This paper describes a set of tools allowing experimentalists insight into the variation present within large serial data sets.
Ultrafast diffraction at X-ray free-electron lasers (XFELs) has the potential to yield new insights into important biological systems that produce radiation-sensitive crystals. An unavoidable feature of the ‘diffraction before destruction’ nature of these experiments is that images are obtained from many distinct crystals and/or different regions of the same crystal. Combined with other sources of XFEL shot-to-shot variation, this introduces significant heterogeneity into the diffraction data, complicating processing and interpretation. To enable researchers to get the most from their collected data, a toolkit is presented that provides insights into the quality of, and the variation present in, serial crystallography data sets. These tools operate on the unmerged, partial intensity integration results from many individual crystals, and can be used on two levels: firstly to guide the experimental strategy during data collection, and secondly to help users make informed choices during data processing.
Data Exploration Toolkit; ultrafast diffraction; X-ray free-electron lasers
In synaptic terminals, complexin is thought to have inhibitory and activating roles for spontaneous mini release and evoked synchronized neurotransmitter release, respectively. We used single vesicle-vesicle microscopy imaging to study the effect of complexin-1 on the on-rate of docking between vesicles that mimic synaptic vesicles and the plasma membrane, respectively. We found that complexin-1 enhances the on-rate of docking of synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma membrane mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of complexin-1, which binds to the membrane, the presence of PS in the membrane, and the core region of complexin-1, which binds to the SNARE complex.
An overview of applications of the deformable elastic network (DEN) refinement method is presented together with recommendations for its optimal usage.
Crystals of membrane proteins and protein complexes often diffract to low resolution owing to their intrinsic molecular flexibility, heterogeneity or the mosaic spread of micro-domains. At low resolution, the building and refinement of atomic models is a more challenging task. The deformable elastic network (DEN) refinement method developed previously has been instrumental in the determinion of several structures at low resolution. Here, DEN refinement is reviewed, recommendations for its optimal usage are provided and its limitations are discussed. Representative examples of the application of DEN refinement to challenging cases of refinement at low resolution are presented. These cases include soluble as well as membrane proteins determined at limiting resolutions ranging from 3 to 7 Å. Potential extensions of the DEN refinement technique and future perspectives for the interpretation of low-resolution crystal structures are also discussed.
deformable elastic network refinement; low resolution
Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion.
synaptic vesicle fusion; neurotransmitter release; complexin; synaptotagmin; SNARE; membrane fusion; none
synaptic terminals, complexin is thought to have inhibitory
and activating roles for spontaneous “mini” release
and evoked synchronized neurotransmitter release, respectively. We
used single vesicle–vesicle microscopy imaging to study the
effect of complexin-1 on the on-rate of docking between vesicles that
mimic synaptic vesicles and the plasma membrane. We found that complexin-1
enhances the on-rate of docking of synaptic vesicle mimics containing
full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma
membrane-mimicking vesicles containing full-length syntaxin-1A and
SNAP-25A. This effect requires the C-terminal domain of complexin-1,
which binds to the membrane, the presence of PS in the membrane, and
the core region of complexin-1, which binds to the SNARE complex.
A protein structure has been refined with electron diffraction data obtained by using a very weak electron beam to collect large numbers of diffraction patterns from a few sub-micron-sized three-dimensional crystals.
electron crystallography; electron diffraction; electron cryomicroscopy (cryo EM); method development; protein structure; None
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.
morphing; model building; sequence assignment; model–map correlation; loop-building
Background: NSF and α-SNAP disassemble all SNARE complexes.
Results: The disassembly kinetics is conserved for different ternary and binary SNARE complexes. α-SNAP and the ternary SNARE complex form a 1:1 complex.
Conclusion: NSF uses a conserved mechanism to disassemble all SNARE complexes, starting from a 1:1 SNAP-SNARE complex interaction.
Significance: We illuminate a broad mechanism allowing NSF to support SNARE-mediated exocytosis.
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.
ATPases; Biophysics; Biosensors; Circular Dichroism (CD); Enzyme Kinetics; Fluorescence; Membrane Fusion; Protein-Protein Interactions; SNARE Proteins; Structural Biology
This Protocol describes a single vesicle-vesicle microscopy system to study Ca2+-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and SNAP-25, and are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at zero Ca2+-concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca2+-injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-hundred millisecond timescale. Other factors, such as complexin, can be easily added. Our system is unique by monitoring both content and lipid mixing, and by starting from a metastable state of interacting vesicle pairs prior to Ca2+-injection.
