Macromolecular crystallography

Gene expression is an essential function which level, fidelity and efficiency are tightly controlled. During my thesis I have focused on the actor of translation termination efficiency Tpa1, on its partner Ett1 and on the complex Dom34-Hbs1 involved in the RNA quality- control mechanisms NGD and NRD. We have solved the crystal structure of each of these proteins and realized a biochemical functional characterization.

A number of commercially available waxes in the form of thin disc samples have been investigated as possible diffraction intensity standards for macromolecular crystallography synchrotron beamlines. Synchrotron X-ray powder diffraction measurements show that beeswax offers the best performance of these waxes owing to its polycrystallinity. Crystallographic lattice parameters and diffraction intensities were examined between 281 and 309 K, and show stable and predictable thermal behaviour. Using an X-ray beam of known incident flux at ! = 1 A ̊ , the diffraction power of two strong Bragg reflections for beeswax were quantified as a function of sample thickness and normalized to 1010 photons s1. To demonstrate its feasibility as a diffraction intensity standard, test measurements were then performed on a new third-generation macromolecular crystallography synchrotron beamline.

An ab initio method is described for solving protein structures for which atomic resolution (better than 1.2 A Ê ) data are available. The problem is divided into two stages. Firstly, a substructure composed of a small percentage ($5%) of the scattering matter of the unit cell is positioned. This is used to generate a starting set of phases that are slightly better than random. Secondly, the full structure is developed from this phase set. The substructure can be a constellation of atoms that scatter anomalously, such as metal or S atoms. Alternatively, a structural fragment such as an idealized -helix or a motif from some distantly related protein can be orientated and sometimes positioned by an extensive molecular-replacement search, checking the correlation co-ef®cient between observed and calculated structure factors for the highest normalized structure-factor amplitudes |E|. The top solutions are further ranked on the correlation coef®cient for all E values. The phases generated from such fragments are improved using Patterson superposition maps and Sayreequation re®nement carried out with fast Fourier transforms. Phase re®nement is completed using a novel density-modi®cation process referred to as dynamic density modi®cation (DDM). The method is illustrated by the solution of a number of known proteins. It has proved fast and very effective, able in these tests to solve proteins of up to 5000 atoms. The resulting electron-density maps show the major part of the structures at atomic resolution and can readily be interpreted by automated procedures.

The reaction of ytterbocene (C5H4Me)2Yb(THF) with 2 equiv of N-2,6-diisopropylphenylimino- 2-pyridine (Ipy) occurs with oxidative cleavage of one Yb-Cp bond and oxidation of the ytterbium(II) ion and affords the half-sandwich Yb(III) complex [Yb(C5H4Me){(2,6-i-Pr2C6H3NCH(C5H4N)•-}2], containing two Ipy radical-anions. The indenyl-containing analogue [Yb(C9H7){(2,6-i-Pr2C6H3NCH- (C5H4N)•-}2] was unexpectedly obtained in the metathesis reaction of (C9H7)2Yb(μ-Cl)2Li(Et2O)2 with an equimolar amount of [Ipy•-]Kþ instead of the sandwich Yb(III) complex [Yb(C9H7)2{(2,6- i-Pr2C6H3NCH(C5H4N)•-}]. The gadolinium complex [Gd(C5Me5){(2,6-i-Pr2C6H3NCH(C5H5N)•-}2] was synthesized by the metathesis reaction of (C5Me5)GdCl2(THF)3 with a 2-fold molar excess of [Ipy•-]Kþ in THF. The structures of all complexes were proved by the X-ray single-crystal diffraction studies. The investigation of magnetic properties of complexes [LnCp{(2,6-i-Pr2C6H3NCH(C5H4N)•-}2] (Cp = C5H4Me, C5Me5, C9H7; Ln = Yb, Gd) in the temperature region 2-300 K revealed the presence of two types of antiferromagnetic interactions between the paramagnetic centers: between the Ln3þ ion and both radicals and between two radicals.

