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Several results published since the work described above for static, nucleotide-bound states of the Lethocerus flight muscle appeared to bear on their interpretation. These results include X-ray crystal structures of myosin II intermediate states obtained from MD constructs of Dictyostelium discoideum , high-resolution structures of actomyosin by cryoEM, and the structure of relaxed striated muscle thick filaments from several species. The crystal structure of the D. Numerous crystal structures of complete myosin heads obtained from molluscan sources have similar features [ 78 , 79 , 80 , 81 ].

How do these results impact the tomographic reconstructions of the Lethocerus flight muscle in the presence of AMPPNP with or without ethylene glycol? The free-head backbone attachment in the relaxed Lethocerus thick filaments is primarily through the RLC [ 6 ]; the position of the MD may be less important for this backbone attachment unless it clashes.

Molluscan post-rigor myosin head structures, when aligned using the RLC and its bound heavy chain segment to the interacting heads motif of relaxed Lethocerus thick filaments, fit with no or only minor clashes between the MD and the thick filament backbone Figure 5.

The pyrophosphate addition to rigor the Lethocerus flight muscle also produces a structure similar to that of AMPPNP plus ethylene glycol with an enhanced Fitting of a post rigor myosin head conformation into the relaxed Lethocerus thick filament. The atomic model of Doryteuthis pealeii squid , PDB 3i5f, a post-rigor conformation [ 82 ] fit using only the RLC portion of the lever arm onto the free head of atomic model of [ 6 ].

Arrows point to the space between the free head and the myosin rods in the backbone; there are no clashes between the MD and the backbone. The related rigor-like structure, PDB 3i5g, fits equally well. The degree of clash observed with other post rigor crystal structures having complete lever arms, e.

Note that the blocked head density and atomic model have been removed for clarity. The first studies attempting to visualize active cross-bridges in the rapidly frozen, freeze substituted muscle were done on the vertebrate striated muscle [ 24 , 85 , 86 , 87 , 88 ], but none of these efforts led to 3D imaging and atomic model building.

The first ET studies of the rapidly frozen Lethocerus muscle were done on stretched rigor fibers [ 33 ]. Myosin cross-bridges in the active muscle act as independent force generators so that all steps in the ATPase cycle are present, though not in equal proportion.

The active muscle when rapidly frozen provides a snapshot in time of the dynamics of cross-bridge interaction with the thin filament and ATP. The specimen preparation methods used for frozen active flight muscle fibers have been described [ 35 ] and so will not be elaborated on here except to say that fibers muscle cells from the glycerinated muscle are mounted on a tension transducer, tested for activation and relaxation and then smashed into a liquid-helium-cooled copper block while simultaneously measuring tension.

The frozen muscle tissue is then freeze-substituted, embedded, sectioned and stained for ET data collection. The tilt series were initially single-axis, but later became dual-axis.

The efforts on the Lethocerus muscle started with what was then described as a state of High Static Tension, which would now be interpreted as an isometric contraction iso-HST. At the time, the flight muscle, which operates in stretch activation mode during flight, was deemed incapable of an isometric contraction. The first tomograms of the active Lethocerus flight muscle used the column averaging method described above in which individual filament averages were produced with no averaging between filaments [ 58 ].

This process produced a richer population of cross-bridge conformations than could be obtained using spatial averages. In column averages, all of the cross-bridges were bound to the region midway between Tn complexes, which is the same location where rigor lead bridges bind; the raw tomograms also showed cross-bridges at other locations along the thin filament.

That most of the cross-bridges in active contraction occurred in the rigor, lead-bridge target zone was presaged by results with AMPPNP described above as well as by X-ray diffraction that showed, in active contraction of Lethocerus fibers, the Each target zone cross-bridge was consistent in size with a single myosin head.

Atomic model building at the time was limited by the number of myosin head structures that had been determined with the complete lever arm present one [ 27 ]; most structures were of the MD alone, sometimes with only the ELC. In the year , the transition state structure of scallop myosin II appeared [ 78 ]; all the S1 crystal structures obtained from the molluscan muscle have a complete light chain-binding domain and thus complete lever arms. In lieu of a transition state crystal structure, the iso-HST cross-bridges were modeled by rebuilding the post-rigor skeletal S1 structure using G as the pivot point, comparing the result to a model transition state structure [ 90 ].

However, even with that degree of added flexibility in the skeletal atomic structure, reasonable fits could not be obtained without also moving the MD away from its position in rigor acto-S1. Large azimuthal changes in the lever arm position were necessary in the majority of cases. Taken together, the models suggested a 2-stage power stroke. In stage-1, the axial orientations of the motor and light chain domains change together; in stage-2 the MD does not change its orientation and only the lever arm changes its axial angle.

The total interaction distance amounted to 13 nm of which 10 nm produced positive work and 3 nm negative work. The axial lever arm change with the MD fixed on actin, i. Developments in ET produced methods to align and classify the heterogeneous individual motifs For the first time, individual actin subunits and myosin heads could be resolved in class averages facilitating the detailed study of myosin head conformations in the fast frozen, active muscle.

The ability to fit the thin filament atomic model independently of the cross-bridges combined with the availability of crystal structures of rigor, post rigor and transition states of S1 produced improved atomic models. The new methodology, not only provided resolution to distinguish individual myosin heads and actin subunits, but was sufficient to develop a criterion for differentiating weak from strong myosin attachments and for quantitating the relative numbers of the different structures [ 68 ].

Weak actin attachments were differentiated from strong actin attachments by whether or not a MD in the well-known strong binding position fit the density. If it fit, the actin attachment was strong; if not, and the MD of myosin had to be moved to fit the density, the actin attachment was weak. The lever arms invariably required either axial or azimuthal changes, or both, as previously required in the earlier atomic models of rigor and AMPPNP.

Once a thin filament attachment in a class average was determined to be weak or strong, the number of class members comprising that class average could be used as a measure of the number of occurrences of that structure on each of the 14 actin subunits in the Quantification of each different actin-bound state of myosin on an individual actin subunit of the thin filament motif has not been duplicated in any other system.

Myosin head binding to specific actin subunits on the On the left weak attachments are shown; on the right are strong attachments. Actin subunits on the two long pitch strands are colored green and blue. The two target-zone actin subunits are in darker shades. Actin subunit designations correspond to the chain names in the coordinate files deposited in the Protein Data Bank, PDB—2w From [ 34 ].

Surprisingly perhaps, in iso-HST, cross-bridging density attributable to myosin heads was found on all actin subunits in the Strong-binding attachments were found on only the four-actin subunits exactly midway between successive Tn complexes, i. Weak attachments were found everywhere else, including within the target zone of strong-binding cross-bridges. These subunits apparently present a very unfavorable actin azimuth for any myosin head attachment. Weak-binding attachments were grouped into three types; 1, 2 and Tn bridges.

Type 1 weak attachments were restricted to the target zone and thus interpreted as precursors of strong-binding cross-bridges, or pre-power stroke cross-bridges Figure 7 E,F. Type 2 weak attachments were found both in the target zone and just M-ward of the target zone, often as part of the mask-motif structure Figure 7 E,F. Their MD contact with the thin filament was generally through TM and not actin.

Hence they were later dubbed TM bridges. Their means of actin attachment was not clear, perhaps being through a long N-terminal extension of the RLC as seems to occur in the Drosophila flight muscle [ 82 , 83 ]. They had no clear path to strong binding as long as they were positioned M-ward of the target zone. Class averages and quasi-atomic models of a selection of class averages from the Lethocerus flight muscle in an isometric contraction. Small panels to the left are the central section top and an opaque isodensity surface view bottom of the larger panel.

In the translucent larger panel, actin long pitch strands are cyan and green with the target-zone actins in darker shades, TM is yellow and Tn orange. Heavy chains of strongly bound myosin heads are colored red, weak binding myosin heads magenta, ELC dark blue and RLC light blue.

A Single headed cross-bridge on the left and a 2-headed, strong-binding cross-bridge on the right; B a pair of 1-headed, strong-binding cross-bridges on actin subunits H and I; C , D a strongly bound 2-headed cross-bridge on the left and a strongly bound 1-headed cross-bridge on the right. E , F are mask motifs with Tn-bridges.

Tn-bridges have not been fit with a myosin head; E the right, M-ward side, cross-bridge is a weak binding Type 2 bridge outside of the target zone contacting TM near actin subunit F, while on the left is a Type 1 weak-binding cross-bridge within the target zone on actin subunit I. F On the M-ward left side is a Type 2 weak-binding, cross-bridge contacting TM outside the target zone near actin subunit G, while the Type 1, weak-binding cross-bridge on the right is contacting target-zone actin subunit H.

Tn bridges formed a highly heterogeneous set of attachments on either Tn or the actin subunits in the same location Figure 7 E,F. They approach this region of the thin filament from a wide variety of thick filament origins as did cross-bridges in the same axial position when AMPPNP was present. Their contacts on the thin filament are at least as variable as those found in other non-target zone locations.

