme, glucose isomerase and thermolysin based on the signal of weak anomalous scatterers such as sulfur atom and chloride ion have been carried out. This analysis also proves that even the anomalous signal provided or present naturally in a macromolecule is good enough to solve crystal structures successfully using lab source chromium-generated X-ray radiation.
文摘Obtaining phase information for the solution of macromolecular structures is a bottleneck in X-ray crystallography. Anomalous dispersion was recognized as a powerful tool for phasing macromolecular structures. It was used mainly to supplement the isomorphous replacement or to locate the anomalous scatterer itself. The first step in solving macromolecular structures by SAD (single-wavelength anomalous diffraction) is the location of the anomalous scatterers. The SAD method for experimental phasing has evolved substantially in the recent years. A phasing tool, 5-amino-2,4,6- triiodoisophthalic acid (I3C—magic triangle), was incorporated into three proteins, lysozyme, glucose isomerase and thermolysin using quick-soaking and co-crystallization method in order to understand the binding of metal ion with proteins. The high quality of the diffraction data, the use of chromium anode X-ray radiation and the required amount of anomalous signal enabled way for successful structure determination and automated model building. An analysis and/or comparison of the sulfur and iodine anomalous signals at the Cr Kα wavelength are discussed.