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic band c-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle a-actin, cytoplasmic b-actin, and cytoplasmic c-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of a-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by b-actin and myosin-7A by c-actin. Our results indicate that band c-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. Citation: Mü ller M, Diensthuber RP, Chizhov I, Claus P, Heissler SM, et al. (2013) Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins. PLoS ONE 8(7): e70636.

The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e.≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e.≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to trans...

The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4 D251N and CYP199A4 T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4 D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4 T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.

A reconstructed precession image. Each point of light is interpreted as a 'reflection' from a plane of atoms. Thousands of these reflections are collected and how bright they are and the angle at which they are collected is used to determine the position of the atoms in the crystal. Reflections further away from the centre (at higher angle) are used for highresolution data.

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the "off" and "on" allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to wellordered locations that should be easier to target.

Assembling data from multiple crystals with BLEND

In recent times, in macromolecular crystallography, it has become increasingly common at synchrotrons to obtain complete merged datasets using partial data wedges from multiple crystals. There are several reasons for the emergence of this recent trend, all pointing to the fact that completeness and redundancy from many crystals are, in general, superior to those coming from single crystals. When dealing with multiple crystals, though, users face two problems: crystals isomorphism and the large number of possible datasets combinations. Both problems can be addressed using cluster analysis. The program BLEND [1], developed at the Diamond Light Source synchrotron and available in CCP4 [2], makes use of cluster analysis and of the POINTLESS [3] and AIMLESS [4] programs to help users assembling useful and complete data from multiple crystals. BLEND acts by default in a semi-automated way, but it is most effective when used interactively in repeated sessions. BLEND has been employed successfully to assemble data for a variety of macromolecules, especially membrane proteins and protein complexes.

[1] Foadi J, Aller P, Alguel Y, Cameron A, Axford D (2013) Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 69(8):1617-1632.

[2] Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P et al (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67(4): 235-242.

[3] Evans PR (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62: 72-82.

[4] Evans PR.and Murshudov GN (2013) How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr 69(7):1204-1214.

Villin-type headpiece domains are small modular, autonomously folding motifs found at the extreme C-terminus of several families of cytoskeletal proteins. Although the majority of these domains bind F-actin, there are some that lack this functionality. Here, the high-resolution NMR solution structure of supervillin headpiece is presented, a villin-type headpiece natively devoid of F-actin binding activity. By comparing the electrostatic surface potential computed from the structure of supervillin headpiece, reported here, to other villin-type headpiece domains that bind F-actin, a positive surface potential was identified that is conserved among the latter but absent from supervillin headpiece. When this positive patch was cloned into supervillin headpiece, it endowed it with the ability to bind F-actin. Using a novel methodology for quantitating F-actin sedimentation assays, described here, it was demonstrated that this point mutant of supervillin headpiece competes with villin headpiece for a single binding site along the microfilament. In order to gain greater insight into the F-actin binding activity of villin headpiece, a complete atomic model of the microvillar cytoskeleton was constructed from published crystallographic and microscopic structures reported by several groups over the last three decades. These efforts resulted in a single, unique, self-consistent arrangement of actin, fimbrin, villin, brush border myosin (Myo1A), calmodulin, and non-erythrocytic spectrin. The proposed model accounts for all major proteinaceous components, reproduces the experimentally determined stoichiometry, and is consistent with the size and morphology of the biological brush border membrane. In building this model, a novel quaternary structure for spectrin was derived. This “Chinese Finger Trap” model explains spectrin’s biologically functional length (~65-80 nm) and provides a mechanism by which spectrin is able to undergo a three-fold extension while maintaining linearity, an experimentally observed property. The dissertation concludes with a discussion of the experiments that will test the models of the microvillar cytoskeleton and the spectrin “Chinese Finger Trap.”

