Current approaches for the fitting and refinement of atomic models into cryo-EM maps usingCCP-EM

Recent advances in instrumentation and software have resulted in cryo-EM rapidly becoming the method of choice for structural biologists, especially for those studying the three-dimensional structures of very large macromolecular complexes. In this contribution, the tools available for macromolecular structure refinement into cryo-EM reconstructions that are availablevia CCP-EMare reviewed, specifically focusing onREFMAC5 and related tools. Whilst originally designed with a view to refinement against X-ray diffraction data, some of these tools have been able to be repurposed for cryo-EM owing to the same principles being applicable to refinement against cryo-EM maps. Since both techniques are used to elucidate macromolecular structures, tools encapsulating prior knowledge about macromolecules can easily be transferred. However, there are some significant qualitative differences that must be acknowledged and accounted for; relevant differences between these techniques are highlighted. The importance of phases is considered and the potential utility of replacing inaccurate amplitudes with their expectations is justified. More pragmatically, an upper bound on the correlation between observed and calculated Fourier coefficients, expressed in terms of the Fourier shell correlation between half-maps, is demonstrated. The importance of selecting appropriate levels of map blurring/sharpening is emphasized, which may be facilitated by considering the behaviour of the average map amplitude at different resolutions, as well as the utility of simultaneously viewing multiple blurred/sharpened maps. Features that are important for the purposes of computational efficiency are discussed, notably theDivide and Conquerpipeline for the parallel refinement of large macromolecular complexes. Techniques that have recently been developed or improved inCootto facilitate and expedite the building, fitting and refinement of atomic models into cryo-EM maps are summarized. Finally, a tool for symmetry identification from a given map or coordinate set,ProSHADE, which can identify the point group of a map and thus may be used during deposition as well as during molecular visualization, is introduced.

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Structure Refinement of CePtSi and Hydrogenation Behavior of CePdGe, CePtSi and CePtGe

Zeitschrift für Naturforschung B ◽

10.1515/znb-2007-0423 ◽

2007 ◽

Vol 62 (4) ◽

pp. 613-616 ◽

Cited By ~ 8

Author(s):

Wilfried Hermes ◽

Ute Ch. Rodewald ◽

Bernard Chevalier ◽

Rainer Pötgena

Keyword(s):

Single Crystal ◽

Diffraction Data ◽

Structure Refinement ◽

Three Dimensional ◽

Subsequent Annealing ◽

X Ray Diffraction ◽

X Ray ◽

Hydride Formation ◽

Arc Melting ◽

Cerium Compounds

The intermetallic cerium compounds CePdGe, CePtSi, and CePtGe were synthesized from the elements by arc-melting and subsequent annealing. The structure of CePtSi was refined from single crystal X-ray diffraction data: LaPtSi-type (ordered α-ThSi2 version), 141md, a = 419.6(1) and c = 1450.0(5) pm, wR2 = 0.0490, 362 F2 values and 16 variables. The Pt-Si distances within the three-dimensional [PtSi] network are 242 pm, indicating strong Pt-Si interactions. Hydrogenation of the three compounds at 623 K and 4 MPa H2 gave no indication for hydride formation.

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Structure refinement of the layered composite crystal Sc2B1.1C3.2 in a five-dimensional formalism

Acta Crystallographica Section B Structural Science ◽

10.1107/s0108768101006607 ◽

2001 ◽

Vol 57 (4) ◽

pp. 449-457 ◽

Cited By ~ 10

Author(s):

Mitsuko Onoda ◽

Ying Shi ◽

A. Leithe-Jasper ◽

Takaho Tanaka

Keyword(s):

Structural Parameters ◽

Structure Refinement ◽

Three Dimensional ◽

Layered Composite ◽

Layered Compound ◽

X Ray Diffraction ◽

X Ray ◽

Composite Crystal ◽

The Difference ◽

Insight Into

The crystal structure of a layered compound Sc2B1.1C3.2, scandium boride carbide (M r = 140.43), has been re-refined as a commensurate composite crystal using 1795 single-crystal X-ray diffraction intensities with I > 2\sigma(I) collected by Shi, Leithe-Jasper, Bourgeois, Bando & Tanaka [(1999), J. Solid State Chem. 148, 442–449]. The crystal is composed of two layered subsystem structures, i.e. Sc—C—Sc sandwiches and graphite-like layers of the composition B1/3C2/3. The structure refinement was performed in a five-dimensional formalism based on the trigonal superspace group P\bar{3}m1(p00)(0p0)0m0. The unit cell and other crystal data are a = b = 3.387 (1), c = 6.703 (2) Å, V = 66.59 (1) Å3, \boldsigma_{1} = (9/7 0 0), \boldsigma_{2} = (0 9/7 0), Z = 1, D x = 3.501 Mg m−1. Two different three-dimensional sections through the superspace were analyzed, corresponding to two different superstructure models, one with P\bar{3}m1 and the other with P\bar{3}m1. A random distribution of B and C was assumed in the graphite-like layer and 41 structural parameters were introduced. R F /wR F } were 0.0533/0.0482 and 0.0524/0.0476, respectively, for the first and second models. Although the difference between these R F or wR F values was too fine to exclude one of the models definitely, the advantages of using a superspace group were obvious. It not only brought about better convergence of refinement cycles by virtue of fewer parameters, but also gave an insight into the problem of symmetry of the superstructure.

