scholarly journals Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7

2008 ◽  
Vol 3 (7) ◽  
pp. 1171-1179 ◽  
Author(s):  
Gerrit Langer ◽  
Serge X Cohen ◽  
Victor S Lamzin ◽  
Anastassis Perrakis
Keyword(s):  
Model Building ◽  
X Ray ◽  
X Ray Crystallography

Using cryo-electron microscopy maps for X-ray structure determination of homologues

2020 ◽  
Vol 76 (1) ◽  
pp. 63-72
Author(s):  
Lingxiao Zeng ◽  
Wei Ding ◽  
Quan Hao
Keyword(s):  
Electron Microscopy ◽  
Hybrid Method ◽  
Model Building ◽  
Test Cases ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity ◽  
Whole Process ◽  
Cryo Electron Microscopy

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable R work and R free values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

scholarly journals The dimensions and shapes of the furanose rings in nucleic acids

1972 ◽  
Vol 130 (2) ◽  
pp. 453-465 ◽  
Author(s):  
S. Arnott ◽  
D. W. L. Hukins
Keyword(s):  
Nucleic Acids ◽  
Model Building ◽  
Molecular Model ◽  
Relative Orientation ◽  
Ring Conformation ◽  
Mean Values ◽  
Pyrimidine Base ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ring Puckering

A survey was made of the geometry of furanose rings in β-nucleotides and β-nucleosides (as monomers related to nucleic acids) for which structures have been determined by X-ray crystallography. Mean values, and estimated standard deviations from them, were calculated for bond-lengths, bond-angles and conformation-angles. For parameters with values dependent on ring-puckering, separate calculations were made for each ring type. (The rings are puckered in one of three conformations: C-2- or C-3-endo or C-3-exo; C-2-exo has not been observed.) The results were used to compute standard furanose rings with C-2-endo, C-3-endo and C-3-exo conformations for use in nucleic acid molecular model-building. The survey also showed that the only other conformation-angle in nucleotides dependent on the furanose ring conformation corresponds to the relative orientation of the purine (or pyrimidine) base and the ring.

scholarly journals Solving a new R2lox protein structure by microcrystal electron diffraction

2019 ◽  
Vol 5 (8) ◽  
pp. eaax4621 ◽  
Author(s):  
Hongyi Xu ◽  
Hugo Lebrette ◽  
Max T. B. Clabbers ◽  
Jingjing Zhao ◽  
Julia J. Griese ◽  
...  
Keyword(s):  
Protein Structure ◽  
Electron Diffraction ◽  
Model Building ◽  
Protein Structures ◽  
X Ray Diffraction ◽  
X Ray ◽  
X Ray Crystallography ◽  
Potential Map ◽  
Metal Cofactor ◽  
And Function

Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables the study of biomolecules from micrometer-sized 3D crystals that are too small to be studied by conventional x-ray crystallography. However, to date, MicroED has only been applied to redetermine protein structures that had already been solved previously by x-ray diffraction. Here, we present the first new protein structure—an R2lox enzyme—solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0-Å resolution was of sufficient quality to allow accurate model building and refinement. The dinuclear metal cofactor could be located in the map and was modeled as a heterodinuclear Mn/Fe center based on previous studies. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function.

scholarly journals Current approaches for automated model building into cryo-EM maps using Buccaneer with CCP-EM

2020 ◽  
Vol 76 (6) ◽  
pp. 531-541
Author(s):  
Soon Wen Hoh ◽  
Tom Burnley ◽  
Kevin Cowtan
Keyword(s):  
Model Building ◽  
Data Sets ◽  
Structural Interpretation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Model ◽  
Reference Map ◽  
Automated Model Building ◽  
Better Than

This work focuses on the use of the existing protein-model-building software Buccaneer to provide structural interpretation of electron cryo-microscopy (cryo-EM) maps. Originally developed for application to X-ray crystallography, the necessary steps to optimise the usage of Buccaneer with cryo-EM maps are shown. This approach has been applied to the data sets of 208 cryo-EM maps with resolutions of better than 4 Å. The results obtained also show an evident improvement in the sequencing step when the initial reference map and model used for crystallographic cases are replaced by a cryo-EM reference. All other necessary changes to settings in Buccaneer are implemented in the model-building pipeline from within the CCP-EM interface (as of version 1.4.0).

scholarly journals A practical method for efficient and optimal production of selenomethionine-labeled recombinant protein complexes in the insect cells

2018 ◽  
Author(s):  
Sabine Wenzel ◽  
Tsuyoshi Imasaki ◽  
Yuichiro Takagi
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Model Building ◽  
Insect Cells ◽  
Protein Complexes ◽  
Practical Method ◽  
Optimal Production ◽  
X Ray ◽  
E Coli ◽  
X Ray Crystallography

AbstractThe use of Selenomethionine (SeMet) incorporated protein crystals for single or multiwavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in E. coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allows for incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared to native protein expression.

scholarly journals Solving the first novel protein structure by 3D micro-crystal electron diffraction

2019 ◽  
Author(s):  
H. Xu ◽  
H. Lebrette ◽  
M.T.B. Clabbers ◽  
J. Zhao ◽  
J.J. Griese ◽  
...  
Keyword(s):  
Protein Structure ◽  
Electron Diffraction ◽  
Model Building ◽  
Protein Structures ◽  
X Ray ◽  
X Ray Crystallography ◽  
Unknown Protein ◽  
Potential Map ◽  
Crystal Electron ◽  
And Function

AbstractMicro-crystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables studying biomolecules from micron-sized 3D crystals that are too small to be studied by conventional X-ray crystallography. However, to the best of our knowledge, MicroED has only been applied to re-determine protein structures that had already been solved previously by X-ray diffraction. Here we present the first unknown protein structure – an R2lox enzyme – solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0 Å resolution was of sufficient quality to allow accurate model building and refinement. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function, opening up new opportunities for structural biologists.

