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26.  Automating crystallographic structure solution and refinement of protein–ligand complexes 
A software system for automated protein–ligand crystallography has been implemented in the Phenix suite. This significantly reduces the manual effort required in high-throughput crystallographic studies.
High-throughput drug-discovery and mechanistic studies often require the determination of multiple related crystal structures that only differ in the bound ligands, point mutations in the protein sequence and minor conformational changes. If performed manually, solution and refinement requires extensive repetition of the same tasks for each structure. To accelerate this process and minimize manual effort, a pipeline encompassing all stages of ligand building and refinement, starting from integrated and scaled diffraction intensities, has been implemented in Phenix. The resulting system is able to successfully solve and refine large collections of structures in parallel without extensive user intervention prior to the final stages of model completion and validation.
PMCID: PMC3919266  PMID: 24419387
protein–ligand complexes; automation; crystallographic structure solution and refinement
27.  Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives 
The level of structural detail around the metal sites in Ni2+ and Cu2+ T6 insulin derivatives was significantly improved by using a combination of single-crystal X-ray crystallography and X-ray absorption spectroscopy. Photoreduction and subsequent radiation damage of the Cu2+ sites in Cu insulin was followed by XANES spectroscopy.
Using synchrotron radiation (SR), the crystal structures of T6 bovine insulin complexed with Ni2+ and Cu2+ were solved to 1.50 and 1.45 Å resolution, respectively. The level of detail around the metal centres in these structures was highly limited, and the coordination of water in Cu site II of the copper insulin derivative was deteriorated as a consequence of radiation damage. To provide more detail, X-ray absorption spectroscopy (XAS) was used to improve the information level about metal coordination in each derivative. The nickel derivative contains hexacoordinated Ni2+ with trigonal symmetry, whereas the copper derivative contains tetragonally distorted hexacoordinated Cu2+ as a result of the Jahn–Teller effect, with a significantly longer coordination distance for one of the three water molecules in the coordination sphere. That the copper centre is of type II was further confirmed by electron paramagnetic resonance (EPR). The coordination distances were refined from EXAFS with standard deviations within 0.01 Å. The insulin derivative containing Cu2+ is sensitive towards photoreduction when exposed to SR. During the reduction of Cu2+ to Cu+, the coordination geometry of copper changes towards lower coordination numbers. Primary damage, i.e. photoreduction, was followed directly by XANES as a function of radiation dose, while secondary damage in the form of structural changes around the Cu atoms after exposure to different radiation doses was studied by crystallography using a laboratory diffractometer. Protection against photoreduction and subsequent radiation damage was carried out by solid embedment of Cu insulin in a saccharose matrix. At 100 K the photoreduction was suppressed by ∼15%, and it was suppressed by a further ∼30% on cooling the samples to 20 K.
PMCID: PMC3919263  PMID: 24419384
bovine insulin; nickel; copper; X-ray absorption spectroscopy; EXAFS; XANES; EPR; photoreduction; radiation damage
28.  The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development 
The structural and biochemical study of Kif15 provides insight into this potential drug target and allows comparison with Eg5, a kinesin that partially shares the functions of Kif15.
Kinesins constitute a superfamily of microtubule-based motor proteins with important cellular functions ranging from intracellular transport to cell division. Some kinesin family members function during the mitotic phase of the eukaryotic cell cycle and are crucial for the successful progression of cell division. In the early stages of mitosis, during prometaphase, certain kinesins are required for the formation of the bipolar spindle, such as Eg5 and Kif15, which seem to possess partially overlapping functions. Because kinesins transform the chemical energy from ATP hydrolysis into mechanical work, inhibition of their function is a tractable approach for drug development. Drugs targeting Eg5 have shown promise as anticancer agents. Kif15 has recently come to the fore because it can substitute the functions of Eg5, and may itself have potential as a prospective drug target. Here, the initial biochemical, kinetic and structural characterization of Kif15 is reported and it is compared with the functionally related motor Eg5. Although Kif15 contains ADP in the catalytic site, its motor-domain structure was captured in the ‘ATP-like’ configuration, with the neck linker docked to the catalytic core. The interaction of Kif15 with microtubules was also investigated and structural differences between these two motors were elucidated which indicate profound differences in their mode of action, in agreement with current models of microtubule cross-linking and sliding.
PMCID: PMC3919264  PMID: 24419385
human kinesins; mitosis; bipolar spindle formation; Kif15; Eg5
29.  Structure–activity correlations of variant forms of the B pentamer of Escherichia coli type II heat-labile enterotoxin LT-IIb with Toll-like receptor 2 binding 
Structural data for the S74D variant of the pentameric B subunit of type II heat-labile enterotoxin of Escherichia coli reveal a smaller pore opening that may explain its reduced Toll-like receptor binding affinity compared to that of the wild type enterotoxin. The explanation for the enhanced Toll-like receptor binding affinity of the S74A variant is more complex than simply being attributed to the pore opening.
