scholarly journals Identification of Mg2+ ions next to nucleotides in cryo-EM maps using electrostatic potential maps

2021 ◽  
Vol 77 (4) ◽  
pp. 534-539
Author(s):  
Jimin Wang ◽  
S. Kundhavai Natchiar ◽  
Peter B. Moore ◽  
Bruno P. Klaholz
Keyword(s):  
Electrostatic Potential ◽  
Charge Compensation ◽  
X Rays ◽  
Charge Effects ◽  
X Ray Crystallography ◽  
Density Maps ◽  
Covalently Bonded ◽  
Structural Details ◽  
Enzymatic Mechanisms ◽  
Phosphate Groups

Cryo electron microscopy (cryo-EM) can produce maps of macromolecules that have resolutions that are sufficiently high that structural details such as chemical modifications, water molecules and bound metal ions can be discerned. However, those accustomed to interpreting the electron-density maps of macromolecules produced by X-ray crystallography need to be careful when assigning features such as these in cryo-EM maps because cations, for example, interact far more strongly with electrons than they do with X-rays. Using simulated electrostatic potential (ESP) maps as a tool led us to re-examine a recent cryo-EM map of the human ribosome, and we realized that some of the ESP peaks originally identified as novel groups covalently bonded to the N7, O6 or O4 atoms of several guanines, adenines or uridines, respectively, in this structure are likely to instead represent Mg2+ ions coordinated to these atoms, which provide only partial charge compensation compared with Mg2+ ions located next to phosphate groups. In addition, direct evidence is provided for a variation in the level of 2′-O ribose methylation of nucleotides in the human ribosome. ESP maps can thus help in identifying ions next to nucleotide bases, i.e. at positions that can be difficult to address in cryo-EM maps due to charge effects, which are specifically encountered in cryo-EM. This work is particularly relevant to nucleoprotein complexes and shows that it is important to consider charge effects when interpreting cryo-EM maps, thus opening possibilities for localizing charges in structures that may be relevant for enzymatic mechanisms and drug interactions.

scholarly journals XFEL diffraction: developing processing methods to optimize data quality

2015 ◽  
Vol 22 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Nicholas K. Sauter
Keyword(s):  
Approximate Model ◽  
Difficult Problem ◽  
Integration Step ◽  
X Rays ◽  
Experimental Conditions ◽  
Structural Details ◽  
Laser Sources ◽  
Serial Crystallography ◽  
Difference Fourier ◽  
Damage Processes

Serial crystallography, using either femtosecond X-ray pulses from free-electron laser sources or short synchrotron-radiation exposures, has the potential to reveal metalloprotein structural details while minimizing damage processes. However, deriving a self-consistent set of Bragg intensities from numerous still-crystal exposures remains a difficult problem, with optimal protocols likely to be quite different from those well established for rotation photography. Here several data processing issues unique to serial crystallography are examined. It is found that the limiting resolution differs for each shot, an effect that is likely to be due to both the sample heterogeneity and pulse-to-pulse variation in experimental conditions. Shots with lower resolution limits produce lower-quality models for predicting Bragg spot positions during the integration step. Also, still shots by their nature record only partial measurements of the Bragg intensity. An approximate model that corrects to the full-spot equivalent (with the simplifying assumption that the X-rays are monochromatic) brings the distribution of intensities closer to that expected from an ideal crystal, and improves the sharpness of anomalous difference Fourier peaks indicating metal positions.

scholarly journals Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography

2019 ◽  
Vol 377 (2145) ◽  
pp. 20170474 ◽  
Author(s):  
Christopher D. M. Hutchison ◽  
Jasper J. van Thor
Keyword(s):  
Structural Dynamics ◽  
Time Domain ◽  
Optical Pumping ◽  
High Repetition Rate ◽  
X Rays ◽  
X Ray Diffraction ◽  
X Ray ◽  
Vibrational Coherence ◽  
X Ray Crystallography ◽  
Ultrafast Optical