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Structure Refinement and Magnetic Properties of Ce2RuAl3 and a Group-Subgroup Scheme for Ce5Ru3Al2

Zeitschrift für Naturforschung B ◽

10.1515/znb-2011-0801 ◽

2011 ◽

Vol 66 (8) ◽

pp. 771-776 ◽

Cited By ~ 7

Author(s):

Trinath Mishra ◽

Rolf-Dieter Hoffmann ◽

Christian Schwickert ◽

Rainer Pöttgen

Keyword(s):

Magnetic Properties ◽

Diffraction Data ◽

Structure Refinement ◽

Three Dimensional ◽

X Ray Diffraction ◽

Space Group ◽

X Ray ◽

Structural Distortions ◽

Dimensional Network ◽

Subgroup Scheme

The hexagonal Laves phase Ce2RuAl3 (≡ CeRu0.5Al1.5) was synthesized by high-frequencemelting of the elements in a sealed tantalum tube and subsequent annealing. The structure was refined from single-crystal X-ray diffraction data: MgZn2 type, P63/mmc, Z = 2, a = 565.38(9), c = 888.3(1) pm, wR2 = 0.0231, 193 F2 values and 13 parameters. The 2a (0.824 Ru + 0.176 Al) and 6h (0.956 Al + 0.044 Ru) Wyckoff positions show mixed occupancies leading to the composition CeRu0.48Al1.52 for the investigated crystal. The aluminum atoms build up Kagomé networks at z = 1/4 and z = 3/4 which are connected to a three-dimensional network by the ruthenium atoms. The cerium atoms fill cavities of coordination number 16 (3 Ru + 9 Al + 4 Ce) within the [RuAl3] network. The Ce2RuAl3 sample orders ferromagnetically at TC = 8.0(1) K. The cerium-rich aluminide Ce5Ru3Al2 shows unusually short Ce-Ru distances of 253 and 260 pm for the Ce1 position as a result of intermediate cerium valence. The structural distortions are discussed on the basis of a group-subgroup scheme for Pr5Ru3Al2 (space group I213) and the superstructure variant Ce5Ru3Al2 (space group R3).

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Neutronen- und Röntgenbeugungsuntersuchungen an Pulvern und Einkristallen von Strukturverwandten des Berliner Blaus: CsMnIICrIII(CN)6 · D2O , NMe4MnII(Cr0,06,Mn0,94)III(CN)6 · 8 H2O , NMe4MnIICoIII(CN)6 · 8 H20 , Mn3II[MnIII(CN)6]2 · 15 H2O und Cd3[FeIII(CN)6]2 · 15 H2O / Neutron and X-Ray Diffraction Studies on Powders and Single Crystals of Compounds Structurally Related to Prussian Blue: CsMnIICrIII(CN)6 · D2O, NMe4MnII(Cr0,06Mn0,94)III(CN)6 · 8 H2O, NMe4MnIICoIII(CN)6 · 8 H2O, Mn3II[MnIII(CN)6]2 · 15 H2O and Cd3[FeIII(CN)6]2 · 15 H2O

Zeitschrift für Naturforschung B ◽

10.1515/znb-1999-0708 ◽

1999 ◽

Vol 54 (7) ◽

pp. 870-876 ◽

Cited By ~ 19

Author(s):

Bernd Ziegler ◽

Michael Witzel ◽

Martin Schwarten ◽

Dietrich Babel

Keyword(s):

Crystal Structure ◽

Ambient Temperature ◽

Single Crystal ◽

Single Crystals ◽

Structure Refinement ◽

Three Dimensional ◽

Crystal Structure Refinement ◽

X Ray Diffraction ◽

One Dimensional ◽

X Ray

The results of a Rietveld refinement of CsMnCr(CN)6 · D2O neutron powder data (a = 1084.3(1) pm, F4̄3m, Z = 4) and of a neutron single crystal structure refinement of tetragonal NMe4MnII(Cr0,06Mn0,94)III(CN)6 · 8 H2O (a = 1065.8(21), c = 1064.6(26) pm, P4/n, Z = 2) at ambient temperature are reported. Single crystal X-ray analyses of the isostructural octahydrate NMe4MnCo(CN)6 · 8 H20 (a = 1062.1 (1), c = 1046.2( 1) pm) and of gel-grown crystals of cubic Mn3II[MnIII(CN)6]2 - 15 H2O (a = 1062.6(3) pm, Fm3̄m, Z = 4/3) and Cd3[Fe(CN)6]2 · 15 H2O (a = 1067.7(3) pm) were performed as well. The latter “Prussian Blues” are highly disordered and intermediate with respect to cyano-bridging between the above three-dimensional cesium and one-dimensional tetramethylammonium compounds.