The pentameric B subunit of the type II heat-labile enterotoxin of Escherichia coli (LT-IIb-B5) is a potent signaling molecule capable of modulating innate immune responses. It has previously been shown that LT-IIb-B5, but not the LT-IIb-­B5 Ser74Asp variant [LT-IIb-B5(S74D)], activates Toll-like receptor (TLR2) signaling in macrophages. Consistent with this, the LT-IIb-B5(S74D) variant failed to bind TLR2, in contrast to LT-IIb-B5 and the LT-IIb-B5 Thr13Ile [LT-IIb-B5(T13I)] and LT-IIb-B5 Ser74Ala [LT-IIb-B5(S74A)] variants, which displayed the highest binding activity to TLR2. Crystal structures of the Ser74Asp, Ser74Ala and Thr13Ile variants of LT-­IIb-B5 have been determined to 1.90, 1.40 and 1.90 Å resolution, respectively. The structural data for the Ser74Asp variant reveal that the carboxylate side chain points into the pore, thereby reducing the pore size compared with that of the wild-type or the Ser74Ala variant B pentamer. On the basis of these crystallographic data, the reduced TLR2-binding affinity of the LT-IIb-B5(S74D) variant may be the result of the pore of the pentamer being closed. On the other hand, the explanation for the enhanced TLR2-binding activity of the LT-IIb-B5(S74A) variant is more complex as its activity is greater than that of the wild-type B pentamer, which also has an open pore as the Ser74 side chain points away from the pore opening. Data for the LT-IIb-B5(T13I) variant show that four of the five variant side chains point to the outside surface of the pentamer and one residue points inside. These data are consistent with the lack of binding of the LT-IIb-B5(T13I) variant to GD1a ganglioside.
PMCID: PMC3498930  PMID: 23151625
type II heat-labile enterotoxin; LT-IIb; pentameric B subunit; Toll-like receptor signaling
30.  Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio  
The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods.
PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.
PMCID: PMC3498933  PMID: 23151633
PriB protein; OB domains; atomic resolution; direct methods
31.  Timely deposition of macromolecular structures is necessary for peer review 
Deposition of crystallographic structures should be concurrent with or prior to manuscript submission for peer review, enabling validation and increasing reliability of the PDB.
Most of the macromolecular structures in the Protein Data Bank (PDB), which are used daily by thousands of educators and scientists alike, are determined by X-ray crystallography. It was examined whether the crystallographic models and data were deposited to the PDB at the same time as the publications that describe them were submitted for peer review. This condition is necessary to ensure pre-publication validation and the quality of the PDB public archive. It was found that a significant proportion of PDB entries were submitted to the PDB after peer review of the corresponding publication started, and many were only submitted after peer review had ended. It is argued that clear description of journal policies and effective policing is important for pre-publication validation, which is key in ensuring the quality of the PDB and of peer-reviewed literature.
PMCID: PMC3852646  PMID: 24311569
Protein Data Bank; deposition; validation
32.  Comment on Timely deposition of macromolecular structures is necessary for peer review by Joosten et al. (2013) 
A response to the article by Joosten et al. [(2013), Acta Cryst. D69, 2293–2295].
The wwPDB responds to the article by Joosten et al. [(2013), Acta Cryst. D69, 2293–2295].
PMCID: PMC3852647  PMID: 24311570
wwPDB; deposition; macromolecular data
33.  Comment on On the propagation of errors by Jaskolski (2013) 
A response to the article by Jaskolski [(2013), Acta Cryst. D69, 1865–1866].
The wwPDB responds to the article by Jaskolski [(2013), Acta Cryst. D69, 1865–1866].
PMCID: PMC3852648  PMID: 24311571
wwPDB; errors; 
34.  Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies 
Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage, to confirm a multi-track radiation-damage process and to develop a model of that process.
Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.
PMCID: PMC3852651  PMID: 24311579
radiation damage; protein; disulfide bonds; UV–visible absorption microspectrophotometry; electron paramagnetic resonance
35.  LigSearch: a knowledge-based web server to identify likely ligands for a protein target 
LigSearch is a web server for identifying ligands likely to bind to a given protein. It can be accessed at
Identifying which ligands might bind to a protein before crystallization trials could provide a significant saving in time and resources. LigSearch, a web server aimed at predicting ligands that might bind to and stabilize a given protein, has been developed. Using a protein sequence and/or structure, the system searches against a variety of databases, combining available knowledge, and provides a clustered and ranked output of possible ligands. LigSearch can be accessed at
PMCID: PMC3852652  PMID: 24311580
LigSearch; ligand prediction
36.  Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2 
The crystal structures of human nucleosomes containing H2A.Z.1 and H2A.Z.2 have been determined. Structural polymorphisms were found in the L1 loop regions of H2A.Z.1 and H2A.Z.2 in the nucleosomes that are likely to be caused by their flexible nature.
