ABSTRACT
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
Subject(s)
Vesicular Transport Proteins , rab5 GTP-Binding Proteins , Humans , Vesicular Transport Proteins/metabolism , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/metabolism , Endosomes/genetics , Endosomes/metabolismABSTRACT
Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80Sâ¢HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation.
Subject(s)
Internal Ribosome Entry Sites , RNA, Viral/chemistry , Ribosome Subunits/chemistry , Amino Acid Sequence , Base Sequence , Hepatitis C/metabolism , Humans , Molecular Docking Simulation , Molecular Sequence Data , Protein BindingABSTRACT
Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.
Subject(s)
Contactin 1/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Animals , Cells, Cultured , HEK293 Cells , Hippocampus/cytology , Hippocampus/metabolism , Humans , Membrane Proteins/ultrastructure , Mice, Inbred C57BL , Models, Molecular , Nerve Tissue Proteins/ultrastructure , Protein Interaction Maps , Protein Structure, Tertiary , Scattering, Small Angle , X-Ray DiffractionABSTRACT
Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.
Subject(s)
Cryoelectron Microscopy/methods , Ribosomes/chemistry , Cell-Free System , Models, Molecular , Ribosomes/ultrastructureABSTRACT
The ribosome-recycling factor (RRF) and elongation factor-G (EF-G) disassemble the 70S post-termination complex (PoTC) into mRNA, tRNA, and two ribosomal subunits. We have determined cryo-electron microscopic structures of the PoTC·RRF complex, with and without EF-G. We find that domain II of RRF initially interacts with universally conserved residues of the 23S rRNA helices 43 and 95, and protein L11 within the 50S ribosomal subunit. Upon EF-G binding, both RRF and tRNA are driven towards the tRNA-exit (E) site, with a large rotational movement of domain II of RRF towards the 30S ribosomal subunit. During this intermediate step of the recycling process, domain II of RRF and domain IV of EF-G adopt hitherto unknown conformations. Furthermore, binding of EF-G to the PoTC·RRF complex reverts the ribosome from ratcheted to unratcheted state. These results suggest that (i) the ribosomal intersubunit reorganizations upon RRF binding and subsequent EF-G binding could be instrumental in destabilizing the PoTC and (ii) the modes of action of EF-G during tRNA translocation and ribosome-recycling steps are markedly different.
Subject(s)
Peptide Elongation Factor G/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Cryoelectron Microscopy , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Escherichia coli Proteins/metabolism , Protein Binding , Protein Conformation , RNA, Ribosomal/metabolismABSTRACT
Time-resolved cryo electron microscopy (TRCEM) has emerged as a powerful technique for transient structural characterization of isolated biomacromolecular complexes in their native state within the time scale of seconds to milliseconds. For TRCEM sample preparation, microfluidic device [9] has been demonstrated to be a promising approach to facilitate TRCEM biological sample preparation. It is capable of achieving rapidly aqueous sample mixing, controlled reaction incubation, and sample deposition on electron microscopy (EM) grids for rapid freezing. One of the critical challenges is to transfer samples to cryo-EM grids from the microfluidic device. By using microspraying method, the generated droplet size needs to be controlled to facilitate the thin ice film formation on the grid surface for efficient data collection, while not too thin to be dried out before freezing, i.e., optimized mean droplet size needs to be achieved. In this work, we developed a novel monolithic three dimensional (3D) annular gas-assisted microfluidic sprayer using 3D MEMS (MicroElectroMechanical System) fabrication techniques. The microsprayer demonstrated dense and consistent microsprays with average droplet size between 6-9 µm, which fulfilled the above droplet size requirement for TRCEM sample preparation. With droplet density of around 12-18 per grid window (window size is 58×58 µm), and the data collectible thin ice region of >50% total wetted area, we collected ~800-1000 high quality CCD micrographs in a 6-8 hour period of continuous effort. This level of output is comparable to what were routinely achieved using cryo-grids prepared by conventional blotting and manual data collection. In this case, weeks of data collection process with the previous device [9] has shortened to a day or two. And hundreds of microliter of valuable sample consumption can be reduced to only a small fraction.