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic band c-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle a-actin, cytoplasmic b-actin, and cytoplasmic c-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of a-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by b-actin and myosin-7A by c-actin. Our results indicate that band c-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug target for latent tuberculosis treatment. The enzyme is essential for the survival of the mycobacterium in macrophages. However, TBNAT has been very difficult to generate as a soluble protein. In this work we describe production of soluble recombinant TBNAT at a reasonable yield achieved by subcloning the tbnat gene with a purification His-tag into the pVLT31 plasmid, and subsequent optimisation of the induction conditions. The expression system results in soluble protein optimised upon extended (60 h) low level isopropyl b-D-1-thiogalactopyranoside level induction (100 lM) at a temperature of 15°C. The level of TBNAT expression obtained in E. coli has been significantly improved from $2 mg to a final yield of up to 16 mg per litre of culture at a purity level suitable for structural studies. The molecular mass of 31310 Da was confirmed using mass spectroscopy and the oligomerisation state was determined. The stability of TBNAT in different buffer systems was investigated by thermal shift assays and sufficient protein is now available for the screening of chemical libraries for inhibitors.

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (a-helix to p-helix) in order to p...

Non-negativity of the electron density function and constancy of the solvent regions were exploited to improve 2633 phases of crystalline yeast tRNA~ aet (P6422, a = 115.3, c = 137.9 A, z = 12), which had been obtained by multiple isomorphous replacement (MIR) in the resolution zone 14-4.5 A. Phases were also determined for an additional 912 reflections not previously phased by MIR from the resolution range 100 to 4 A, the very limits of the diffraction pattern. An iterative procedure was employed in which phases for each cycle were calculated from a density map modified by imposing the above constraints and were combined with the observed amplitudes to produce a new and improved map. Initially phases calculated in each cycle were merged with the original MIR phase probability curve; convergence was achieved in seven cycles. The phases were then released from the MIR analysis by using just the calculated phases until a second convergence was achieved (four cycles). The average difference between the experimental phases and phases calculated from the refined coordinates was reduced from 68 o for the original MIR analysis to 43 o by the use of these real-space direct methods. Phases determined solely by density modification were as accurate as the original MIR phase set. The map calculated with improved and extended phases was dramatically superior to the MIR map and even approached in quality the map produced with phases calculated from the refined molecular coordinates.

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solventexposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (a-helix to p-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.

To reduce the misfit dislocation density is a great challenge for semiconductor devices. Misfit dislocation generation is harmful for device performance. In this paper, we have used different techniques to reduce misfit dislocation. We have studied and calculated the critical layer thickness by varying In composition and we have compared the result between two models i.e. Matthews-Blakeslee and People-Bean model. Matthews-Blakeslee model shows the better performance than People-Bean model. Then we have analyzed the misfit dislocation generation by varying layer thickness and compared the result between two graded layer i.e. uniform graded layer and step graded layer for different three planes such as 1/3<11-23>{11-22},1/3<1-101>{11-22},1/3<11-23>{0001}. Here it is remarkable that we are also able to show the edge, screw as well as mixed dislocation density by varying layer thickness among these planes.

Abstract Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg 80-Asp 93 as essential for hexamer stability, compensating for the decreased intersubunit contact area.

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Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this
communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (a-helix to p-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein’s fold.

Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug target for latent tuberculosis treatment. The enzyme is essential for the survival of the mycobacterium in macrophages. However, TBNAT has been very difficult to generate as a soluble protein. In this work we describe production of soluble recombinant TBNAT at a reasonable yield achieved by subcloning the tbnat gene with a purification His-tag into the pVLT31 plasmid, and subsequent optimisation of the induction conditions. The expression system results in soluble protein optimised upon extended (60 h) low level isopropyl b-D-1-thiogalactopyranoside level induction (100 lM) at a temperature of 15°C. The level of TBNAT expression obtained in E. coli has been significantly improved from $2 mg to a final yield of up to 16 mg per litre of culture at a purity level suitable for structural studies. The molecular mass of 31310 Da was confirmed using mass spectroscopy and the oligomerisation state was determined. The stability of TBNAT in different buffer systems was investigated by thermal shift assays and sufficient protein is now available for the screening of chemical libraries for inhibitors.