The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.
PMCID: PMC3852653  PMID: 24311584
H2A.Z.1; H2A.Z.2; nucleosomes; chromatin; FRAP
37.  Structural basis for type VI secreted peptidoglycan dl-endopeptidase function, specificity and neutralization in Serratia marcescens  
Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment.
Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.
PMCID: PMC3852654  PMID: 24311588
amidases; cysteine proteases; disulfide linkage; effector; endopeptidases; Gram-negative; immunity protein; peptidoglycan; Serratia marcescens; type VI secretion system
38.  Split green fluorescent protein as a modular binding partner for protein crystallization 
A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants.
A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-­strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization.
PMCID: PMC3852656  PMID: 24311592
protein crystallization; synthetic symmetrization; protein tagging; split GFP; split protein; green fluorescent protein; protein expression; protein-fragment complementation; crystallization reagents
39.  Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration 
The anisotropy of crystals of glypican-1 was significantly reduced by controlled dehydration using the HC1 device, allowing the building of previously disordered parts of the structure.
The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time T inc. Of these, the most important was shown to be T inc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously dis­ordered parts of the structure.
PMCID: PMC3852657  PMID: 24311593
glypican-1; crystal dehydration; HC1; optimization; diffraction anisotropy; crystal packing
40.  Protein energy landscapes determined by five-dimensional crystallography 
Barriers of activation within the photocycle of a photoactive protein were extracted from comprehensive time courses of time resolved crystallographic data collected at multiple temperature settings.
Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012 ▶), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001 ▶), Chem. Rev. 101, 1569–1581; Schmidt et al. (2005 ▶), Methods Mol. Biol. 305, 115–154; Schmidt (2008 ▶), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallo­graphy, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010 ▶), Acta Cryst. A66, 198–206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallo­graphic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes.
PMCID: PMC3852658  PMID: 24311594
five-dimensional crystallography; time-resolved crystallography; time-resolved microspectrophotometry; chemical kinetics; photoactive yellow protein
41.  The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis  
The structure of Rv3717 determined to 1.7 Å resolution by Pt-SAD phasing reveals a unique autolysin that lacks a cell-wall-binding domain. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. Structural analysis reveals that Rv3717 utilizes a β-hairpin turn at its N-terminus to autoregulate its enzymatic activity.
Bacterial N-acetylmuramoyl-l-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and l-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-­SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-l-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.
PMCID: PMC3852659  PMID: 24311595
Rv3717; M. tuberculosis; N-acetylmuramoyl-l-alanine amidase; SAD phasing; LC/MS analysis; α/β-fold
42.  Structure of human apurinic/apyrimidinic endonuclease 1 with the essential Mg2+ cofactor 
Human AP endonuclease 1 (APE1) belongs to the DNase I-like superfamily of enzymes that require divalent cation(s) to catalyze phosphoryl-transfer reactions. A new 1.92 Å resolution crystal structure of APE1 reveals ideal octahedral coordination of a single Mg2+ ion and informs on the role of this essential cofactor.
Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5′-deoxyribose phosphate and the 3′-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3′-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm3+ or Pb2+ in the active site. However, these are poor surrogates for Mg2+ because Sm3+ is not a cofactor and Pb2+ inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg2+ ion in the active site. The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules. One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme–product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.
PMCID: PMC3852660  PMID: 24311596
apurinic/apyrimidinic DNA; base-excision repair; nucleases; phosphoryl transfer
43.  Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation 
Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation.
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.
PMCID: PMC3852661  PMID: 24311597
SUFU; GLI; Hedgehog signalling regulation
44.  Modeling the SHG activities of diverse protein crystals 
The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed.
A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-­magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.
PMCID: PMC3478120  PMID: 23090400
second-harmonic generation; SHG microscopy
45.  Nanoflow electrospinning serial femtosecond crystallography 
A low flow rate liquid microjet method for delivery of hydrated protein crystals to X-ray lasers is presented. Linac Coherent Light Source data demonstrates serial femtosecond protein crystallography with micrograms, a reduction of sample consumption by orders of magnitude.
An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14–3.1 µl min−1 to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min−1 and diffracted to beyond 4 Å resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
PMCID: PMC3478121  PMID: 23090408
serial femtosecond crystallography; nanoflow electrospinning
46.  From lows to highs: using low-resolution models to phase X-ray data 
An unusual example of how virus structure determination pushes the limits of the molecular replacement method is presented.