Ultrafast pump-probe X-ray crystallography has now been established at X-ray free electron lasers that operate at hard X-ray energies. We discuss the performance and development of current applications in terms of the available data quality and sensitivity to detect and analyse structural dynamics. A discussion of technical capabilities expected at future high repetition rate applications as well as future non-collinear multi-pulse schemes focuses on the possibility to advance the technique to the practical application of the X-ray crystallographic equivalent of an impulse time-domain Raman measurement of vibrational coherence. Furthermore, we present calculations of the magnitude of population differences and distributions prepared with ultrafast optical pumping of single crystals in the typical serial femtosecond crystallography geometry, which are developed for the general uniaxial and biaxial cases. The results present opportunities for polarization resolved anisotropic X-ray diffraction analysis of photochemical populations for the ultrafast time domain. This article is part of the theme issue ‘Measurement of ultrafast electronic and structural dynamics with X-rays’.

scholarly journals Closing the gap between electron and X-ray crystallography

2015 ◽  
Vol 71 (6) ◽  
pp. 737-739 ◽  
Author(s):  
Enrico Mugnaioli
Keyword(s):  
Electron Diffraction ◽  
Structure Characterization ◽  
Diffraction Tomography ◽  
X Rays ◽  
Electron Crystallography ◽  
X Ray ◽  
X Ray Crystallography ◽  
Closing The Gap ◽  
Refinement Algorithm

The development of a proper refinement algorithm that takes into account dynamical scattering guarantees, for electron crystallography, results approaching X-rays in terms of precision, accuracy and reliability. The combination of such dynamical refinement and electron diffraction tomography establishes a complete pathway for the structure characterization of single sub-micrometric crystals.

scholarly journals Opportunities and challenges for assigning cofactors in cryo-EM density maps of chlorophyll-containing proteins

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Christopher J. Gisriel ◽  
Jimin Wang ◽  
Gary W. Brudvig ◽  
Donald A. Bryant
Keyword(s):  
Electron Microscopy ◽  
Electrostatic Potential ◽  
Protein Function ◽  
Protein Complexes ◽  
Chemical Environment ◽  
Cryo Electron Microscopy ◽  
Functional Differences ◽  
Density Maps ◽  
Structural Differences

AbstractThe accurate assignment of cofactors in cryo-electron microscopy maps is crucial in determining protein function. This is particularly true for chlorophylls (Chls), for which small structural differences lead to important functional differences. Recent cryo-electron microscopy structures of Chl-containing protein complexes exemplify the difficulties in distinguishing Chl b and Chl f from Chl a. We use these structures as examples to discuss general issues arising from local resolution differences, properties of electrostatic potential maps, and the chemical environment which must be considered to make accurate assignments. We offer suggestions for how to improve the reliability of such assignments.

scholarly journals Temperature-dependent macromolecular X-ray crystallography

2010 ◽  
Vol 66 (4) ◽  
pp. 437-446 ◽  
Author(s):  
Martin Weik ◽  
Jacques-Philippe Colletier
Keyword(s):  
Radiation Damage ◽  
Routine Data ◽  
Dynamical Behaviour ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Structural Details ◽  
Induced Changes ◽  
Structural Insights ◽  
Reaction Initiation

X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.

Crystal structure analysis of molecular dynamics using synchrotron X-rays

CrystEngComm ◽  
2015 ◽  
Vol 17 (46) ◽  
pp. 8786-8795 ◽  
Author(s):  
Manabu Hoshino ◽  
Shin-ichi Adachi ◽  
Shin-ya Koshihara
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Structure Analysis ◽  
Crystal Structure Analysis ◽  
X Rays ◽  
X Ray ◽  
X Ray Crystallography

X-ray crystallography using synchrotron X-rays enables observation of molecular dynamics in a crystal.

scholarly journals Photoreduction and validation of haem–ligand intermediate states in protein crystals byin situsingle-crystal spectroscopy and diffraction

IUCrJ ◽  
2017 ◽  
Vol 4 (3) ◽  
pp. 263-270 ◽  
Author(s):  
Demet Kekilli ◽  
Tadeo Moreno-Chicano ◽  
Amanda K. Chaplin ◽  
Sam Horrell ◽  
Florian S. N. Dworkowski ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Structural Data ◽  
X Rays ◽  
Solvated Electrons ◽  
X Ray ◽  
Redox States ◽  
Density Maps ◽  
Correct Assignment ◽  
Wide Range