Membrane biology; vesicle fusion; membrane fusion; sulforhodamine B; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate; DiIC18(5); DiD; cascade blue; SNARE; Ca2+; fluorescence microscopy; TIR; total internal reflection
α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself.
The central nervous system coordinates many different activities by sending instructions to large numbers of cells and, simultaneously, processing all the signals that are sent back to the brain. All these messages are carried by electrical pulses that travel along chains of neurons, with neurotransmitter molecules enclosed inside synaptic vesicles conveying the messages across the synapses between neurons. A protein called α-synuclein is thought to have a role in the transport of neurotransmitter molecules across synapses, but the details of its involvement are not fully understood.
Mutations in the gene that codes for α-synuclein, and also duplications and triplications of this gene, are known to lead to an increased risk of early onset Parkinson's disease, a condition where the central nervous system degenerates. Moreover, the Lewy bodies found in the neurons of patients with Parkinson's disease contain high concentrations of α-synuclein. Again, however, none of this is fully understood.
Diao et al. have shed new light on these questions by creating synthetic vesicles to mimic what happens in real synapses, and using optical microscopy to observe the behaviour of these vesicles. They found that native α-synuclein (and another set of membrane proteins) increases the availability of synthetic vesicles at the synapse by causing them to cluster together. In a second experiment, Diao et al. showed that native α-synuclein does not decrease calcium-triggered fusion between membranes, the process that releases neurotransmitter into the synaptic cleft. In contrast, it is known that pathogenic α-synuclein aggregates directly interfere with the release of the neurotransmitter molecules. Moreover, when Diao et al. used a particular mutant form of α-synuclein that is associated with Parkinson's disease, the vesicles did not form clusters. If these results are confirmed in vivo, the role played by native α-synuclein in the central nervous system, and the connection between α-synuclein and Parkinson's disease, will be much clearer.
Parkinson's disease; synaptic terminals; alpha-synuclein; Mouse
The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.
The central nervous system relies on electrical signals travelling along neurons and through synapses at high speeds. Signals often have to pass between two neurons, or from a neuron to a muscle fiber, and the nervous system relies on a process called membrane fusion to ensure that the neurotransmitter molecules that carry the signal across the synapses are released as quickly as possible. Membrane fusion is an important process in many areas of biology, including intracellular transport and fertilization, but it occurs much faster (millisecond time scale) in the nervous system than anywhere else in the body. The reasons for this have long been a mystery, although calcium ions are known to trigger the fusion process.
The fusion of two biological membranes is similar in many regards to the way that small soap bubbles merge together to form large bubbles. Just as soap bubbles can form a variety of discernible intermediate structures when they merge, so can biological membranes. This means that it is possible to produce a so-called hemifusion intermediate in which the outer layers of the membranes have merged, but the inner layers have not, so it is not possible for anything—such as serotonin, dopamine and other neurotransmitter molecules—to transfer from one membrane to the other.
Diao et al. have used a combination of advanced optical imaging and cryogenic electron microscopy to explore membrane fusion between synthetic membranes that contained reconstituted synaptic proteins, including synaptotagmin and a family of protein receptors called SNAREs. When calcium ions were injected into the synthetic system, the basic characteristics of neurotransmitter release—such as membrane fusion on a millisecond time scale—was observed. Contrary to some theories of membrane fusion, the fastest fusion events did not begin or proceed via a discernible hemifusion intermediate state. Rather, these events proceeded from a ‘point contact’ state in which the membranes were close to each other (just 1–5 nm apart) without being fused, and were ready to undergo fast fusion once the calcium ions had been injected. And when Diao et al. introduced a protein called complexin, which is known to be important for fast neurotransmitter release in vivo, they observed more immediate fusion events and fewer events that involved a hemifusion intermediate.
With a synthetic system it is possible to perform experiments that are currently not possible with live neurons, and this has allowed Diao et al. to clarify the roles of the individual components in the process of membrane fusion, and could prove useful in efforts to develop novel therapeutic treatments to combat neurological disorders.
neurotransmitter release; synaptic vesicle fusion; SNARE; synaptotagmin; complexin; Other
In X-ray crystallography, molecular replacement and subsequent refinement is challenging at low resolution. We compared refinement methods using synchrotron diffraction data of photosystem I at 7.4 Å resolution, starting from different initial models with increasing deviations from the known high-resolution structure. Standard refinement spoiled the initial models moving them further away from the true structure and leading to high Rfree-values. In contrast, DEN-refinement improved even the most distant starting model as judged by Rfree, atomic root-mean-square differences to the true structure, significance of features not included in the initial model, and connectivity of electron density. The best protocol was DEN-refinement with initial segmented rigid-body refinement. For the most distant initial model, the fraction of atoms within 2 Å of the true structure improved from 24% to 60%. We also found a significant correlation between Rfree-values and the accuracy of the model, suggesting that Rfree is useful even at low resolution.