Because of their heterogeneity, they were not investigated further. However, they may play a yet-to-be-determined role in stretch activation. The positions of Type 1 weak binding myosin heads showed slightly variable axial positions and orientations, but highly variable azimuthal positions, biased almost exclusively to the anticlockwise direction from the strong binding position relative to the thin filament center Figure 8 A.

The alterations to the lever arm relative to the MD of all weak binding bridges Figure 8 B were much smaller than those for strong-binding cross-bridges Figure 8 C. The small axial variations in the MD of pre-powerstroke cross-bridges were distributed on both sides of the strong binding MD position and thus did not indicate a concerted motion that could contribute to the power stroke. Thus, the pre-powerstroke attachments suggested that the weak to strong transition involved mostly the azimuthal movement of the MD across actin subdomain 1 and towards TM in a clockwise direction before actin binding cleft closure.

The changes needed to fit the lever arm of pre-powerstroke cross-bridges were smaller than the azimuthal changes, but biased in the anticlockwise direction relative to the starting crystal structure. Range of lever arm positions for strongly and weakly bound cross-bridges in isometrically contracting Lethocerus flight muscle. Ribbon diagrams of actin subunits are colored blue and green. Ribbon diagrams are shown for only the heavy chains of myosin. A All weak binding cross-bridges superimposed on actin subunit I see Figure 6.

This view illustrates the variations in MD position when referred to a single actin subunit. Chain traces of Type 1 bridges are shown in gray and Type 2 bridges in yellow. The single post-rigor conformation found is colored light brown.

Note the relatively small axial dispersion of the Type 1 MDs compared to the broad dispersion of the Type 2 MDs; B all weak-binding cross-bridges aligned on the scallop transition state MD to illustrate lever arm variations compared with the starting scallop S1 structure.

Coloring scheme is the same as for panel A; C rebuilt models of strong-binding cross-bridges are colored gold superimposed on both starting myosin head structures red and magenta as docked onto actin in the strong binding configuration. Adapted from [ 34 ]. Strong-binding cross-bridges consisted of both single- and double-headed actin attachments and had axial lever arm orientations that covered a range of Axial changes in the MD as previously interpreted for strong-binding attachments [ 58 ] were visible only on Type 1 weak-binding cross-bridges because the criterion for identifying strong-binding bridges precluded it.

Based on the crystal structures available to Wu et al. The overwhelming majority of in situ cross-bridges originated on the anticlockwise side giving the strong-binding cross-bridges a straightened appearance relative to the two crystal structures used as references.

This observation was presaged by the earlier work on flight muscle in rigor, particularly the rear bridges of rigor muscle and the lead bridges of aqueous AMPPNP. Wu et al. These models differed in whether the S2 domain or the myosin lever arm were compliant. In one model, myosin heads are non-compliant and must find an appropriate actin subunit by rapidly attaching and detaching until they contact an actin subunit in an orientation that facilitates strong binding through closure of the actin binding cleft.

If the heads are assumed to be non-compliant, their ability to find an appropriate actin subunit depends on flexibility of the S2 whose origin must be on the clockwise side of the inter-filament axis.

To make the cross-bridge origin appear as if it comes from the anticlockwise side, as observed, requires an azimuthal swing of the lever arm during the power stroke. The other model involved an azimuthal movement of the MD across Subdomain 1 of actin as described above.

In this case, the myosin origin was anticlockwise of the inter-filament axis. When the power stroke ensues, the S2 aligns with the filament axis, further bending the lever arm azimuthally if necessary. An azimuthal component of the power stroke is not necessary to position the S1—S2 junction in the observed region of the thick filament, i.

An axial force transmitted through S2 would be sufficient, provided the myosin head originated from the positions observed in iso-HST. Studies of the rigor Lethocerus flight muscle have characterized the physical dimensions of the S2 as a tether of myosin heads better than for any other striated muscle. The ET of rigor fibers swollen in low ionic strength buffer pulled the S2 tether free of the filament backbone, but revealed only 11 nm of S2 [ 70 ].

If the ionic strength was lowered even further, whole ribbons of myosin rods referred to as subfilaments at the time, but now known to be ribbons were pulled free of the filament backbone.

The 11 nm length of the S2 tether was later confirmed by the high resolution structure of the relaxed thick filament [ 6 ]. The length of the S2 that functions as a tether for active myosin heads searching for actin subunits and its consequences for muscle contraction have not been examined in detail, even in Lethocerus where the structure is well defined.

The axial lever arm changes were within expectations, but the implied thick filament origins and the azimuthal variations were novel, implying an aspect of the power stroke not considered in models current at the time or since. Because rigor lead bridges bind to the same target zone as strong-binding cross-bridges in active contraction, their origins could be investigated from transverse sections of rigor fibers swollen in low ionic strength buffer.

Arakelian et al. This location would be anticlockwise of the inter-filament axis with the thin filament as the center of reference. The distribution was consistent with the hypothesis that the myosin MD moved azimuthally across its actin-binding site towards the strong binding position, bending either the lever arm or the S2 connection or both in the process.

The presence of azimuthal movements of myosin across its actin-binding site during the weak to strong transition, implied that a force produced by purely axial lever arm movements might produce a torque on either the thick filament, the thin filament or both.

X-ray diffraction of muscle fibers are clear on this issue; changes in the helical pitch of the thin filament are not observed, although the axial repeat is altered by a small amount in response to applied tension [ 93 , 94 ].

In vitro motility assays have observed azimuthal movements of actin filaments produced by myosin, referred to as twirling [ 95 ]. If changes in actin filament pitch occur in situ in muscle, they are either 1 local and compensated by changes in the opposite direction in order to maintain the For the thick filament, particularly that from the Lethocerus flight muscle, helical changes observed during active contraction are also observed when the relaxed muscle is stretched [ 96 ] and thus cannot be attributed to the myosin power stroke.

The length changes in Lethocerus thick filaments are only 0. An azimuthal component to the power stroke could dissipate any torque produced by an azimuthal movement of S2 as the myosin head moves across an actin subunit. Alternatively, compliance of the lever arm and S2 might dissipate any torque because they are much smaller physically than the filaments themselves.

After characterizing the head distribution in iso-HST, Wu et al. Length transients were completed within 2. The elapsed time, though fast for this type of specimen preparation, was too slow to capture the length transient itself, or the immediate structural response. Changes observed in the structure of strong-binding cross-bridges were smaller than expected in the lever arm angle, but larger changes were observed in the distribution of cross-bridge types, both weak and strong.

Class averages of both str- and rls-HST showed a large reduction in the numbers of pre-powerstroke cross-bridges, Type 1, within the target zone. The disappearance of pre-powerstroke weak-binding cross-bridges was explained by a recent kinetic model for actomyosin interactions in the muscle [ 98 ]. The number of strong-binding cross-bridges was largely unchanged, although there were fewer following the release as might have been predicted from the lower tension developed at the point of freezing.

After the length transient, when 2-headed cross-bridges were observed, both heads were strongly bound; in iso-HST some 2-headed cross-bridges had a weakly bound head. The number of 2-headed, strong-binding cross-bridges increased following the stretch and decreased following the release. An increase in 2-headed strong-binding cross-bridges had previously been proposed as an explanation for changes in the X-ray diagram of the vertebrate striated muscle following a stretch [ 99 ].

Outside of the target zone, changes were less dramatic. Changes in TM bridges were small. Comparatively more TM bridges are found after a stretch, and fewer after a release. The other type of weak-binding cross-bridge, the Tn bridges, are more frequent after a release and less frequent after a stretch. Following a stretch, one strong-binding class average was found just outside of the target zone on the M-ward side, but it represented a small fraction of all strong-binding heads.

Changes in TM and Tn bridges, though small, are consistent with a role in an active contraction. A stretch would be equivalent to a change toward an earlier stage of shortening, where more cross-bridges need be positioned to bind the target zone and move it M-ward thus shortening the sarcomere length.

A release would be equivalent to a change toward later in the contraction where further shortening is small. If Tn bridges play a role in stretch-activation, an increase in their number would be expected toward the end of the shortening cycle. Changes in the lever arm axial angles of strong-binding cross-bridges were more towards rigor following a release and more towards anti-rigor following a stretch, but the differences were not large, probably reflecting the elapsed time following the length transient.

Azimuthally, strong-binding cross-bridges following a length transient reflected the same highly biased lever arm distribution described above for iso-HST. Asynchronous flight muscles like those in Lethocerus are designed to oscillate rapidly so that the amount of shortening per half sarcomere is small. The muscles generally have very short I-bands consistent with a small amount of shortening. With a half sarcomere length of 1.

Examined from the standpoint of a single thin filament, the shortening of 39 nm would require that a target zone be relayed between two or more successive crowns on the thick filament [ 97 ].

The relay mechanism implied by the presence of TM bridges binding M-wards of the target zone would be consistent with such a mechanism. The precise filament geometry and arrangement in the Lethocerus flight muscle was key to controlling the tension increase and subsequent decrease as the thick and thin filaments slide past each other. The high-resolution reconstruction from Lethocerus thick filaments [ 6 ] showed myosin heads arranged in an IHM oriented perpendicular to the thick filament axis rather than roughly tangential to it as found in other striated muscles Figure 2.

