Transmission electron microscopy as a tool for nanocrystal characterization pre- and post-injector.

Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.

Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins.

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.

Structural features of the single-stranded DNA-binding protein of Epstein-Barr virus.

We report the structural features of a C-terminal deletion construct of the Epstein-Barr virus single-stranded DNA-binding protein, Balf2 (Balf2DeltaC), which like the herpes simplex virus I encoded protein, infected cell protein 8 (ICP8), binds non-sequence specifically to single-stranded DNA (ssDNA). ICP8, in the absence of ssDNA, assembles into long filamentous structures. Removal of the 60 C-terminal amino acids of ICP8 (ICP8DeltaC) prevents the formation of such filaments, whereas addition of circular ssDNA to ICP8DeltaC induces formation of "super helical" filaments. Balf2DeltaC, which we show is a zinc-binding protein, does not form these filaments under the same conditions but does bind ssDNA in a weakly cooperative manner. Further structural comparison of both proteins in solution by small-angle X-ray scattering shows proteins with similar molecular envelopes. One major difference is the tendency of Balf2DeltaC to dimerize on different surfaces to that used for oligomerization when binding to ssDNA, and this may have implications for the mechanism of replication initiation.

Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis.

Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.

Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins.

Rolling circle replication from M13 DNA circles was previously reconstituted in vitro using purified factors encoded by bacteriophage T4. The products are duplex circles with linear tails >100 kb. When T4 DNA polymerase deficient in 3' to 5' exonuclease activity was employed, electron microscopy revealed short single-stranded DNA "flaps" along the replicated tails. This marked the beginning of each Okazaki fragment, allowing an analysis of the lengths of sequential Okazaki fragments on individual replicating molecules. DNAs containing runs of Okazaki fragments of similar length were found, but most showed large length variations over runs of six or more fragments reflecting the broad population distribution.

Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance.

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.

Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding.

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G(1). To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.

Betabellins 15D and 16D, de Novo designed beta-sandwich proteins that have amyloidogenic properties.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta-sheets packed against one another by hydrophobic interactions. d-Amino acid residues are used to energetically favor formation of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p denotes d-Pro, h denotes d-His, and k denotes d-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constrained d-Pro residue at the i + 1 position of each type-I' beta turn. To test whether d-Pro residues are necessary for folding at these positions, the six d-Pro residues of B15D are replaced by d-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D forms unbranched, 35-A wide fibrils that associate into bundles in 5.0 mM 3-(N-morpholino)propanesulfonate and 250 mM NaCl at pH 7; under these conditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils. The present studies show that both B15D and B16D have characteristics of amyloidogenic proteins: the unbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naphthalenesulfonate. Thus, these de novo designed beta-sandwich proteins should provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential inhibitors of amyloidogenesis.

Biophysical characterization of betabellin 16D: a beta-sandwich protein that forms narrow fibrils which associate into broad ribbons.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta sheets packed against one another by hydrophobic interactions. Betabellin 16S (B16S), a 32-residue peptide chain (HSLTAKIakLTFSIAahTYTCAVakYTAKVSH, where a is DAla, h is DHis, and k is DLys), did not have beta structure in water at pH 6.5. Air oxidation of B16S furnished betabellin 16D (B16D), a 64-residue disulfide-bridged two-chain protein, which also did not fold in water at pH 6.5. However, the extent of beta structure observed for B16D increased with pH and ionic strength of the solution and the B16D concentration as observed by circular dichroism spectropolarimetry. Transmission electron microscopy showed that B16D formed narrow fibrils that associated into broad ribbons in 5.0 mM Mops and 0.25 M NaCl at pH 6.9.

Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.

Electron microscopy (EM) was used to visualize intermediates of in vitro replication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass replication of pyrimidine dimers. The DNA substrate was either undamaged or contained a single cis, syn thymine dimer. This lesion was inserted 385 bp downstream from the center of the SV40 origin of replication and sited specifically in the template to the leading strand of the newly synthesized DNA. Products from 30 minute reactions were crosslinked with psoralen and UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules replicated by either bypass-proficient or deficient extracts. These regions could be coated with Escherichia coli single-stranded DNA binding protein. The length of duplex DNA from a unique restriction site to the single-stranded DNA region was that predicted from blockage of leading strand synthesis by the site-specific dimer. These results were confirmed by S1nuclease treatment of replication products linearized with single cutting restriction enzymes, followed by detection of the diagnostic fragments by gel electrophoresis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass replication. These criteria were fulfilled in 17 % of the molecules replicated by the IDH4 extract.

Electron microscopy of DNA-protein complexes and chromatin.

This article focused on a number of aspects of the preparation of chromatin and other DNA-protein complexes for conventional transmission EM that are critical for success but may not have been addressed in a single chapter before. These include the importance of optimizing fixation, the generation of active supporting supports, and the use of negative staining as a means of obtaining higher resolution detail than can be garnered from shadow casting methods.

Human Hsp70 and Hsp40 chaperone proteins facilitate human papillomavirus-11 E1 protein binding to the origin and stimulate cell-free DNA replication.

Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.

Engineering of betabellin-15D: a 64 residue beta sheet protein that forms long narrow multimeric fibrils.

The betabellin target structure is a beta-sandwich protein consisting of two 32 residue beta-sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin-15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p=DPro, k=DLys, and h=DHis) did not fold in water at pH 6.5. Air oxidation of betabellin-15S provided betabellin-15D, the 64 residue disulfide bridged two-chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of beta structure observed for betabellin-15D increased with the pH and ionic strength of the solution and the betabellin-15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin-15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin-15D fibrils are consistent with a single row of beta-sandwich molecules joined by multiple intersheet H-bonds.

Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8.

UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA.

The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures.

The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifically to the HSV-1 oriS and oriL origins of replication, and is a DNA helicase and DNA-dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS. In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex. In the presence of 4 mM ATP, binding at oriS was enhanced 2-fold, DNA loops or stem-loops were extruded from the UL9 protein complex at oriS, and the DNA in them frequently appeared highly condensed into a tight rod. The stem-loops contained from a few hundred to over one thousand base pairs of DNA and in most, oriS was located at their apex, although in some, oriS was at a border. The DNA in the stem-loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound the stem-loops strongly. Thus the DNA strands in the stem-loops exist in a partially paired, partially single-stranded state presumably making them available for ICP8 binding in vivo. These observations provide direct evidence for an origin specific unwinding by the HSV-1 UL9 protein and for the formation of a relatively stable four-stranded DNA in this process.

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells.

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.

Electron microscopic visualization of RecT protein and its complexes with DNA.

Electron microscopy has been used to examine Escherichia coli RecT protein alone and in the complexes it forms with DNA substrates, with which it catalyzes strand exchange in vitro. Negative staining has revealed that the 33 kDa RecT protein monomers form open C-shaped and closed O-shaped particles. RecT protein monomers assemble into donut-shaped oligomers containing seven or eight protein monomers and rod-like structures. When bound to single-stranded DNA, RecT forms highly twisted nucleoprotein filaments that are 18 nm in diameter and have a helical pitch of 10 nm. When added to linear duplex DNA in the presence of active RecE protein (exonuclease VIII), filamentous nucleoprotein complexes are formed on the DNA ends and the DNA molecules are frequently cyclized through protein-protein interactions.

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is an adhesin that binds the bacteria to cells of the respiratory epithelium in whooping-cough infections. Mature FHA is a 220 kDa secretory protein that is highly immunogenic and has been included in acellular vaccines. We have investigated its structure by combining electron microscopy and circular dichroism spectroscopy (CD) with computational analysis of its amino acid sequence. The FHA molecule is 50 nm in length and has the shape of a horseshoe nail: it has a globular head that appears to consist of two domains; a 35 nm-long shaft that averages 4 nm in width, but tapers slightly from the head end; and a small, flexible, tail. Mass measurements by scanning transmission electron microscopy establish that FHA is a monomer. Its sequence contains two regions of tandem 19-residue pseudo-repeats: the first, of 38 cycles, starts at residue 344; the second, of 13 cycles, starts at residue 1440. The repeat motifs are predicted to consist of short beta-strands separated by beta-turns, and secondary structure measurements by CD support this prediction. We propose a hairpin model for FHA in which the head is composed of the terminal domains; the shaft consists mainly of the repeat regions conformed as amphipathic, hyper-elongated beta-sheets, with their hydrophobic faces apposed; and the tail is composed of the intervening sequence. Further support for the model was obtained by immuno-labeling electron microscopy. The 19-residue repeats of FHA have features in common with the leucine-rich repeats (LRRs) that are present in many eukaryotic proteins, including some adhesion factors. The model is also compared with the two other classes of filamentous proteins that are rich in beta-structure, i.e. viral adhesins and two beta-helical secretory proteins. Our proposed structure implies how the functionally important adhesion sites and epitopes of FHA are distributed: its tripeptide (RGD) integrin-binding site is assigned to the tail; the putative hemagglutination site forms part of the head; and two classes of immunodominant epitopes are assigned to opposite ends of the molecule. Possible mechanisms are discussed for two modes of FHA-mediated adhesion.

The short tail-fiber of bacteriophage T4: molecular structure and a mechanism for its conformational transition.

Electron microscopy, image processing and computational sequence analysis were used to investigate the structure of the short tail-fiber of bacteriophage T4. This molecule, an oligomer of gp12, is an adhesin that binds the virion irreversibly to the bacterial surface. Short tail-fibers were isolated from mutant-infected cells in which gp12 is synthesized and assembled correctly, but not incorporated into virions. Visualized in negative stain, these filamentous molecules are approximately 38 nm in total length, with an arrowhead-shaped head (approximately 10 nm long by 6 nm wide), a 24-nm shaft of uniform width (approximately 3.8 nm), and a small, seemingly flexible, tail. The primary sequence contains a domain consisting of tandem quasi-repeats, each about 40 residues long, extending from approximately residue 50 to residue 320. Molecular mass analyses by scanning transmission electron microscopy confirm that the molecule is a trimer. The masses of the head, shaft, and tail domains are consistent with (trimers of) the carboxy-terminus, the repeat region, and the amino-terminus, respectively. When short tail-fibers are visualized extending from baseplates, their heads are distal, i.e., detached, implying that it is the tail that remains in contact with the baseplate. Analysis of the molecules' curvature properties detects three hinge-sites: these suggest how the short tail-fiber may be initially accommodated in a compact conformation in the "hexagon" state of the baseplate, from which it converts to the extended conformation when the baseplate switches into its "star" state.

Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus.

Double-stranded RNA viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A double-stranded RNA virus we have investigated the mechanism by which the virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag-pol fusion protein which is formed by a -1 ribosomal frameshift. Here we show that Gag alone is sufficient for virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A virus encapsidation model: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.