Philip J.B. Koeck

Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin

MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D′D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure

The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus–cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast‐localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single‐particle electron microscopy, and lysine‐specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.

Two-dimensional crystals of reconstituted β-subunits of the chaperonin TF55 from Sulfolobus shibatae

We have obtained 2-dimensional crystals of the beta-subunits of the chaperonin TF55 from Sulfolobus shibatae reconstituted into oligomers in the absence of alpha-subunits. The subunits form rings with 9-fold rotational symmetry which arrange themselves in a trigonal lattice. From electron micrographs of negatively stained specimens we have calculated a projection map in plane group p312 showing the rings in top-view.

Turning of the receptor‐binding domains opens up the murine leukaemia virus Env for membrane fusion

The activity of the membrane fusion protein Env of Moloney mouse leukaemia virus is controlled by isomerization of the disulphide that couples its transmembrane (TM) and surface (SU) subunits. We have arrested Env activation at a stage prior to isomerization by alkylating the active thiol in SU and compared the structure of isomerization‐arrested Env with that of native Env. Env trimers of respective form were isolated from solubilized particles by sedimentation and their structures were reconstructed from electron microscopic images of both vitrified and negatively stained samples. We found that the protomeric unit of both trimers formed three protrusions, a top, middle and a lower one. The atomic structure of the receptor‐binding domain of SU fitted into the upper protrusion. This was formed similar to a bent finger. Significantly, in native Env the tips of the fingers were directed against each other enclosing a cavity below, whereas they had turned outward in isomerization‐arrested Env transforming the cavity into an open well. This might subsequently guide the fusion peptides in extended TM subunits into the target membrane.

Quantization errors in averaged digitized data

Analytic expressions which describe average quantization errors in digitized data with additive noise are derived. The magnitude of this error depends on the noise present in the analog signal, the bin-size (the difference between neighboring quantization levels) and also the signal itself. An iterative process, which corrects for these residual quantization errors after averaging, is proposed and tested in simulations. Alternatively a method for avoiding quantization errors during digitization of signals which will later be averaged is suggested.

Ins and Outs of Digital Electron Microscopy

In order to do any sort of computer processing of the images produced by an electron microscope they have to be digitized. Therefore, any form of microscopy that involves computer processing has to be considered as digital microscopy independently of whether the original images were recorded on film and scanned or recorded directly on a digital medium such as the charge coupled device (CCD). As soon as one digitizes images, one encounters a range of effects and artefacts specific to the digital world such as aliasing, quantization errors, and ambiguities in the modulation transfer function. Even a quick discussion of these effects makes it clear that what comes out of a digitizer is not related in a simple way to what goes in. After introducing the commonly used recording media, I will discuss the most important effects of digitization and then compare the limitations and advantages of the different recording media for various applications. Microsc. Res. Tech. 49:217–223, 2000.

Improved immuno double labelling for cell and structural biology

Colloidal gold is the most widely used electron dense marker in biological electron microscopy. The development of procedures for making gold particles of very defined sizes has made double or even multiple labelling possible using gold of two or more different sizes. Lately a new type of electron dense marker has been developed consisting of ligand-stabilized metal atom clusters rather than colloidal particles. The differences between these two types of markers are highlighted and the advantages of using metal atom clusters for immuno labelling of certain biological specimens are discussed. Possible methods of distinguishing two such cluster labels in double labelling experiments are reviewed.

Unconventional immuno double labelling by energy filtered transmission electron microscopy

A new method of immuno double labelling of biological specimens with a very high spatial resolution is presented. The advantage over conventional techniques is the possibility of using two very small labels leading to higher labelling efficiency, better penetration into the specimen and reduced steric hindrance between labels at closely spaced sites. The two labels are distinguished by their electron energy loss spectra using principal component analysis and then identified by comparison with an external standard using discriminant function analysis. The method is tested on samples of insect flight muscle labelled with 8 nm colloidal gold and silver and the statistical reliability of the classification is assessed. Extensions of the method are suggested and its potential for biological research is discussed.