Structure determination using convergent-beam electron diffraction

1992 ◽  
Vol 50 (2) ◽  
pp. 1162-1163
Author(s):  
J W Steeds ◽  
R Vincent
Keyword(s):  
Electron Diffraction ◽  
Model Building ◽  
Diffraction Theory ◽  
Bloch Wave ◽  
X Ray ◽  
X Ray Crystallography ◽  
Local Perturbations ◽  
Fitting Procedures ◽  
Convergent Beam ◽  
Kinematical Approach

There are many different approaches in quantitative electron diffraction which are being vigorously pursued at present. The approach we adopt is based on the insights provided by the Bloch-wave formulation of dynamical electron diffraction theory into the physics of dynamical scattering. This insight is used to select diffraction situations where a pseudo-kinematical approximation may be made. A forwards route is then possible directly from the experimental observations to the structural implications. This contrasts with the model-building, multi-parameter fitting procedures used in many other approaches where a problem of uniqueness inevitably arises.Because the pseudo-kinematical approach ignores many of the detailed dynamical interactions which occur locally over small angular ranges we do not attempt to make accurate measurements, and wherever possible average (visually at least) along Bragg lines to eliminate local perturbations. In a sense the work resembles early X-ray crystallography where reflections were put in one of six or so classes from very weak to very strong.

scholarly journals Strategies for carbohydrate model building, refinement and validation

2017 ◽  
Vol 73 (2) ◽  
pp. 171-186 ◽  
Author(s):  
Jon Agirre
Keyword(s):  
Model Building ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Manual Intervention ◽  
Cryo Electron Microscopy ◽  
Model Components ◽  
Complete Protein ◽  
New Generation ◽  
Generally True

Sugars are the most stereochemically intricate family of biomolecules and present substantial challenges to anyone trying to understand their nomenclature, reactions or branched structures. Current crystallographic programs provide an abstraction layer allowing inexpert structural biologists to build complete protein or nucleic acid model components automatically either from scratch or with little manual intervention. This is, however, still not generally true for sugars. The need for carbohydrate-specific building and validation tools has been highlighted a number of times in the past, concomitantly with the introduction of a new generation of experimental methods that have been ramping up the production of protein–sugar complexes and glycoproteins for the past decade. While some incipient advances have been made to address these demands, correctly modelling and refining carbohydrates remains a challenge. This article will address many of the typical difficulties that a structural biologist may face when dealing with carbohydrates, with an emphasis on problem solving in the resolution range where X-ray crystallography and cryo-electron microscopy are expected to overlap in the next decade.

scholarly journals Using cryo-electron microscopy maps for X-ray structure determination

IUCrJ ◽  
2018 ◽  
Vol 5 (4) ◽  
pp. 382-389 ◽  
Author(s):  
Lingxiao Zeng ◽  
Wei Ding ◽  
Quan Hao
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Hybrid Method ◽  
Structure Determination ◽  
Model Building ◽  
Initial Model ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Starting Point

X-ray crystallography and cryo-electron microscopy (cryo-EM) are complementary techniques for structure determination. Crystallography usually reveals more detailed information, while cryo-EM is an extremely useful technique for studying large-sized macromolecules. As the gap between the resolution of crystallography and cryo-EM data narrows, the cryo-EM map of a macromolecule could serve as an initial model to solve the phase problem of crystal diffraction for high-resolution structure determination. FSEARCH is a procedure to utilize the low-resolution molecular shape for crystallographic phasing. The IPCAS (Iterative Protein Crystal structure Automatic Solution) pipeline is an automatic direct-methods-aided dual-space iterative phasing and model-building procedure. When only an electron-density map is available as the starting point, IPCAS is capable of generating a completed model from the phases of the input map automatically, without the requirement of an initial model. In this study, a hybrid method integrating X-ray crystallography with cryo-EM to help with structure determination is presented. With a cryo-EM map as the starting point, the workflow of the method involves three steps. (1) Cryo-EM map replacement: FSEARCH is utilized to find the correct translation and orientation of the cryo-EM map in the crystallographic unit cell and generates the initial low-resolution map. (2) Phase extension: the phases calculated from the correctly placed cryo-EM map are extended to high-resolution X-ray data by non-crystallographic symmetry averaging with phenix.resolve. (3) Model building: IPCAS is used to generate an initial model using the phase-extended map and perform model completion by iteration. Four cases (the lowest cryo-EM map resolution being 6.9 Å) have been tested for the general applicability of the hybrid method, and almost complete models have been generated for all test cases with reasonable R work/R free. The hybrid method therefore provides an automated tool for X-ray structure determination using a cryo-EM map as the starting point.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

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