Background Supercapsular percutaneously-assisted total hip (SuperPath) arthroplasty has been proposed to be minimally invasive and tissue sparing, with possible superior postoperative outcomes compared with conventional approaches for total hip arthroplasty (THA). However, previous studies have underlined the shortcomings of conventional THA approaches, including higher dislocation, more blood loss, longer incisions, more tissue damage, and delayed postoperative rehabilitation. In the present study, we compared the short-term outcomes of unilateral THA with those of SuperPath and the mini-incision posterolateral approach (PLA) for hip osteoarthritis (OA). Methods Patients with unilateral hip OA were prospectively recruited and underwent either SuperPath (SuperPath group) or mini-incision PLA THA (PLA group). Perioperative status [operative time, incision length, intraoperative blood loss, soft tissue damage, and length of hospital stay (LOS)], and postoperative function outcomes, including range of motion (ROM), pain visual analog scale (VAS), and Harris Hip Score (HHS), were evaluated and compared between the groups at scheduled time points within 12 months postoperatively. Results Compared with the PLA group, the SuperPath group yielded a significantly shorter incision length (7.83 vs. 12.45 cm, P<0.001), longer operative time (102.72 vs. 66.22 min, P<0.001), more blood loss (1,007.38 vs. 844.55 mL, P=0.005), and more soft tissue damage (creatine kinase: 1,056.05 vs. 821.50 U/L, P=0.006) on postoperative day 3. The SuperPath group also showed deficient acetabular cup positioning (abduction angle: 36.94° vs. 42.66°, P=0.004) and a greater decrease in ROM (flexion: 107.66° vs. 114.44°, P=0.004; 109.83° vs. 116.11°, P=0.002; 111.66° vs. 118.88°, P<0.001) on postoperative days 1, 3, and 14, as well as severe early-term pain symptoms (pain VAS on postoperative day 3: 7.05 vs. 6.55, P=0.041). However, the LOS, C-reactive protein levels, erythrocyte sedimentation rate (within 2 weeks postoperatively), and HHS were comparable between the groups during the 12 months postoperatively. Conclusions SuperPath may be a promising, minimally invasive technique for the treatment of OA in the future. Further investigation is necessary to evidence the possible superiority of SuperPath over other conventional mini-incision THA approaches.

To the editor: To classify the intertrochanteric fractures, Ianiv Klaber et al. compared the inter- and intraobserver agreement by using a new AO/OTA classification (2018) or the original AO/OTA classification (1996), revealing that the new classification allowed better agreement than the original version when distinguishing stable from unstable patterns [ [1] Klaber I Besa P Sandoval F Lobos D Zamora T Schweitzer D et al. The new AO classification system for intertrochanteric fractures allows better agreement than the original AO classification. An inter- and intra-observer agreement evaluation. Injury. 2020; Google Scholar ]. However, these results should be interpreted with caution. Authors’ reply to letter by Dr. Meng et al.InjuryPreviewWe read with interest the letter by Dr. Meng et al. Full-Text PDF

Abstract Background The supercapsular percutaneously-assisted total hip arthroplasty (SuperPath) was proposed to be minimally invasive and tissue sparing with possible superior postoperative outcomes to traditional approaches of total hip arthroplasty (THA). Here, we compared the short-term outcomes of staged THA with the SuperPath or through posterolateral approach (PLA) for bilateral osteonecrosis of the femoral head (ONFH). Methods Patients with bilateral late-stage ONFH were prospectively recruited from our department from March 2017 to March 2018. Staged bilateral THAs with one side SuperPath and the other side PLA were performed consecutively in the same patients with right and left hips alternating within approaches. The average time interval between the staged THAs was 3 months. Perioperative status (operation time, incision length, intraoperative blood loss, soft tissue damage, and length of hospital stay) and postoperative function (range of motion, pain, and hip function) were recorded and compared between the SuperPath and PLA approaches within 12-month postoperatively. Results Four male patients (age, 51.00 ± 4.54; BMI, 21.49 ± 1.73) with bilateral alcohol-induced ONFH (Ficat III/IV) were followed up over 12 months postoperatively. Compared with the PLA, the SuperPath yielded shorter incision length (7.62 vs. 11.12 cm), longer operation time (103.25 vs. 66.50 min), more blood loss (1108.50 vs. 843.50 ml), deficient abduction angle of the acetabular cup (38.75° vs. 44.50°), and inferior early-term hip function (Harris hip score, 72.50 vs. 83.25) at 12-month postoperatively. Soft tissue damage, length of hospital stay, postoperative pain, postoperative range of motion, and 12-month patient satisfaction were comparable between both approaches. Conclusion The SuperPath may be a minimally invasive technique but the present study shows less favorable short-term outcomes than PLA for total hip arthroplasty in osteonecrosis of the femoral head. More investigations are required to provide convincing favorable evidences of the SuperPath over other traditional THA approaches. Trial registration information The trial was retrospectively registered in https://www.researchregistry.com (No. Researchregistry4993) on July 04, 2019. The first participant was enrolled on March 13, 2017.

Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I,-II and-V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a similar to 6 A resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 A) and lowest (similar to 6-7 A) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin n bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.

The SPHIRE software package embodies a cryo-EM single-particle analysis (SPA) pipeline supported by reworked modules of the SPARX system and by newly developed graphical user interface. It is designed for easy immersion into cryo-EM SPA with emphasis on validation and reproducibility of results by using concepts of statistical resampling. SPHIRE modules realize all steps of a typical SPA structure determination process: (1) drift analysis, (2) CTF analysis, (3) particle extraction, (4) validated 2-D clustering, (5) validated ab initio reconstruction, (6) novel rapid maximum likelihood based structure refinement, (7) 3-D variability-guided sorting, and (8) local resolution analysis. With an extensive step-by-step tutorial and sample dataset, users new to SPA and the package can experience determination of a 3.5 A resolution structure using the pipeline. Distinctive characteristic of SPHIRE is its open design that permits experienced users to fine-tune the performance and contribute their own Python code, in order to target uniquely challenging cases of structure analysis. Finally, optimization of computationally demanding steps reduced the time to process dataset of typical size from days to hours. Now, a near-atomic resolution structure can be obtained within one or a few days using SPHIRE SPA pipeline.

Objective To evaluate the effectiveness of lumbopelvic fixation using the combination of closed multi-axial screws (CMAS) iliosacral fixation system and the posterior segmental spinal fixation for unstable sacral fractures. Methods Between January 2013 and November 2014, 25 patients (39 sides) with unstable sacral fractures were treated with lumbopelvic fixation using the combination of CMAS iliosacral fixation system and the posterior segmental spinal fixation. There were 17 males and 8 females, aged 19-55 years (mean, 33.9 years). The causes were traffic accident injury in 15 cases, falling injury from height in 8 cases, and crushing injury in 2 cases. The interval of injury and operation was 1-13 days (mean, 3.5 days). Fracture was classified as Denis type I in 2 sides, type II in 20 sides, and type III in 17 sides; nerve injury was rated as Gibbons grade I in 2 cases, grade II in 2 cases, grade III in 7 cases, and grade IV in 9 cases. The reduction quality was evaluated by Matta criterion, the clinical function outcome by Majeed, and nerve function by Gibbons criterion. Results The average operation time was 110 minutes (range, 80-150 minutes). The average blood loss was 570 mL (range, 250-1 400 mL). Superficial wound infection occurred in 2 patients, and was cured after debridement and antibiotic therapy. All patients were followed up for an average of 18 months (range, 15-22 months). Postoperative X-ray and CT examination showed clinical healing of sacral fractures at 8-12 weeks after operation (mean, 10 weeks). The mean removal time of internal fixation was 13 months (range, 12-20 months). No screw loosening and fracture, adhesion of internal fixation to surrounding tissue, and obvious electrolysis phenomenon occurred. According to Matta criterion, reduction was rated as excellent in 32 sides, good in 6 sides, fair in 1 side, and the excellent and good rate was 97.5%. According to Majeed functional scoring at last follow-up, the mean score was 84.7 (range, 64-98); the results were excellent in 18 cases, good in 5 cases, and fair in 2 cases, and the excellent and good rate was 92.0%. The nerve function was significantly improved when compared with preoperative one; nerve injury was rated as Gibbons grade I in 8 cases, grade II in 8 cases, grade III in 3 cases, and grade IV in 1 case. Conclusion Lumbopelvic fixation using the combination of CMAS iliosacral fixation system and the posterior segmental spinal fixation is a relatively effective fixation for unstable sacral fractures. Not only is the fracture fixation rigid for early full weight-bearing, but also nerve decompression can be performed which facilitates nerve function recovery.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