The blocked head was comparatively poorly ordered, but visible in the reconstruction. The helical angle of A subsequent reconstruction from filaments with poorly ordered heads showed a 0.

These results impact models for contracting the Lethocerus muscle in several ways. This idea has yet to be tested against the rich X-ray diffraction pattern of contracting Lethocerus flight muscle. The orientation of the IHM in the Lethocerus flight muscle and the filament separation means that large radial movements of blocked heads are not necessary to contact the target zone or the Tn complex [ , ]. Azimuthal movements may be more important. We note that in the work described above, azimuthal changes in the lever arm are consistently required to fit myosin head crystal structures into 3D images of actin-myosin interactions in situ.

If IHMs reform after each contraction, free head rebinding to the thick filament backbone could highly restrict blocked head rebinding to the thin filament thereby contributing to shortening deactivation. Several differences between the vertebrate muscle and Lethocerus flight muscle are worth keeping in mind. The filament arrangement in the vertebrate striated muscle is much less favorable for thin section EM, particularly for viewing of longitudinal sections.

The filament arrangement in flight muscle places the thin filament between two thick filaments, i. The most favorable orientation for cross-bridge viewing in the vertebrate muscle is a section cut parallel to the planes of the hexagonal lattice Figure 1 B , which places two thin filaments between successive thick filaments, but the arrangement permits cross-bridges to approach and bind the thin filament from both the front and back sides of the section. Between relaxed and active contractions in Lethocerus , the thick filament axial repeat changes by only 0.

In the activated and rigor vertebrate muscle, the relaxed axial period of Measurements of the rigor and contracting muscle rescaled assuming the myosin meridional has a spacing of The F-actin period and the Tn period of the Lethocerus flight muscle are congruent, which results in target zone binding by myosin enhancing the inner parts of the Thus, two types of target zones could be observed.

If myosin head binding were defined by actin azimuth alone, it would enhance either or both of the nm layer line and the inner parts of its second order at 18— Changes in the TM position would affect the outer parts of the second layer line. Enhancement of the inner parts of a The first application of fast freezing and freeze substitution to active the muscle utilized a rabbit psoas muscle held in a tension transducer to measure tension followed by the exchange from a rigor solution to an activating solution with ATP and calcium, which was frozen by smashing into a copper mirror cooled to liquid helium temperature [ 24 ].

Myosin head distribution in the frozen active fibers was distinctly different from rigor fibers in that heads were largely perpendicular to the fiber axis, instead of showing the characteristic arrow head appearance of rigor cross-bridges. Intensity enhancement of the 37 nm layer line corresponding to the thin filament half repeat was seen in both rigor and active fibers, but not in relaxed fibers, consistent with heavy myosin head decoration of the thin filament in the active muscle.

An Hirose and Wakabayashi [ 85 ] investigated the frozen, isometrically contracting, rabbit psoas muscle utilizing similar techniques to Tsukita and Yano, but notably examining thin sections cut transverse to the filament axis. Rigor cross-bridges appeared triangular in longitudinal sections with a tilted appearance, a large contact on the thin filament and a narrow attachment to the thick filament.

Few active cross-bridges had a triangular shape; most had uniform width between thick and thin filaments. They classified visually the different cross-bridge forms observed in transverse sections and found that rigor cross-bridges were predominately bent and active cross-bridges remarkably straight, similar to the observations described above for the Lethocerus flight muscle. Target zone marking was not observed in active contraction, though it was observed in their rigor images.

Hirose et al. After flash photolysis, fibers were frozen after 20, 50, 80 and ms. A weak 19 nm layer line was reported in power spectra from micrographs of fibers following ATP photolysis, which might suggest the presence of target zones between successive Tn complexes marked by myosin heads. At a 20 ms time point following photolysis, some rigor cross-bridges identifiable by their 2-headed appearance are seen but none are seen after 50 ms.

Note that in vertebrate thin filaments, the actin crossover spacing is 36 nm, whereas the Tn period is This work was further advanced by the application of correspondence analysis, a form of the multivariate data analysis used for the Lethocerus muscle, to quantitatively characterize the differences in cross-bridge shape in transverse sections [ 86 ]. By determining the direction of view of the transverse sections from serial sections, they identified three basic types of cross-bridge from sections 20 nm thick.

Using the centerline connecting thick and thin filaments as the point of reference, they observed bridging density coming off the thin filament to the left or right as well as along the inter-filament axis.

In rigor, most bridging density extended off the left or along the inter-filament axis, with generally a distinct bend from right to left as it approached the thick filament. Although rigor-like forms were seen at all time points after photolysis, they were lowest at 50 ms, replaced by more straightened forms that also extended off the thin filaments to the left, right or center line and with less curvature as they approached the thick filament.

Although the interpretation of the results is complicated, since they are based on projections alone, one conclusion stands out clearly; active cross-bridges are different from rigor cross-bridges. From studies using caged ATP in a rigor solution, Lenart et al. The authors did not attempt to classify individual cross-bridge types, but instead examined the changes in the summed power spectrum of longitudinal sections at different time points after calcium release as well as in rigor and relaxed fibers.

Many of the features known to change in X-ray fiber diffraction of the active muscle were reproduced. However, some features were apparently novel. The 36 nm layer line increases in intensity as expected, most likely due to the marking of actin subunits by myosin heads.

Unexpectedly, the peak of the off-meridional intensity moved radially outward indicating that the myosin head mass was moving radially inward on the thin filament. Several observations were made about the appearance of off-meridional layer line intensity at an axial spacing of 19 nm, but the source was unknown.

A clear identification of myosin head binding to target zones in the contracting vertebrate muscle by X-ray fiber diffraction has not been shown. However, modeling studies to explain the X-ray diagram of rigor fish muscle [ ] concluded that intensity increases in the 36 nm layer line could be explained by myosin head binding to target zones of actin subunits in length, comparable to those observed for rigor Lethocerus flight muscle. Increases in the 36 nm layer line imply that actin azimuth is the parameter defining myosin head binding.

The difference in spacing between The target zones in Lethocerus shrank from actin subunits in rigor to two subunits once nucleotide was added. A similar effect likely occurs in the vertebrate striated muscle, which would define target-zone position more precisely.

Because the Lethocerus flight muscle work concentrated on 3D imaging and classification while the vertebrate muscle results generally were confined to projections and analysis of power spectra, the two sets of results are not easily compared.

However, we find it intriguing that several studies observed the buildup of intensity on the inner part of the 19 nm layer line, which might indicate that the target zone marking similar to that observed in the active flight muscle and others noted azimuthal changes in the appearance of the myosin heads when compared to rigor.

The chief limitation of the work described above on imaging actin-myosin interactions in situ is the necessity of cutting thin sections of plastic embedded tissue and improving the contrast using heavy metal stains.

Ideally, the sections should be cut through the frozen-hydrated tissue and visualized without addition of heavy metals. Generally, improvements in resolution of at least a factor of 2, from 5 nm to 2. A technique to do this, called CEMOVIS [ ] has been under development for a number of years, but not yet applied to muscle tissue in any systematic way. First, sectioning of frozen, unfixed tissue produces several artefacts, such as knife marks and crevasses [ ].

These are mostly confined to one surface and are not by themselves limiting since part of the section depth appears unaffected. Second, frozen-hydrated sections suffer much more from section compression than plastic sections, which would significantly affect atomic models.

There is no reason in principle that the multivariate data analysis could not be used to produce class averages free of staining artefacts. In principle, CEMOVIS could be applied to smash frozen, active muscle fibers, though it may be technically challenging due to the relatively small depth of good freezing.

In FIB-SEM, a beam of heavy ions is used to literally carve out a thin lamella in the tissue as mounted or cultured on the EM grid which can then be transferred to a TEM for subsequent tilt series data collection.

The technique could be applied to a myofibril preparation spread over an EM grid. Although mechanical effects would be difficult to monitor, all of the AMPPNP induced states as well as the relaxed and rigor muscle would be accessible to this technique. Importantly, one state of active contraction, the so-called calcium poised state, in which the muscle is bathed in a MgATP solution with submaximal calcium has not been studied.

Calcium poised muscle is primed to contract but requires a stretch to fully activate. Calcium poised myofibrils could yield details about how the muscle positions itself for its next contraction. Is calcium poised muscle rich in Tn bridges and weak Type I target zone attachments? There are many challenging technical issues that must be solved before active force bearing cross-bridges can be imaged in the muscle.

There are still many questions remaining before a complete picture is obtained for the actively contracting muscle. This paper is dedicated to the memory of Mary C.

Reedy, whose expertise in preparing, evaluating and selecting thin sections for our 3D reconstruction work was invaluable to the project. She contributed to all aspects of the work, including write up and interpretation. The authors declare no conflict of interest.

The funders had no role in the design of the studies reported here either in the collection, analysis, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.

Int J Mol Sci. Published online Apr 5. Kenneth A. Edwards , 2 and Michael K. Reedy 2. Robert J. Find articles by Robert J. Michael K. Find articles by Michael K. Author information Article notes Copyright and License information Disclaimer. Received Mar 6; Accepted Apr 1. Abstract Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy cryoEM of F-actin, decorated with myosin heads.