ABSTRACT
Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.
Subject(s)
Introns , RNA Precursors/chemistry , RNA, Catalytic/chemistry , Ribonucleoproteins/chemistry , Lactococcus lactis/genetics , Molecular Conformation , RNA Precursors/isolation & purification , RNA, Catalytic/isolation & purification , Ribonucleoproteins/isolation & purification , Ribonucleoproteins/ultrastructureABSTRACT
Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.
Subject(s)
Hydrolases , Cryoelectron Microscopy/methods , Crystallography, X-Ray , Substrate Specificity , Hydrolases/chemistryABSTRACT
In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-A cryo-electron microscopy map of the aminoacyl-tRNA x EF-Tu x GDP x kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.
Subject(s)
Escherichia coli/metabolism , Guanosine Triphosphate/metabolism , Peptide Elongation Factor Tu/chemistry , Ribosomes/metabolism , Cryoelectron Microscopy , Enzyme Activation , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Histidine/metabolism , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Peptide Elongation Factor Tu/ultrastructure , Protein Structure, Secondary , RNA, Transfer/metabolism , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , Signal TransductionABSTRACT
A widely used approach to analyze single particles in electron microscopy data is 2D classification. This process is very computationally expensive, especially when large data sets are analyzed. In this paper we present GPU ISAC, a newly developed, GPU-accelerated version of the established Iterative Stable Alignment and Clustering (ISAC) algorithm for 2D images and generating class averages. While the previously existing implementation of ISAC relied on a computer cluster, GPU ISAC enables users to produce high quality 2D class averages from large-scale data sets on a single desktop machine equipped with affordable, consumer-grade GPUs such as Nvidia GeForce GTX 1080 TI cards. With only two such cards GPU ISAC matches the performance of twelve high end cluster nodes and, using high performance GPUs, is able to produce class averages from a million particles in between six to thirteen hours, depending on data set quality and box size. We also show GPU ISAC to scale linearly in all input dimensions, and thereby capable of scaling well with the increasing data load demand of future data sets. Further user experience improvements integrate GPU ISAC seamlessly into the existing SPHIRE GUI, as well as the TranSPHIRE on-the-fly processing pipeline. It is open source and can be downloaded at https://gitlab.gwdg.de/mpi-dortmund/sphire/cuISAC/.
ABSTRACT
The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.
Subject(s)
Bacterial Proteins/chemistry , Salmonella typhimurium/chemistry , Bacterial Proteins/ultrastructure , Computer Simulation , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/ultrastructure , Pliability , Protein Structure, Quaternary , Protein Subunits/chemistryABSTRACT
We report the investigation of a novel microfluidic mixing device to achieve submillisecond mixing. The micromixer combines two fluid streams of several microliters per second into a mixing compartment integrated with two T- type premixers and 4 butterfly-shaped in-channel mixing elements. We have employed three dimensional fluidic simulations to evaluate the mixing efficiency, and have constructed physical devices utilizing conventional microfabrication techniques. The simulation indicated thorough mixing at flow rate as low as 6 µL/s. The corresponding mean residence time is 0.44 ms for 90% of the particles simulated, or 0.49 ms for 95% of the particles simulated, respectively. The mixing efficiency of the physical device was also evaluated using fluorescein dye solutions and FluoSphere-red nanoparticles suspensions. The constructed micromixers achieved thorough mixing at the same flow rate of 6 µL/s, with the mixing indices of 96% ± 1%, and 98% ± 1% for the dye and the nanoparticle, respectively. The experimental results are consistent with the simulation data. The device demonstrated promising capabilities for time resolved studies for macromolecular dynamics of biological macromolecules.