The study of virus structures has contributed to methodo­logical advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.
PMCID: PMC3817700  PMID: 24189238
virus structure; phasing methods; data collection; noncrystallographic symmetry
47.  Molecular replacements 
An introduction to the proceedings of the CCP4 Study Weekend held at the East Midlands Conference Centre of the University of Nottingham, England, in January 2013.
PMCID: PMC3817688  PMID: 24189226
CCP4 Study Weekend 2013
48.  Model morphing and sequence assignment after molecular replacement 
A procedure for model building is described that combines morphing a model to match a density map, trimming the morphed model and aligning the model to a sequence.
A procedure termed ‘morphing’ for improving a model after it has been placed in the crystallographic cell by molecular replacement has recently been developed. Morphing consists of applying a smooth deformation to a model to make it match an electron-density map more closely. Morphing does not change the identities of the residues in the chain, only their coordinates. Consequently, if the true structure differs from the working model by containing different residues, these differences cannot be corrected by morphing. Here, a procedure that helps to address this limitation is described. The goal of the procedure is to obtain a relatively complete model that has accurate main-chain atomic positions and residues that are correctly assigned to the sequence. Residues in a morphed model that do not match the electron-density map are removed. Each segment of the resulting trimmed morphed model is then assigned to the sequence of the molecule using information about the connectivity of the chains from the working model and from connections that can be identified from the electron-density map. The procedure was tested by application to a recently determined structure at a resolution of 3.2 Å and was found to increase the number of correctly identified residues in this structure from the 88 obtained using phenix.resolve sequence assignment alone (Terwilliger, 2003 ▶) to 247 of a possible 359. Additionally, the procedure was tested by application to a series of templates with sequence identities to a target structure ranging between 7 and 36%. The mean fraction of correctly identified residues in these cases was increased from 33% using phenix.resolve sequence assignment to 47% using the current procedure. The procedure is simple to apply and is available in the Phenix software package.
PMCID: PMC3817698  PMID: 24189236
morphing; model building; sequence assignment; model–map correlation; loop-building
49.  Extending molecular-replacement solutions with SHELXE  
Under favourable circumstances, density modification and polyalanine tracing with SHELXE can be used to improve and validate potential solutions from molecular replacement.
Although the program SHELXE was originally intended for the experimental phasing of macromolecules, it can also prove useful for expanding a small protein fragment to an almost complete polyalanine trace of the structure, given a favourable combination of native data resolution (better than about 2.1 Å) and solvent content. A correlation coefficient (CC) of more than 25% between the native structure factors and those calculated from the polyalanine trace appears to be a reliable indicator of success and has already been exploited in a number of pipelines. Here, a more detailed account of this usage of SHELXE for molecular-replacement solutions is given.
PMCID: PMC3817699  PMID: 24189237
molecular replacement; density modification; autotracing; SHELX
50.  Molecular replacement then and now 
A brief overview, with examples, of the evolution of molecular-replacement methods and models over the past few years is presented.
The ‘phase problem’ in crystallography results from the inability to directly measure the phases of individual diffracted X-ray waves. While intensities are directly measured during data collection, phases must be obtained by other means. Several phasing methods are available (MIR, SAR, MAD, SAD and MR) and they all rely on the premise that phase information can be obtained if the positions of marker atoms in the unknown crystal structure are known. This paper is dedicated to the most popular phasing method, molecular replacement (MR), and represents a personal overview of the development, use and requirements of the methodology. The first description of noncrystallographic symmetry as a tool for structure determination was explained by Rossmann and Blow [Rossmann & Blow (1962 ▶), Acta Cryst. 15, 24–31]. The term ‘molecular replacement’ was introduced as the name of a book in which the early papers were collected and briefly reviewed [Rossmann (1972 ▶), The Molecular Replacement Method. New York: Gordon & Breach]. Several programs have evolved from the original concept to allow faster and more sophisticated searches, including six-dimensional searches and brute-force approaches. While careful selection of the resolution range for the search and the quality of the data will greatly influence the outcome, the correct choice of the search model is probably still the main criterion to guarantee success in solving a structure using MR. Two of the main parameters used to define the ‘best’ search model are sequence identity (25% or more) and structural similarity. Another parameter that may often be undervalued is the quality of the probe: there is clearly a relationship between the quality and the correctness of the chosen probe and its usefulness as a search model. Efforts should be made by all structural biologists to ensure that their deposited structures, which are potential search probes for future systems, are of the best possible quality.
PMCID: PMC3817701  PMID: 24189239
molecular replacement; models; accuracy; quality

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