Keywords: striated muscle, image reconstruction, muscle physiology. Introduction Electron microscopy EM of the flight muscles of large water bugs of the Lethocerus genus has informed the evolution of models of muscle contraction since Reedy, Holmes and Tregear showed pronounced changes in the axial orientation of myosin heads in rigor flight muscle when ATP is added [ 1 ].

Open in a separate window. Figure 1. Figure 2. Table 1 Timeline of Lethocerus flight muscle 3D imaging. Electron Microscopy of Thin Sections 2. Methods for 3D Imaging of Muscle Tissue In the early s, the high degree of order within myac layer thin sections of the Lethocerus flight muscle suggested that 3D images could be obtained if the muscle lattice was treated as if it were a 2D protein array and images processed using methods developed for 2D crystals such as bacteriorhodopsin [ 38 ].

Early 3D Reconstruction Work 3. Rigor Muscle Spatial Average Reconstructions The earliest 3D reconstruction work on muscle was confined to the rigor state.

Figure 3. Figure 4. Impact of New Information Several results published since the work described above for static, nucleotide-bound states of the Lethocerus flight muscle appeared to bear on their interpretation. Assuming the AMPPNP addition to Lethocerus myosin also produces a post-rigor conformation, the head would have its lever arm down and its actin binding cleft open. That sometimes they appeared to be single headed would reflect an effect of actin subunit azimuth in relationship to the lead bridge origin on the filament backbone.

An open actin binding cleft with its correspondingly lower actin-binding affinity when AMPPNP is bound would explain the systematic loss of the highly distorted rigor rear bridges. On the other hand, with the lever arm down, the M-ward cross-bridge of mask motifs and other thin filament attachments outside of the target zone must differ from similar structures identified subsequently from the active muscle described below.

The mask motifs of the active muscle have a lever-arm up conformation characteristic of the myosin II transition state, not a lever arm down conformation, and they seem to be contacting TM rather than actin in the active muscle. Conceivably, they might be held near the thin filament by a structure other than the myosin MD, perhaps a long N-terminal extension of the RLC of myosin, in which case, the up or down state of the lever arm would be relatively unimportant.

A connection between the RLC and actin seems to occur in the Drosophila flight muscle [ 82 , 83 ]. Ordering of the RLC at the head rod junction probably contributes too little mass to account for such an effect. So why does it take on a relaxed appearance in the glycol-stiff state and ultimately take on the appearance of fully relaxed muscle? Figure 5. Electron Tomography of Frozen Active Muscle The first studies attempting to visualize active cross-bridges in the rapidly frozen, freeze substituted muscle were done on the vertebrate striated muscle [ 24 , 85 , 86 , 87 , 88 ], but none of these efforts led to 3D imaging and atomic model building.

Figure 6. Figure 7. Figure 8. Impact of Recent Results on the Interpretation of Frozen Active Muscle Imaging The high-resolution reconstruction from Lethocerus thick filaments [ 6 ] showed myosin heads arranged in an IHM oriented perpendicular to the thick filament axis rather than roughly tangential to it as found in other striated muscles Figure 2.

An X-ray fiber diffraction of the relaxed Lethocerus flight muscle shows that tension applied sinusoidally causes changes in the helical angle between crowns as well as changes in the Under tension, relaxed fibers showed a The changes in the If tension applied to the relaxed muscle can disorder the IHM, thereby producing a change in the helical angle, a conjecture at this point, then ordering the IHM in isolated filaments should increase the helical angle, which is what is observed.

In other words, the rod structure, which defines the filaments helical structure, and the IHM structure appear to be coupled. Highly restricted movements of the blocked head would require a rather precisely placed target zone actin subunit to initiate force production.

Reformation of the interacting heads motif after a contraction must be fast enough to sequester the myosin heads from further interaction with the thin filament. The kinetics of ATP cleavage in the Lethocerus myosin in this context have been discussed previously [ 6 ], indicating that the ATPase speed is sufficient. Free-head rebinding to the thick filament backbone would be a first order reaction and potentially faster than the ATP cleavage step that recocks the lever arm because it does not involve a covalent bond cleavage.

Results from Fast Frozen Vertebrate Muscle Fibers Several differences between the vertebrate muscle and Lethocerus flight muscle are worth keeping in mind. Prospects for Future Improvements The chief limitation of the work described above on imaging actin-myosin interactions in situ is the necessity of cutting thin sections of plastic embedded tissue and improving the contrast using heavy metal stains.

Acknowledgments This paper is dedicated to the memory of Mary C. Conflicts of Interest The authors declare no conflict of interest. References 1. Reedy M. Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Huxley H. The mechanism of muscular contraction. Holmes K. Interpretation of the low angle X-ray diffraction from insect muscle in rigor.

B Biol. Ultrastructure of insect flight muscle. Screw sense and structural grouping in the rigor cross-bridge lattice. Miller A. Structure of insect fibrillar flight muscle in the presence and absence of ATP. Wray J. Structure of the backbone in myosin filaments of muscle. How Many Myosins per Cross-Bridge? Flight Muscle Myofibrils from the Blowfly, Sarcophaga bullata.

Cold Spring Harb. Morris E. The 4-stranded helical arrangement of myosin heads on insect Lethocerus flight muscle thick filaments. Schmitz H. Oblique section 3-D reconstruction of relaxed insect flight muscle reveals cross bridge lattice in helical registration.

Pringle J. The Croonian Lecture, Stretch activation of muscle: Function and mechanism. Bullard B. Regulating the contraction of insect flight muscle. Muscle Res. Cell Motil. Luther P. Evolution of myosin filament arrangements in vertebrate skeletal muscle. Hirose K. Thin filaments of rabbit skeletal muscle are in helical register. Woodhead J.

Atomic model of a myosin filament in the relaxed state. Zoghbi M. Three-dimensional structure of vertebrate cardiac muscle myosin filaments. Al-Khayat H. Atomic model of the human cardiac muscle myosin filament. Wendt T. Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2. Henderson R. Three-dimensional model of purple membrane obtained by electron microscopy.

Taylor K. Three-dimensional reconstruction of rigor insect flight muscle from tilted thin sections. Crowther R. Three-dimensional reconstruction from a single oblique section of fish muscle M-band. Tsukita S. Actomyosin structure in contracting muscle detected by rapid freezing.

Three-dimensional image reconstruction of insect flight muscle. The rigor myac layer. Cell Biol. A protocol for 3D image reconstruction from a single image of an oblique section.

Rayment I. Three-dimensional structure of myosin subfragment A molecular motor. Winkler H. Three-dimensional structure of nucleotide-bearing crossbridges in situ: Oblique section reconstruction of insect flight muscle in AMPPNP at 23 degrees C.

Braunfeld M. Cryo automated electron tomography: Towards high-resolution reconstructions of plastic-embedded structures.

The use of electron tomography for structural analysis of disordered protein arrays. Multivariate statistical analysis of three-dimensional cross-bridge motifs in insect flight muscle. Chen L. Real space refinement of acto-myosin structures from sectioned muscle. Liu J. Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges.

Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions. Imaging actomyosin in situ. In: McIntosh J. Cellular Electron Microscopy. Volume Lehrer S. Damage to actin filaments by glutaraldehyde: Protection by tropomyosin. Maupin-Szamier P. Actin filament destruction by osmium tetroxide. NOTE: Also note that the utility is going to provide a report to you at the end of execution. Within that report it includes the running processes on the target computer.

I like to know which of my applications are talking and to who. This is performed on the backend by the application to map PIDS to executables. Well, the capture file might not tell me the executable, but it does give me the PID. So, by looking at the report I can identify which PID to focus on and then use that when looking at the network trace file in Message Analyzer.

As you can see, it states the location. On the target computer we can even see the temporary files which are put in place for the capture:. Once the specified time is reached, the utility sends a stop command to the target computer to end the network capture:.

NOTE: In the event that the utility is disconnected from the target computer prior to the stop command being issued, you can issue the commands locally at the target computer itself:. Finally, the tool will move the files used for the trace to the specified network share, and then remove them from the target computer. Lots of goodies. Topic 5: What are the limitation of the tool? Topic 6: How can I customize the tool?

Well, we do need to address some customization options. The function names are called out below. To do so, execute netsh trace show scenarios :. Well, what if I wanted to configure that to be higher or lower. There are plenty of other options as well. I strongly recommend that you review Netsh Commands for Network Trace:. In this case, we are going to focus on two aspects.

Configuring the NetEventSession: This overall is simple. Now, the real meat of the capture. The NetEventProvider. However, there are quite a few others available. You may want to output to a file as there will be several. What you should notice is that the providers are all set with a default configuration. You can adjust these as necessary as well using:. By adding an additional Invoke-Command line within the Start-NetEvent function, you can easily customize the provider s which you wish to use within the network capture session.