ABSTRACT
We describe here the implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. A previously designed computer-controlled cryo-plunging apparatus [White, H.D., Thirumurugan, K., Walker, M.L., Trinick, J., 2003. A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J. Struct. Biol. 144, 246-252] was used as a hardware platform, onto which a xenon flash lamp and liquid light pipe were mounted. The irradiation initiates a reaction through cleavage of the photolabile blocking group from a biologically active compound. The timespan between flashing and freezing in cryogen is on the order of milliseconds, and defines the fastest observable reaction. Blotting of excess fluid, which takes on the order of 1s, is done before irradiation and thus does not represent a rate-limiting step. A specimen-heating problem, identified by measurements with a thermocouple, was alleviated with the use of thick, aluminum-coated grids.
Subject(s)
Cryoelectron Microscopy/methods , Photolysis , Acetates/chemistry , Cryoelectron Microscopy/instrumentation , Ethylenediamines/chemistry , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/chemistry , Spectrophotometry, Ultraviolet , Temperature , Thionucleotides/chemistry , Time FactorsABSTRACT
The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.
Subject(s)
Cryoelectron Microscopy/instrumentation , Cryoelectron Microscopy/methods , Kinetics , Ribosomes/ultrastructureABSTRACT
Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.
Subject(s)
Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods , Software , Datasets as Topic , Deep Learning , Neural Networks, ComputerABSTRACT
As collection of electron microscopy data for single-particle reconstruction becomes more efficient, due to electronic image capture, one of the principal limiting steps in a reconstruction remains particle-verification, which is especially costly in terms of user input. Recently, some algorithms have been developed to window particles automatically, but the resulting particle sets typically need to be verified manually. Here we describe a procedure to speed up verification of windowed particles using multivariate data analysis and classification. In this procedure, the particle set is subjected to multi-reference alignment before the verification. The aligned particles are first binned according to orientation and are binned further by K-means classification. Rather than selection of particles individually, an entire class of particles can be selected, with an option to remove outliers. Since particles in the same class present the same view, distinction between good and bad images becomes more straightforward. We have also developed a graphical interface, written in Python/Tkinter, to facilitate this implementation of particle-verification. For the demonstration of the particle-verification scheme presented here, electron micrographs of ribosomes are used.
Subject(s)
Artificial Intelligence , Image Processing, Computer-Assisted , Microscopy, Electron/methods , Algorithms , Classification , Image Enhancement/methods , Image Processing, Computer-Assisted/methods , Multivariate Analysis , Ribosomes/ultrastructureABSTRACT
A number of image processing parameters in the 3D reconstruction of a ribosome complex from a cryo-EM data set were varied to test their effects on the final resolution. The parameters examined were pixel size, window size, and mode of Fourier amplitude enhancement at high spatial frequencies. In addition, the strategy of switching from large to small pixel size during angular refinement was explored. The relationship between resolution (in Fourier space) and the number of particles was observed to follow a lin-log dependence, a relationship that appears to hold for other data, as well. By optimizing the above parameters, and using a lin-log extrapolation to the full data set in the estimation of resolution from half-sets, we obtained a 3D map from 131,599 ribosome particles at 6.7A resolution (FSC=0.5).
Subject(s)
Cryoelectron Microscopy/methods , Escherichia coli/ultrastructure , Ribosomes/ultrastructure , Cryoelectron Microscopy/standards , Image Enhancement/methods , Image Processing, Computer-Assisted/methods , Ribosomes/chemistryABSTRACT
It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome.
Subject(s)
Alcohol Dehydrogenase/metabolism , Aldehyde Oxidoreductases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Ribosomes/metabolism , Alcohol Dehydrogenase/chemistry , Aldehyde Oxidoreductases/chemistry , Binding Sites , Escherichia coli Proteins/chemistry , Models, Molecular , Porins/chemistry , Porins/metabolism , Protein Binding , Protein Conformation , Ribosomes/chemistry , Ribosomes/genetics , Structure-Activity RelationshipABSTRACT
This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
Subject(s)
Image Processing, Computer-Assisted , Microscopy, Electron , Software , Models, Molecular , Molecular Structure , User-Computer InterfaceABSTRACT
The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The N- and C-terminal sequences of the eight axial proteins were predicted to form interlocking alpha-domains generating an axial tube. We report on an approximately 1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an approximately 2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.