DEN refinement; membrane protein; low-resolution refinement; simulated annealing; free R value
A density-based procedure is described for improving a homology model that is locally accurate but differs globally. The model is deformed to match the map and refined, yielding an improved starting point for density modification and further model-building.
An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Å radius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Å r.m.s.d. representing 65–100% of the structure.
molecular replacement; automation; macromolecular crystallography; structure similarity; modeling; Phenix; morphing
DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols.
Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
The deformable elastic network (DEN) method for reciprocal-space crystallographic refinement improves crystal structures, especially at resolutions lower than 3.5 Å. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements.
Deformable elastic network (DEN) restraints have proved to be a powerful tool for refining structures from low-resolution X-ray crystallographic data sets. Unfortunately, optimal refinement using DEN restraints requires extensive calculations and is often hindered by a lack of access to sufficient computational resources. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements in parallel on the Open Science Grid, the US cyberinfrastructure. Access to the grid is provided through a simple and intuitive web interface integrated into the SBGrid Science Portal. Using this portal, refinements combined with full parameter optimization that would take many thousands of hours on standard computational resources can now be completed in several hours. An example of the successful application of DEN restraints to the human Notch1 transcriptional complex using the grid resource, and summaries of all submitted refinements, are presented as justification.
deformable elastic network restraints; low-resolution refinement; DEN refinement
A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis
Crystallographic structures encompassing GPCR autoproteolytic sequences (GPS) delineate a novel conserved structural domain called GAIN, which is found in cell-adhesion GPCRs, polycystic kidney disease proteins conserved throughout evolution.
The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40-residue GPS motif represents an integral part of a much larger ∼320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.
adhesion GPCRs; autoproteolysis; latrotoxin; polycystic kidney disease-1; synapse
Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in non-neuronal cells, neurexins induce formation of postsynaptic specializations in co-cultured neurons, suggesting that neurexins are synaptogenic. However, we now find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at sub-micromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and with purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABAA-receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABAA-receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABAA-receptors, which may contribute to regulate the excitatory/inhibitory balance in brain.
Background: A key step in intoxication by botulinum neurotoxins is the translocation of the protease domain by the translocation domain (TD) across endosomes. The requirements for translocation remain poorly understood.
Results: A construct encompassing the TD yet devoid of the belt embodies a minimum channel-forming unit.
Conclusion: The belt is dispensable for channel formation.
Significance: The belt restricts cargo dissociation from channel during translocation.
Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the “beltless” TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.
Membrane Proteins; Membrane Reconstitution; Neurotoxin; Patch Clamp; Protein Translocation; Botulinum Neurotoxin; Membranes; Protein Domains; Protein Translocases; Single Channels
Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms.
This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.
► Validation criteria used by the PDB for X-ray crystal structures have been reassessed ► Key scores should be presented prominently in an easily understood format ► A concise validation report should be available to referees of papers on crystal structures
Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.
single molecule fluorescence; FRET; molecular dynamics; protein-protein interactions
X-ray diffraction plays a pivotal role in understanding of biological systems by revealing atomic structures of proteins, nucleic acids, and their complexes, with much recent interest in very large assemblies like the ribosome. Since crystals of such large assemblies often diffract weakly (resolution worse than 4 Å), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, while others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex1. Determining such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution below 5 Å generally exceed the number of degrees of freedom. Here we introduce a new method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with Rfree determines the optimum deformation and influence of the homology model. For test cases at 3.5 – 5 Å resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model coordinate accuracy, the definition of secondary structure, and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the PDB, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to studying weakly diffracting crystals using X-ray micro-diffraction2 as well as data from new X-ray light sources3. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to sub-nanometer resolution4,5, it can use similar tools.
X-ray crystallography; homology modeling; cross-validation; Rfree value; refinement
Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1, a synaptic vesicle protein that interacts with SNAREs and membranes. We used single-molecule FRET between synaptotagmin’s two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intra-synaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive synaptotagmin 1–SNARE complex by using a multi-body docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they could interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+ bound synaptotagmin 3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs plays a role in Ca2+-triggered fusion.
membrane fusion; neurotransmitter release; protein-protein interactions; synaptic vesicle; single molecule FRET