Once you know the command syntax is correct and the output is what you desire then incorporate that customization back into the tool as necessary. Topic 7: References and Recommendations for Additional Reading:. Hello there! As a reminder, the Noteworthy News series covers various areas, to include interesting news, announcements, links, tips and tricks from Windows, Azure, and Security worlds on a monthly basis. Microsoft on November 21, , announced new services to facilitate your VMware migration to Azure.

Effective November, 16th. Azure RIs give you price predictability and help improve your budgeting and forecasting. Azure RIs also provide unprecedented flexibility should your business needs change.

Stay current with a constantly growing scope of Azure services and features. Learn how to manage and protect your Azure resources efficiently and how to solve common design challenges. Azure Active Directory Azure AD Pass-through Authentication allows your users to sign in to both on-premises and cloud-based applications using the same passwords.

This feature provides your users a better experience — one less password to remember, and reduces IT helpdesk costs because your users are less likely to forget how to sign in. Storage Replica may allow you to decommission existing file replication systems such as DFS Replication that were pressed into duty as low-end disaster recovery solutions.

While DFS Replication works well over extremely low bandwidth networks, its latency is very high — often measured in hours or days. This is caused by its requirement for files to close and its artificial throttles meant to prevent network congestion. With those design characteristics, the newest and hottest files in a DFS Replication replica are the least likely to replicate. Storage Replica operates below the file level and has none of these restrictions.

The new build features an ability to mute a tab that is playing media in Microsoft Edge, an ability to wirelessly share files and URLs to nearby PCs using the Near Share feature, improvements to Windows Update, and more. Since Windows 10 originally released we have continued to make significant investments to Windows Hello for Business, making it easier to deploy and easier to use, and we are seeing strong momentum with adoption and usage of Windows Hello. As we shared at Ignite conference, Windows Hello is being used by over 37 million users, and more than commercial customers have started deployments of Windows Hello for Business.

One of its features, Controlled folder access , stops ransomware in its tracks by preventing unauthorized access to your important files. Many of the risks associated with ransomware and worm malware can be alleviated through systems design. Referring to our now codified list of vulnerabilities, we know that our solution must:. Planning and implementing a security strategy to protect a hybrid of on-premises and cloud assets against advanced cybersecurity threats is one of the greatest challenges facing information security organizations today.

Join Lex Thomas as he welcomes back Mark Simos to the show as they discuss how Microsoft has built a robust set of strategies and integrated capabilities to help you solve these challenges so that you can build a better understanding how to build an identity security perimeter around your assets.

On November 10, , a vulnerability called AVGater was discovered affecting some antivirus products. The vulnerability requires a non-administrator-level account to perform a restore of a quarantined file.

Windows Defender Antivirus is not affected by this vulnerability. Technical Preview Branch releases give you an opportunity to try out new Configuration Manager features in a test environment before they are made generally available. Microsoft on November 14, , released security updates to provide additional protections against malicious attackers. By default, Windows 10 receives these updates automatically, and for customers running previous versions, Microsoft recommends that they turn on automatic updates as a best practice.

Hello, Paul Bergson, back with some great new information regarding the recent release of Fall Creators Update FCU for Windows 10, Microsoft released some great new security features that can protect you from unwanted Malware. A young scientist was trying to get the generals attention on newly developed battlefield equipment, a machine gun. The general was dismissing him, telling him he was too busy to be bothered and to leave him alone. I sometimes worry this is occurring and, so I try evangelizing the latest tools Microsoft provides to help protect our customers.

These newly built-in, mitigations are even more comprehensive than EMET. This lets you see a record of what would have happened if you had enabled the feature. While the features will not block or prevent apps, scripts, or files from being modified, the Windows Event Log will record events as if the features were fully enabled. This means you can enable audit mode and then review the event log to see what impact the feature would have had were it enabled.

Thereby allowing a measured rollout of rules. It helps prevent actions and apps that are typically used by exploit-seeking malware to infect machines. Ransomware has become one of the biggest security threats facing our customers today.

Enabling CFA can be managed locally, configuring locally requires the user to manage the settings within Windows Defender Security Center. The folder and application configuration settings can also be managed by your desktop administrator with Group Policy. Prior to rolling out CFA, Microsoft has created a demo tool that allows the administrator to trial the impact of an application that has not been granted permission to update an authorized location.

This error is what users would see if this protective feature was enabled. CFA also provides the ability to audit impact prior to enabling this feature, thereby providing the administrator the ability to find any application compatibility issues.

End users are the weakest link in the chain. All the protections can be put in place but if a user clicks on a link that might result in them going to a location that will attack them. This will protect any browser loaded on the device as well as any application, such as a malicious app attempting to contact a command and control server residing on the internet. There is nothing to configure it is all built into the product. So how do we know if a site is untrustworthy? Everything should work the same for the user, but any corruptive changes made to the operating system are dropped once the virtualized session has been shut down.

The innocent user, not noticing anything suspicious about the mail, clicks on the link to an untrusted location. In order to proactively keep the user and enterprise resources safe, Application Guard coordinates with Microsoft Edge to open that site in a temporary and isolated copy of Windows.

The attack is completely disrupted. As soon as the user is done, whether or not they are even aware of the attack having taken place, this temporary container is thrown away, and any malware is discarded along with it. After deletion, a fresh new container is created for future browsing sessions. To manage the enterprise, we do provide new Group Policy settings, so the desktop administrator can ensure security and conformity for all of the enterprises users.

Well there you have it, some great new security features and they are provided as free updates. Look through the links and try out some of the demos. If you are already convinced that you need to get off the older operating system but need help justifying, hopefully this will help you convince the decision makers to move forward. Hello everyone!

Here in the fall, in the Ozark Mountains area the colors of the trees are just amazing! If only it was that easy! Kerberos plays a huge role in server authentication so feel free to take advantage of it. The Kerberos authentication protocol provides a mechanism for authentication — and mutual authentication — between a client and a server, or between one server and another server.

This is the underlying authentication that takes place on a domain without the requirement of certificates. Why not you ask? Well for one thing, using sniffing tools attackers can successfully extrapolate every single key stroke you type in to an RDP session, including login credentials. And given that, often customers are typing in domain admin credentials…which means you could have just given an attacker using a Man-in-the-Middle MTM attack the keys to the kingdom.

Granted, current versions of the Remote Desktop Client combined with TLS makes those types of attacks much more difficult, but there are still risks to be wary of. However, what should be done is making sure the remote computers are properly authorized in the first place.

Read the following quick links, and pick which one applies for your situation: or read them all. Although technically achievable, using self-signed certificates is normally NOT a good thing as it can lead to a never-ending scenario of having to deploy self-signed certs throughout a domain. Talk about a management overhead nightmare! Additionally, security risk to your environment is elevated…especially in public sector or government environments.

Needless to say, any security professional would have a field day with this practice an ANY environment. Jacob has also written a couple of awesome guides that will come in handy when avoiding this scenario. Both of course feature the amazing new Windows Server , and they are spot on to help you avoid this first scenario. Just remember they are guides for LAB environments.

Sure, it works…but guess what? Neither can Kerberos for that matter. Main security reason: Someone could have hijacked it. You can stop reading now. Think of a Root CA Certificate and the chain of trust.

RDP is doing the same thing. So how do we remedy that? You still must connect using the correct machine names. The idea is to get rid of the warning message the right way…heh. Okay this scenario is a little like the previous one, except for a few things. Normally when deploying ADCS, certificate autoenrollment is configured as a good practice. But RDS is a bit different since it can use certificates that not all machines have.

Remember, by default the local Remote Desktop Protocol will use the self-signed certificate…not one issued by an internal CA…even if it contains all the right information. Basically, the right certificate with appropriate corresponding GPO settings for RDS to utilize…and that should solve the warning messages. How do we do that? Remember, certificates you deploy need to have a subject name CN or subject alternate name SAN that matches the name of the server that a user is connecting to!

Manual enrollment is a bit time consuming, so I prefer autoenrollment functionality here. To mitigate the CA from handing out a ton of certs from multiple templates, just scope the template permissions to a security group that contains the machine s you want enrollment from.

I always recommend configure certificate templates use specific security groups. Where certificates are deployed is all dependent upon what your environment requires. Next, we configure Group Policy.

This is to ensure that ONLY certificates created by using your custom template will be considered when a certificate to authenticate the RD Session Host Server or machine is automatically selected. Translation: only the cert that came from your custom template will be used when someone connects via RDP to a machine…not the self-signed certificate.

As soon as this policy is propagated to the respective domain computers or forced via gpupdate. I updated group policy on a member server, and tested it. Of course, as soon as I try to connect using the correct machine name, it connected right up as expected. Warning went POOF! Another way of achieving this result, and forcing machines to use a specific certificate for RDP…is via a simple WMIC command from an elevated prompt, or you can use PowerShell.

The catch is that you must do it from the individual machine. Quick, easy, and efficient…and unless you script it out to hit all machines involved, you’ll only impact one at a time instead of using a scoped GPO. Now we get to the meaty part as if I haven’t written enough already. Unlike the above 2 scenarios, you don’t really need special GPO settings to deploy certificates, force RDS to use specific certs, etc.

