Bacteriophage PRD1 Capsid Structure: Iterative Combination of Threedimensional Electron Microscopy and X-Ray Crystallography

2000 ◽  
Vol 6 (S2) ◽  
pp. 284-285
Author(s):  
Carmen San Martin ◽  
Roger M. Burnett ◽  
Felix de Haas ◽  
Ralph Heinkel ◽  
Twan Rutten ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Cyclic Process ◽  
Particle Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Bacteriophage Prd1 ◽  
Cryo Electron Microscopy ◽  
Moderate Resolution ◽  
Protein Rich

PRD1 is a ds-DNA bacteriophage from the Tectiviridae family with an unusual structural feature: the viral genome is enclosed by a protein-rich membrane, which is in turn enclosed by an external icosahedral protein shell (capsid). Three-dimensional reconstructions from cryo-electron microscopy (cryo-EM) images have revealed the structure of the PRD1 capsid at moderate resolution (28 Å), while X-ray crystallographic studies have recently provided a high resolution (1.85 Å) picture of the major coat protein, P3. We have now combined these results from different imaging methods to obtain a more detailed understanding of the virion organization. The combination has been made in a cyclic process: a preliminary fitting of the atomic structure of P3 to each one of its independent positions in the cryo-EM maps of the capsids provided initial models that could be used to improve the reconstructions; the refined maps then served as a base frame for an optimized fit. This process allows us to study the viral particle structure at “quasi-atomic” resolution.

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 Resolution in X-ray Crystallography and Single-Particle Cryogenic Electron Microscopy

Crystals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 580
Author(s):  
Victor R.A. Dubach ◽  
Albert Guskov
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Fourier Transform

X-ray crystallography and single-particle analysis cryogenic electron microscopy are essential techniques for uncovering the three-dimensional structures of biological macromolecules. Both techniques rely on the Fourier transform to calculate experimental maps. However, one of the crucial parameters, resolution, is rather broadly defined. Here, the methods to determine the resolution in X-ray crystallography and single-particle analysis are summarized. In X-ray crystallography, it is becoming increasingly more common to include reflections discarded previously by traditionally used standards, allowing for the inclusion of incomplete and anisotropic reflections into the refinement process. In general, the resolution is the smallest lattice spacing given by Bragg’s law for a particular set of X-ray diffraction intensities; however, typically the resolution is truncated by the user during the data processing based on certain parameters and later it is used during refinement. However, at which resolution to perform such a truncation is not always clear and this makes it very confusing for the novices entering the structural biology field. Furthermore, it is argued that the effective resolution should be also reported as it is a more descriptive measure accounting for anisotropy and incompleteness of the data. In single particle cryo-EM, the situation is not much better, as multiple ways exist to determine the resolution, such as Fourier shell correlation, spectral signal-to-noise ratio and the Fourier neighbor correlation. The most widely accepted is the Fourier shell correlation using a threshold of 0.143 to define the resolution (so-called “gold-standard”), although it is still debated whether this is the correct threshold. Besides, the resolution obtained from the Fourier shell correlation is an estimate of varying resolution across the density map. In reality, the interpretability of the map is more important than the numerical value of the resolution.

scholarly journals The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0146457 ◽  
Author(s):  
Noella Silva-Martin ◽  
María I. Daudén ◽  
Sebastian Glatt ◽  
Niklas A. Hoffmann ◽  
Panagiotis Kastritis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Insight Into

scholarly journals From X-ray crystallography to cryo-electron microscopy: computing infrastructure in structural biology

2018 ◽  
Vol 74 (a1) ◽  
pp. a224-a224
Author(s):  
Jason Key ◽  
Peter A. Meyer ◽  
Carol Herre ◽  
Michael Timony ◽  
Dimitry Filonov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Computing Infrastructure

scholarly journals A basic introduction to single particles cryo-electron microscopy

2021 ◽  
Vol 9 (1) ◽  
pp. 5-20
Author(s):  
Vittoria Raimondi ◽  
◽  
Alessandro Grinzato ◽  
◽  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Protein Complexes ◽  
Computational Power ◽  
Single Particles ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
First Choice ◽  
New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

scholarly journals GPU-accelerated analysis and visualization of large structures solved by molecular dynamics flexible fitting

2014 ◽  
Vol 169 ◽  
pp. 265-283 ◽  
Author(s):  
John E. Stone ◽  
Ryan McGreevy ◽  
Barry Isralewitz ◽  
Klaus Schulten
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Hybrid Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Interactive Computation ◽  
Biomolecular Complexes ◽  
Quality Of Fit

Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithmsvis-à-visexisting tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.

scholarly journals Functional implications of quasi-equivalence in a T=3 icosahedral animal virus established by cryo-electron microscopy and X-ray crystallography

Structure ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 271-282 ◽  
Author(s):  
R Holland Cheng ◽  
Vijay S Reddy ◽  
Norman H Olson ◽  
Andrew J Fisher ◽  
Timothy S Baker ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
X Ray ◽  
Animal Virus ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Functional Implications

scholarly journals Prospects for antimicrobial development in the cryo-EM era – a focus on the ribosome

2020 ◽  
Vol 44 (6) ◽  
pp. 793-803
Author(s):  
Alba Herrero del Valle ◽  
C Axel Innis
Keyword(s):  
Electron Microscopy ◽  
Drug Development ◽  
Health Concern ◽  
Parasitic Infections ◽  
Antimicrobial Drugs ◽  
Major Health ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Novel Drugs

ABSTRACT Resistance to antimicrobial drugs used to treat bacterial, viral, fungal and parasitic infections is a major health concern requiring a coordinated response across the globe. An important aspect in the fight against antimicrobial resistance is the development of novel drugs that are effective against resistant pathogens. Drug development is a complex trans-disciplinary endeavor, in which structural biology plays a major role by providing detailed functional and mechanistic information on an antimicrobial target and its interactions with small molecule inhibitors. Although X-ray crystallography and nuclear magnetic resonance have until now been the methods of choice to characterize microbial targets and drive structure-based drug development, cryo-electron microscopy is rapidly gaining ground in these areas. In this perspective, we will discuss how cryo-electron microscopy is changing our understanding of an established antimicrobial target, the ribosome, and how methodological developments could help this technique become an integral part of the antimicrobial drug discovery pipeline.

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