Powerful synergies are available from the combination of multiple methods to study proteins in the crystalline form. Spectroscopies which probe the same region of the crystal from which X-ray crystal structures are determined can give insights into redox, ligand and spin states to complement the information gained from the electron-density maps. The correct assignment of crystal structures to the correct protein redox and ligand states is essential to avoid the misinterpretation of structural data. This is a particular concern for haem proteins, which can occupy a wide range of redox states and are exquisitely sensitive to becoming reduced by solvated electrons generated from interactions of X-rays with water molecules in the crystal. Here, single-crystal spectroscopic fingerprinting has been applied to investigate the laser photoreduction of ferric haem in cytochromec′. Furthermore,in situX-ray-driven generation of haem intermediates in crystals of the dye-decolourizing-type peroxidase A (DtpA) fromStreptomyces lividansis described.

scholarly journals Identification of ions in experimental electrostatic potential maps

IUCrJ ◽  
2018 ◽  
Vol 5 (4) ◽  
pp. 375-381 ◽  
Author(s):  
Jimin Wang ◽  
Zheng Liu ◽  
Joachim Frank ◽  
Peter B. Moore
Keyword(s):  
Electron Scattering ◽  
Electrostatic Potential ◽  
Neutral Atom ◽  
Scattering Length ◽  
Atomic Charge ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Electron Distributions ◽  
The Difference

Cryo-electron microscopy (cryo-EM) directly images the distribution of electrostatic potential (ESP) within macromolecules, and thus can provide much more information about atomic charge than X-ray crystallography. The electron-scattering length of an isolated ion is quite different from that of the corresponding neutral atom. The difference is very large at small scattering angles where the effects of electron distributions are largest, but becomes smaller at high scattering angles where nuclear charge determines outcomes. For this reason, in cryo-EM maps that have been solved at resolutions lower than ∼2.5 Å, peaks corresponding to anions will always be less prominent than those of cations, and may even be negative. Furthermore, if a map of this kind is smeared computationally after the fact, which reduces its effective resolution, anion peaks will diminish in size, cation peaks will grow and peaks that represent uncharged atoms will remain about the same. These effects can be used to determine the sign of the charges carried by the ions associated with a macromolecule and even estimate their magnitudes. The ESP value for a cation in a cation–anion pair is smaller than the value of the cation in isolation, but the ESP value for the anion in the ionic pair is greater than the value of the anion in isolation. The experimental range of ESP values for Mg2+ relative to that of the closest C1′ atom is found to be between 0.57 and 1.27.

scholarly journals Structural flexibility revealed by ultra-high resolution data

2014 ◽  
Vol 70 (a1) ◽  
pp. C1199-C1199
Author(s):  
Zbigniew Dauter
Keyword(s):  
High Resolution ◽  
Data Bank ◽  
Geometrical Parameters ◽  
Protein Chain ◽  
X Rays ◽  
High Resolution Data ◽  
Density Maps ◽  
Target Values ◽  
Multiple Conformations ◽  
Dna Dodecamer

Not many macromolecular crystals diffract X-rays to ultra-high resolution, defined usually as higher than 0.8 Å, and in the Protein Data Bank there are currently 43 such submissions. These structures range in size from antibiotics of about a hundred atoms to proteins with more than 3,000 independent atoms in the asymmetric unit of the crystal cell. The unprecedented data resolution reveals a great wealth of structural details, which cannot be visualized by analyses at lower resolution. The accuracy of the refined stereochemical and geometrical parameters is then comparable with values typical for small-molecular crystallography and exceeds the accuracy of the library of the standard restraint target values, routinely used in refinement of proteins and nucleotides. Somewhat unexpectedly, the very high resolution diffraction does not necessarily relates to extreme stability of the crystallized molecules, so that the obtained electron density maps reveal significant parts of the atomic models existing in multiple conformations, slightly differing from each other. For example, about 1/3 of the protein chain in the 0.65 Å structure of lysozyme [1] and majority of phosphate groups in the 0.75 Å structure of Z-DNA dodecamer [2] could be modeled in double conformations.

Sign in / Sign up

Export Citation Format

Share Document