The roles themselves handle all that. Let’s say Remote Desktop Services has been fully deployed in your environment.

Doesn’t matter…or does it? Kristin Griffin wrote an excellent TechNet Article detailing how to use certificates and more importantly, why for every RDS role service. Just remember the principals are the same. First thing to check if warnings are occurring, is yep, you guessed it …are users connecting to the right name? Next, check the certificate s that are being used to ensure they contain the proper and accurate information.

Referring to the methods mentioned in. The following information is from this TechNet Article :. The certificates you deploy need to have a subject name CN or subject alternate name SAN that matches the name of the server that the user is connecting to. For example, for Publishing, the certificate needs to contain the names of all the RDSH servers in the collection.

If you have users connecting externally, this needs to be an external name it needs to match what they connect to. If you have users connecting internally to RDWeb, the name needs to match the internal name. For Single Sign On, the subject name needs to match the servers in the collection. Go and read that article thoroughly.

Now that you have created your certificates and understand their contents, you need to configure the Remote Desktop Server roles to use those certificates. This is the cool part! Or you will use multiple certs if you have both internal and external requirements. Note : even if you have multiple servers in the deployment, Server Manager will import the certificate to all servers, place the certificate in the trusted root for each server, and then bind the certificate to the respective roles.

Told you it was cool! You don’t have to manually do anything to each individual server in the deployment! You can of course, but typically not mandatory. DO use the correct naming. DO use custom templates with proper EKUs. DO use RDS. You don’t have an internal PKI, then use the self-signed certs The other takeaway is just have an internal PKI And for all our sanity, do NOT mess with the security level and encryption level settings!

The default settings are the most secure. Just leave them alone and keep it simple. Thank you for taking the time to read through all this information. I tried to think of all the scenarios I personally have come across in my experiences throughout the past 25 years, and I hope I didn’t miss any.

If I did, please feel free to ask! Happy RDP’ing everyone! Understanding the differences will make it much easier to understand what and why settings are configured and hopefully assist in troubleshooting when issues do arise. A cryptographic protocol is leveraged for security data transport and describes how the algorithms should be used.

What does that mean? Simply put, the protocol decides what Key Exchange, Cipher, and Hashing algorithm will be leveraged to set up the secure connection. Transport Layer Security is designed to layer on top of a transport protocol i. TCP encapsulating higher level protocols, such the application protocol. An example of this would be the Remote Desktop Protocol. The main difference is where the encryption takes place. Just like the name implies, this is the exchange of the keys used in our encrypted communication.

For obvious reasons, we do not want this to be shared out in plaintext, so a key exchange algorithm is used as a way to secure the communication to share the key.

Diffie-Hellman does not rely on encryption and decryption rather a mathematical function that allows both parties to generate a shared secret key. This is accomplished by each party agreeing on a public value and a large prime number. Then each party chooses a secret value used to derive the public key that was used.

Both ECDH and its predecessor leverage mathematical computations however elliptic-curve cryptography ECC leverages algebraic curves whereas Diffie-Hellman leverages modular arithmetic.

In an RSA key exchange, secret keys are exchanged by encrypting the secret key with the intended recipients public key. The only way to decrypt the secret key is by leveraging the recipients private key. Ciphers have existed for thousands of years. In simple terms they are a series of instructions for encrypting or decrypting a message.

We could spend an extraordinary amount of time talking about the different types of ciphers, whether symmetric key or asymmetric key, stream ciphers or block ciphers, or how the key is derived, however I just want to focus on what they are and how they relate to Schannel.

Symmetric key means that the same key is used for encryption and decryption. This requires both the sender and receiver to have the same shared key prior to communicating with one another, and that key must remain secret from everyone else.

The use of block ciphers encrypts fixed sized blocks of data. RC4 is a symmetric key stream cipher. As noted above, this means that the same key is used for encryption and decryption.

The main difference to notice here is the user of a stream cipher instead of a block cipher. In a stream cipher, data is transmitted in a continuous steam using plain-text combined with a keystream. Hashing Algorithms, are fixed sized blocks representing data of arbitrary size.

They are used to verify the integrity of the data of the data being transmitted. When the message is created a hash of the original message is generated using the agreed upon algorithm i. That hash is used by the receiver to ensure that the data is the same as when the sender sent it. MD5 produces a bit hash value. Notice the length difference? NOTE: Both hash algorithms have been found to be vulnerable to attacks such as collision vulnerabilities and are typically not recommended for use in cryptography.

Again, see the noticeable size difference? Now that everything is explained; what does this mean? Remember that a protocol simply defines how the algorithms should be used. This is a where the keys will be exchanged that are leveraged for encrypting and decrypting our message traffic.

This is the algorithm, in this instance the Elliptic-Curve Digital Signature Algorithm, used to create the digital signature for authentication. GCM Again…… what? This is the mode of operation that the cipher leverages. The purpose is to mask the patterns within the encrypted data.

SHA indicates that the hashing algorithm used for message verification and in this example is SHA2 with a bit key. Hopefully this helps to further break down the barriers of understanding encryption and cipher suites.

We decided to round up a few customer stories for you, to illustrate the various real-world benefits being reported by users of Shielded VMs in Windows Server To all of you that have downloaded the Technical Preview and provided feedback via UserVoice, thank you.

On December 1st we released the first public update to the Technical Preview. Windows Defender Antivirus uses a layered approach to protection: tiers of advanced automation and machine learning models evaluate files in order to reach a verdict on suspected malware. While Windows Defender AV detects a vast majority of new malware files at first sight, we always strive to further close the gap between malware release and detection. We look at advanced attacks perpetrated by the highly skilled KRYPTON activity group and explore how commodity malware like Kovter abuses PowerShell to leave little to no trace of malicious activity on disk.

From there, we look at how Windows Defender ATP machine learning systems make use of enhanced insight about script characteristics and behaviors to deliver vastly improved detection capabilities. Backdoor user accounts are those accounts that are created by an adversary as part of the attack, to be used later in order to gain access to other resources in the network, open new entry points into the network as well as achieve persistency.

MITRE lists the create account tactic as part of the credentials access intent of stage and lists several toolkits that uses this technique. And, now that the celebrations are mostly over, I wanted to pick all your brains to learn what you would like to see from us this year…. As you all know, on AskPFEPlat, we post content based on various topics in the realms of the core operating system, security, Active Directory, System Center, Azure, and many services, functions, communications, and protocols that sit in between.

Christopher Scott, Premier Field Engineer. I have recently transitioned into an automation role and like most people my first thought was to setup a scheduled task to shutdown and startup Virtual Machines VMs to drive down consumption costs. Now, the first thing I did, much like I am sure you are doing now, is look around to see what and how other people have accomplished this. So, I came up with the idea of using Tags to shutdown or startup a filtered set of resources and that is what I wanted to show you all today.

The first thing you will need to do is setup an Automation Account. From the Azure portal click more actions and search for Automation.

By clicking the star to the right of Automation Accounts you can add it to your favorites blade. Now you will be prompted to fill in some values required for the creation.

Now is the time to create the Azure Run as Accounts so click the Yes box in the appropriate field and click create. From within the Automation Accounts blade select Run as Accounts.

After the accounts and connections have been verified we want to update all the Azure Modules. We can also review the job logs to ensure no errors were encountered. Now that the Automation Accounts have been created and modules have been updated we can start building our runbook.

But before we build the runbooks I want to walk you through tagging the VMs with custom tags that can be called upon later during the runbook. From the Assign Tags callout blade, you can use the text boxes to assign custom a Name known as the Key property in Powershell and a custom Value. If you have already used custom tags for other resources they are also available from the drop-down arrow in the same text box fields. Click Assign to accept the tags.

To start building the runbook we are going to select the Runbook option from the Automation Account Pane and click Add a Runbook. When the Runbook Creation blade comes up click Create a Runbook , In the callout blade Give the runbook a name, select Powershell from the dropdown, and finally click Create. At this point you will brought to the script pane of the Runbook.

You can paste the attached script directly into the pane and it should look something like this. Once the script has been pasted in, click the Test Pane button on the ribbon bar to ensure operability.

If we go back to the Virtual Machine viewing pane we can verify the results. Since the script processed correctly and is working as intended we can proceed to publishing the runbook. Click Publish and confirm with Yes. But what are we using to invoke the runbooks?

Well we could add a webhook, or manually call the runbook from the console, we could even create a custom application with a fancy GUI Graphical User Interface to call the runbook, for this article we are going to simply create a schedule within our automation account and use it to initiate our runbook. To build our schedule we select Schedules from the Automation Account then click Add a schedule. Create a Schedule Name, Give it a description, assign a Start date and Time, set the Reoccurrence schedule and expiration and click Create.

Now that the schedule has been created click OK to link it to the Runbook. Originally, I used this runbook to shutdown VMs in an order so at the end of the Tier 2 Runbook would call the Tier 1 Runbook and finally the Tier 0 runbook. For Startup I would reverse the order to ensure services came up correctly.

By splitting the runbooks, I ensured the next set of services did not start or stop until the previous set had finished. However, by utilizing the custom tags and making minor changes to the script you can customize your runbooks to perform whatever suits your needs.