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Measuring and modeling diffuse scattering in protein X-ray crystallography

10.1101/033746 ◽

2015 ◽

Author(s):

Andrew H. Van Benschoten ◽

Lin Liu ◽

Ana Gonzalez ◽

Aaron S. Brewster ◽

Nicholas K. Sauter ◽

...

Keyword(s):

Data Collection ◽

Diffuse Scattering ◽

Structure Refinement ◽

Three Dimensional ◽

Coarse Grained ◽

X Ray Diffraction ◽

Diffuse Intensity ◽

X Ray ◽

Protein Motions ◽

Extra Effort

AbstractX-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations must be measured. Until now measurement of diffuse scattering from protein crystals has been scarce, due to the extra effort of collecting diffuse data. Here, we present three-dimensional measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested Translation-Libration-Screw (TLS), Liquid-Like Motions (LLM), and coarse-grained Normal Modes (NM) models of protein motions. The LLM model provides a global picture of motions and were refined against the diffuse data, while the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that NM-based refinement can generate dynamics-inspired structural models that simultaneously agree with both Bragg and diffuse scattering.SignificanceThe structural details of protein motions are critical to understanding many biological processes, but they are often hidden to conventional biophysical techniques. Diffuse X-ray scattering can reveal details of the correlated movements between atoms; however, the data collection historically has required extra effort and dedicated experimental protocols. We have measured three-dimensional diffuse intensities in X-ray diffraction from CypA and trypsin crystals using standard crystallographic data collection techniques. Analysis of the resulting data is consistent with the protein motions resembling diffusion in a liquid or vibrations of a soft solid. Our results show that using diffuse scattering to model protein motions can become a component of routine crystallographic analysis through the extension of commonplace methods.

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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An investigation of polyhedral deformation in two mixed-metal diarsenates: SrZnAs2O7and BaCuAs2O7

Acta Crystallographica Section C Structural Chemistry ◽

10.1107/s2053229615005616 ◽

2015 ◽

Vol 71 (4) ◽

pp. 330-337 ◽

Cited By ~ 2

Author(s):

Sabina Kovač ◽

Ljiljana Karanović ◽

Tamara Đorđević

Keyword(s):

Crystal Structure ◽

Single Crystal ◽

General Formula ◽

Three Dimensional ◽

X Ray Diffraction ◽

Hydrothermal Conditions ◽

X Ray ◽

Open Framework ◽

Geometrical Characteristics ◽

Barium Copper

Two isostructural diarsenates, SrZnAs2O7(strontium zinc diarsenate), (I), and BaCuAs2O7[barium copper(II) diarsenate], (II), have been synthesized under hydrothermal conditions and characterized by single-crystal X-ray diffraction. The three-dimensional open-framework crystal structure consists of corner-sharingM2O5(M2 = Zn or Cu) square pyramids and diarsenate (As2O7) groups. Each As2O7group shares its five corners with five differentM2O5square pyramids. The resulting framework delimits two types of tunnels aligned parallel to the [010] and [100] directions where the large divalent nine-coordinatedM1 (M1 = Sr or Ba) cations are located. The geometrical characteristics of theM1O9,M2O5and As2O7groups of known isostructural diarsenates, adopting the general formulaM1IIM2IIAs2O7(M1II= Sr, Ba, Pb;M2II= Mg, Co, Cu, Zn) and crystallizing in the space groupP21/n, are presented and discussed.

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A new model for packing of type-I collagen molecules in the native fibril

Bioscience Reports ◽

10.1007/bf01114803 ◽

1981 ◽

Vol 1 (10) ◽

pp. 801-810 ◽

Cited By ~ 49

Author(s):

Karl A. Piez ◽

Benes L. Trus

Keyword(s):

Type I Collagen ◽

Hexagonal Lattice ◽

Three Dimensional ◽

Equatorial Region ◽

Type I ◽

X Ray Diffraction ◽

X Ray ◽

Left Handed ◽

Crystal Model ◽

Collagen Molecules

A specific fibril model is presented consisting of bundles of five-stranded microfibrils, which are usually disordered (except axially) but under lateral compression become ordered. The features are as follows (where D = 234 residues or 67 nm): (1) D-staggered collagen molecules 4.5 D long in the helical microfibril have a left-handed supercoil with a pitch of 400–700 residues, but microfibrils need not have helical symmetry. (2) Straight-tilted 0.5-D overlap regions on a near-hexagonal lattice contribute the discrete x-ray diffraction reflections arising from lateral order, while the gap regions remain disordered. (3) The overlap regions are equivalent, but are crystallographically distinguished by systematic displacements from the near-hexagonal lattice. (4) The unit cell is the same as in a recently proposed three-dimensional crystal model, and calculated intensities in the equatorial region of the x-ray diffraction pattern agree with observed values.

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