For example, if you wanted to shutdown just John Smiths machines every night all you would need to do is tag the VMs accordingly Ex. I have also attached the startup script that was mentioned earlier in the article for your convenience. Thank you for taking the time to read through this article, I hope you can adapt it to you found it helpful and are able to adapt it your environment with no issues. Please leave a comment if you come across any issues or just want to leave some feedback.

Disclaimer The sample scripts are not supported under any Microsoft standard support program or service. The sample scripts are provided AS IS without warranty of any kind. Microsoft further disclaims all implied warranties including, without limitation, any implied warranties of merchantability or of fitness for a particular purpose.

The entire risk arising out of the use or performance of the sample scripts and documentation remains with you. In no event shall Microsoft, its authors, or anyone else involved in the creation, production, or delivery of the scripts be liable for any damages whatsoever including, without limitation, damages for loss of business profits, business interruption, loss of business information, or other pecuniary loss arising out of the use of or inability to use the sample scripts or documentation, even if Microsoft has been advised of the possibility of such damages.

Azure Automation — Custom Tagged Scripts. Hi, Matthew Walker again. Recently I worked with a few of my co-workers to present a lab on building out Shielded VMs and I thought this would be useful for those of you out there wanting to test this out in a lab environment. Shielded VMs, when properly configured, use Bitlocker to encrypt the drives, prevent access to the VM using the VMConnect utility, encrypt the data when doing a live migration, as well blocking the fabric admin by disabling a number of integration components, this way the only access to the VM is through RDP to the VM itself.

With proper separation of duties this allows for sensitive systems to be protected and only allow those who need access to the systems to get the data and prevent VMs from being started on untrusted hosts.

In my position I frequently have to demo or test in a number of different configurations so I have created a set of configurations to work with a scripted solution to build out labs. At the moment there are some differences between the two and only my fork will work with the configurations I have. Now, to setup your own environment I should lay out the specs of the environment I created this on.

All of the above is actually a Hyper-V VM running on my Windows 10 system, I leverage nested virtualization to accomplish this, some of my configs require Windows Server. Extract them to a directory on your system you want to run the scripts from. Once you have extracted each of the files from GitHub you should have a folder that is like the screenshot below. By default these files should be marked as blocked and prevent the scripts from running, to unblock the files we will need to unblock them.

If you open an administrative PowerShell prompt and change to the directory the files are in you can use the Unblock-File cmdlet to resolve this. This will require you to download the ADKSetup and run it and select to save the installer files. The Help folder under tools is not really necessary, however, to ensure I have the latest PowerShell help files available I will run the Save-Help PowerShell cmdlet to download and save the files so I can install them on other systems.

Next, we move back up to the main folder and populate the Resources Folder, so again create a new folder named Resources. While these are not the latest cumulative updates they were the latest I downloaded and tested with, and are referenced in the config files. I also include the WMF 5. I know it seems like a lot, but now that we have all the necessary components we can go through the setup to create the VMs.

You may receive a prompt to run the file depending on your execution policy settings, and you may be prompted for Admin password as the script is required to be run elevated.

First it will download any DSC modules we need to work with the scripts. You may get prompted to trust the NuGet repository to be able to download the modules — Type Y and hit enter.

It will then display the current working directory and pop up a window to select the configuration to build. The script will then verify that Hyper-V is installed and if it is server it will install the Failover Clustering feature if not installed not needed for shielded VMs, sorry I need to change the logic on that. The Script may appear to hang for a few minutes, but it is actually copying out the.

Net 3. The error below is normal and not a concern. Creating the Template files can take quite a long time, so just relax and let it run. Once the first VM Domain Controller is created, I have set up the script to ensure it is fully configured before the other VMs get created. You will see the following message when that occurs.

Periodically during this time you will see message such as the below indicating the status. Once all resources are in the desired state the next set of VMs will be created.

Once the script finishes however those VMs are not completely configured, DSC is still running in them to finish out the configuration such as Joining the domain or installing roles and features.

So, there you have it, a couple of VMs and DC to begin working on creating a virtualized environment that you can test and play with shielded VMs a bit. So now grab the documentation linked at the top and you can get started without having to build out the base.

I hope this helps you get started playing with some of the new features we have in Windows Server Data disk drives do not cache writes by default. Data disk drives that are attached to a VM use write-through caching. It provides durability, at the expense of slightly slower writes. As of January 10 th , PowerShell Core 6.

For the last two decades, changing the domain membership of a Failover Cluster has always required that the cluster be destroyed and re-created. This is a time-consuming process, and we have worked to improve this. Howdy folks! Before going straight to the solution, I want to present a real scenario and recall some of the basic concepts in the Identity space.

Relying Party signature certificate is rarely used indeed. Signing the SAML request ensures no one modifies the request. COM wants to access an expense note application ClaimsWeb. COM purchasing a license for the ClaimsWeb application. Relying party trust:. Now that we have covered the terminology with the entities that will play the role of the IdP or IP, and RP, we want to make it perfectly clear in our mind and go through the flow one more time. Step : Present Credentials to the Identity Provider.

The URL provides the application with a hint about the customer that is requesting access. Assuming that John uses a computer that is already a part of the domain and in the corporate network, he will already have valid network credentials that can be presented to CONTOSO. These claims are for instance the Username, Group Membership and other attributes. Step : Map the Claims. The claims are transformed into something that ClaimsWeb Application understands.

We have now to understand how the Identity Provider and the Resource Provider can trust each other. When you configure a claims provider trust or relying party trust in your organization with claim rules, the claim rule set s for that trust act as a gatekeeper for incoming claims by invoking the claims engine to apply the necessary logic in the claim rules to determine whether to issue any claims and which claims to issue. The Claim Pipeline represents the path that claims must follow before they can be issued.

The Relying Party trust provides the configuration that is used to create claims. Once the claim is created, it can be presented to another Active Directory Federation Service or claim aware application. Claim provider trust determines what happens to the claims when it arrives. COM IdP. COM Resource Provider. Properties of a Trust Relationship. This policy information is pulled on a regular interval which is called trust monitoring. Trust monitoring can be disabled and the pulling interval can be modified.

Signature — This is the verification certificate for a Relying Party used to verify the digital signature for incoming requests from this Relying Party. Otherwise, you will see the Claim Type of the offered claims. Each federation server uses a token-signing certificate to digitally sign all security tokens that it produces. This helps prevent attackers from forging or modifying security tokens to gain unauthorized access to resources. When we want to digitally sign tokens, we will always use the private portion of our token signing certificate.

When a partner or application wants to validate the signature, they will have to use the public portion of our signing certificate to do so. Then we have the Token Decryption Certificate. Encryption of tokens is strongly recommended to increase security and protection against potential man-in-the-middle MITM attacks that might be tried against your AD FS deployment.

Use of encryption might have a slight impact on throughout but in general, it should not be usually noticed and in many deployments the benefits for greater security exceed any cost in terms of server performance.

Encrypting claims means that only the relying party, in possession of the private key would be able to read the claims in the token. This requires availability of the token encrypting public key, and configuration of the encryption certificate on the Claims Provider Trust same concept is applicable at the Relying Party Trust.

By default, these certificates are valid for one year from their creation and around the one-year mark, they will renew themselves automatically via the Auto Certificate Rollover feature in ADFS if you have this option enabled. This tab governs how AD FS manages the updating of this claims provider trust. You can see that the Monitor claims provider check box is checked.

ADFS starts the trust monitoring cycle every 24 hours minutes. This endpoint is enabled and enabled for proxy by default. The FederationMetadata. Once the federation trust is created between partners, the Federation Service holds the Federation Metadata endpoint as a property of its partners, and uses the endpoint to periodically check for updates from the partner.

For example, if an Identity Provider gets a new token-signing certificate, the public key portion of that certificate is published as part of its Federation Metadata.

All Relying Parties who partner with this IdP will automatically be able to validate the digital signature on tokens issued by the IdP because the RP has refreshed the Federation Metadata via the endpoint. The Federation Metadata. XML publishes information such as the public key portion of a token signing certificate and the public key of the Encryption Certificate. What we can do is creating a schedule process which:. You can create the source with the following line as an Administrator of the server:.

Signing Certificate. Encryption Certificate. As part of my Mix and Match series , we went through concepts and terminologies of the Identity metasystem, understood how all the moving parts operates across organizational boundaries.

We discussed the certificates involvement in AD FS and how I can use PowerShell to create a custom monitor workload and a proper logging which can trigger further automation. I hope you have enjoyed and that this can help you if you land on this page. Hi everyone, Robert Smith here to talk to you today a bit about crash dump configurations and options.

With the wide-spread adoption of virtualization, large database servers, and other systems that may have a large amount or RAM, pre-configuring the systems for the optimal capturing of debugging information can be vital in debugging and other efforts. Ideally a stop error or system hang never happens. But in the event something happens, having the system configured optimally the first time can reduce time to root cause determination.

The information in this article applies the same to physical or virtual computing devices. You can apply this information to a Hyper-V host, or to a Hyper-V guest. You can apply this information to a Windows operating system running as a guest in a third-party hypervisor. If you have never gone through this process, or have never reviewed the knowledge base article on configuring your machine for a kernel or complete memory dump , I highly suggest going through the article along with this blog.

When a windows system encounters an unexpected situation that could lead to data corruption, the Windows kernel will implement code called KeBugCheckEx to halt the system and save the contents of memory, to the extent possible, for later debugging analysis. The problem arises as a result of large memory systems, that are handling large workloads.

Even if you have a very large memory device, Windows can save just kernel-mode memory space, which usually results in a reasonably sized memory dump file. But with the advent of bit operating systems, very large virtual and physical address spaces, even just the kernel-mode memory output could result in a very large memory dump file. When the Windows kernel implements KeBugCheckEx execution of all other running code is halted, then some or all of the contents of physical RAM is copied to the paging file.

On the next restart, Windows checks a flag in the paging file that tells Windows that there is debugging information in the paging file. Please see KB for more information on this hotfix. Herein lies the problem. One of the Recovery options is memory dump file type. There are a number of memory.

For reference, here are the types of memory dump files that can be configured in Recovery options:. Anything larger would be impractical. For one, the memory dump file itself consumes a great deal of disk space, which can be at a premium. Second, moving the memory dump file from the server to another location, including transferring over a network can take considerable time. The file can be compressed but that also takes free disk space during compression. The memory dump files usually compress very well, and it is recommended to compress before copying externally or sending to Microsoft for analysis.

On systems with more than about 32 GB of RAM, the only feasible memory dump types are kernel, automatic, and active where applicable. Kernel and automatic are the same, the only difference is that Windows can adjust the paging file during a stop condition with the automatic type, which can allow for successfully capturing a memory dump file the first time in many conditions.

A 50 GB or more file is hard to work with due to sheer size, and can be difficult or impossible to examine in debugging tools. In many, or even most cases, the Windows default recovery options are optimal for most debugging scenarios. The purpose of this article is to convey settings that cover the few cases where more than a kernel memory dump is needed the first time.

Nobody wants to hear that they need to reconfigure the computing device, wait for the problem to happen again, then get another memory dump either automatically or through a forced method.

The problem comes from the fact that the Windows has two different main areas of memory: user-mode and kernel-mode. User-mode memory is where applications and user-mode services operate. Kernel-mode is where system services and drivers operate. This explanation is extremely simplistic. More information on user-mode and kernel-mode memory can be found at this location on the Internet:. User mode and kernel mode. What happens if we have a system with a large amount of memory, we encounter or force a crash, examine the resulting memory dump file, and determine we need user-mode address space to continue analysis?

This is the scenario we did not want to encounter. We have to reconfigure the system, reboot, and wait for the abnormal condition to occur again. The secondary problem is we must have sufficient free disk space available. If we have a secondary local drive, we can redirect the memory dump file to that location, which could solve the second problem. The first one is still having a large enough paging file. If the paging file is not large enough, or the output file location does not have enough disk space, or the process of writing the dump file is interrupted, we will not obtain a good memory dump file.

In this case we will not know until we try. Wait, we already covered this. The trick is that we have to temporarily limit the amount of physical RAM available to Windows. The numbers do not have to be exact multiples of 2. The last condition we have to meet is to ensure the output location has enough free disk space to write out the memory dump file. Once the configurations have been set, restart the system and then either start the issue reproduction efforts, or wait for the abnormal conditions to occur through the normal course of operation.

Note that with reduced RAM, there ability to serve workloads will be greatly reduced. Once the debugging information has been obtained, the previous settings can be reversed to put the system back into normal operation.

This is a lot of effort to go through and is certainly not automatic. But in the case where user-mode memory is needed, this could be the only option. Figure 1: System Configuration Tool. Figure 2: Maximum memory boot configuration. Figure 3: Maximum memory set to 16 GB.

With a reduced amount of physical RAM, there may now be sufficient disk space available to capture a complete memory dump file. In the majority of cases, a bugcheck in a virtual machine results in the successful collection of a memory dump file. The common problem with virtual machines is disk space required for a memory dump file. The default Windows configuration Automatic memory dump will result in the best possible memory dump file using the smallest amount of disk space possible.

The main factors preventing successful collection of a memory dump file are paging file size, and disk output space for the resulting memory dump file after the reboot. These drives may be presented to the VM as a local disk, that can be configured as the destination for a paging file or crashdump file.

The problem occurs in case a Windows virtual machine calls KeBugCheckEx , and the location for the Crashdump file is configured to write to a virtual disk hosted on a file share. Depending on the exact method of disk presentation, the virtual disk may not be available when needed to write to either the paging file, or the location configured to save a crashdump file.

It may be necessary to change the crashdump file type to kernel to limit the size of the crashdump file. Either that, or temporarily add a local virtual disk to the VM and then configure that drive to be the dedicated crashdump location.

How to use the DedicatedDumpFile registry value to overcome space limitations on the system drive when capturing a system memory dump. The important point is to ensure that a disk used for paging file, or for a crashdump destination drive, are available at the beginning of the operating system startup process.

Virtual Desktop Infrastructure is a technology that presents a desktop to a computer user, with most of the compute requirements residing in the back-end infrastructure, as opposed to the user requiring a full-featured physical computer. Usually the VDI desktop is accessed via a kiosk device, a web browser, or an older physical computer that may otherwise be unsuitable for day-to-day computing needs. Non-persistent VDI means that any changes to the desktop presented to the user are discarded when the user logs off.

Even writes to the paging file are redirected to the write cache disk. Typically the write cache disk is sized for normal day-to-day computer use. The problem occurs that, in the event of a bugcheck, the paging file may no longer be accessible. Even if the pagefile is accessible, the location for the memory dump would ultimately be the write cache disk. Even if the pagefile on the write cache disk could save the output of the bugcheck data from memory, that data may be discarded on reboot.

Even if not, the write cache drive may not have sufficient free disk space to save the memory dump file. In the event a Windows operating system goes non-responsive, additional steps may need to be taken to capture a memory dump. Setting a registry value called CrashOnCtrlScroll provides a method to force a kernel bugcheck using a keyboard sequence.

This will trigger the bugcheck code, and should result in saving a memory dump file. A restart is required for the registry value to take effect. This situation may also help in the case of accessing a virtual computer and a right CTRL key is not available.

For server-class, and possibly some high-end workstations, there is a method called Non-Maskable Interrupt NMI that can lead to a kernel bugcheck. The NMI method can often be triggered over the network using an interface card with a network connection that allows remote connection to the server over the network, even when the operating system is not running. In the case of a virtual machine that is non-responsive, and cannot otherwise be restarted, there is a PowerShell method available. This command can be issued to the virtual machine from the Windows hypervisor that is currently running that VM.

The big challenge in the cloud computing age is accessing a non-responsive computer that is in a datacenter somewhere, and your only access method is over the network. In the case of a physical server there may be an interface card that has a network connection, that can provide console access over the network.

Other methods such as virtual machines, it can be impossible to connect to a non-responsive virtual machine over the network only. The trick though is to be able to run NotMyFault. If you know that you are going to see a non-responsive state in some amount of reasonable time, an administrator can open an elevated.

Some other methods such as starting a scheduled task, or using PSEXEC to start a process remotely probably will not work, because if the system is non-responsive, this usually includes the networking stack.

Hopefully this will help you with your crash dump configurations and collecting the data you need to resolve your issues. Hello Paul Bergson back again, and I wanted to bring up another security topic. There has been a lot of work by enterprises to protect their infrastructure with patching and server hardening, but one area that is often overlooked when it comes to credential theft and that is legacy protocol retirement.

To better understand my point, American football is very fast and violent. Professional teams spend a lot of money on their quarterbacks. Quarterbacks are often the highest paid player on the team and the one who guides the offense.

There are many legendary offensive linemen who have played the game and during their time of play they dominated the opposing defensive linemen. Over time though, these legends begin to get injured and slow down do to natural aging. Unfortunately, I see all too often, enterprises running old protocols that have been compromised, with in the wild exploits defined, to attack these weak protocols.

TLS 1. The WannaCrypt ransomware attack, worked to infect a first internal endpoint. The initial attack could have started from phishing, drive-by, etc… Once a device was compromised, it used an SMB v1 vulnerability in a worm-like attack to laterally spread internally. A second round of attacks occurred about 1 month later named Petya, it also worked to infect an internal endpoint.

Once it had a compromised device, it expanded its capabilities by not only laterally moving via the SMB vulnerability it had automated credential theft and impersonation to expand on the number devices it could compromise. This is why it is becoming so important for enterprises to retire old outdated equipment, even if it still works! The above listed services should all be scheduled for retirement since they risk the security integrity of the enterprise.

The cost to recover from a malware attack can easily exceed the costs of replacement of old equipment or services. Improvements in computer hardware and software algorithms have made this protocol vulnerable to published attacks for obtaining user credentials. As with any changes to your environment, it is recommended to test this prior to pushing into production. If there are legacy protocols in use, an enterprise does run the risk of services becoming unavailable.

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