OBJECTIVE:To compare the effectiveness of two lumbopelvic fixation procedures for treating unstable sacral fractures. METHODS:The clinical data of 47 patients were treated for unstable sacral fractures in the West China Hospital of Sichuan University from January 2010 to December 2014 were reviewed. Twentytwo patients (28 sides) were treated with USS combined with iliosacral screw (group A),while 25 patients (39 sides) were treated with closed multiaxial screws (CMAS) iliosacral fixation system combined with Posterior Segmental Spinal Fixation system (group B). The outcomes of the two procedures were compared using the following indicators: length of operations,amount of intraoperative blood loss,MATTA score of fracture reduction,MAJEED function score one year postoperation,postoperative complications,and GIBBONS Classification of sacral nerve injury in patients with sacral nerve symptoms. RESULTS:Group A had longer operations [(121.4±5.1) min] than group B [(110.6±4.5) min, P<0.05]. Group A had larger intraoperative blood loss [(618±45) mL] than group B [(570±40) mL, P<0.05]. Both groups had two cases of wound infection after operations that were cured by debridement and antibiotic therapy. According to the MATTA scoring criteria,group A had 92.9% excellent and good fracture reduction,compared with 97.5% in group B ( P<0.05). According to the MAJEED functional scoring criteria,group A had 86.4% excellent and good clinical functions,compared with 92.0% in group A ( P<0.05). The GIBBONS criteria indicated that neurological functions of both groups improved significantly after operations ( P<0.05),but no significant difference appeared between the two groups ( P>0.05). CONCLUSION:CMAS iliosacral fixation system is better for treating unstable sacral fractures compared with USS combined with iliosacral screws.

OBJECTIVE:To evaluate the effectiveness of autologous vein nerve conduit supported by vascular stent in repairing a 10 mm gap peroneal nerve in white New Zealand rabbits.METHODS:30 New Zealand rabbits were randomly divided into three groups: autologous nerve group (group A),conventional autologous vein nerve conduit group (group B),autologous vein nerve conduit supported by vascular stent group (group C). 10 mm common peroneal nerve was cut off. In groups A,the peroneal nerve was turned 180 ° before suturing. In group B and group C,20 mm long external jugular vein was cut and removed. After dilution of venous retraction,the venous bridge filled the gap of the nerve defect in group B. In group C,a blood vessel stent was placed for accessing the external jugular vein,and then connected to the nerve defect. Ulnar ulcer was observed after operations. Reflex score of left foot toe was recorded. The nerve regeneration and functional recovery was assessed through electrophysiological examinations,comparison of wet mass ratio between the left and right hind limb gastrocnemius,morphological observations,transmission electron microscopy 12 weeks after operations.RESULTS:Group B had the lowest scoring of toespreading reflex,whereas Group A had the highest scoring of toespreading reflex. There was a statistically significant difference in the scoring of toespreading reflex between group A and group C. In terms of the diameter of regenerated nerve fiber and the thickness of regenerated myelin sheath,no statistically significant ( P>0.05) difference was found between group A and group C,whereas the difference was significant ( P<0.05) between groups A/C and group B. The presence of peripheral nerves found in light microscopic examinations revealed normal characteristics of myelinated fibers in all groups. The myelinated axon profile was almost equal between group B and group C under electron microscopic examinations. However,more degenerated axons with disturbed contoursin were found in group B compared with group C.CONCLUSION:Autologous vein nerve conduit supported by vascular stent increases regeneration of nerves.

OBJECTIVE:To develop a renewed classification and treatment regimen for sacroiliac joint dislocation.METHODS:According to the direction of dislocation of sacroiliac joint,combined iliac,sacral fractures,and fracture morphology,sacroiliac joint dislocation was classified into 4 types. Type Ⅰ (sacroiliac anterior dislocation): main fracture fragments of posterior iliac wing dislocated in front of sacroiliac joint. Type Ⅱ (sacroiliac posterior dislocation): main fracture fragments of posterior iliac wing dislocated in posterior of sacroiliac joint. Type Ⅲ (Crescent fracturedislocation of the sacroiliac joint): upward dislocation of posterior iliac wing with oblique fracture through posterior iliac wing. Type ⅢA: a large crescent fragment and dislocation comprises no more than onethird of sacroiliac joint,which is typically inferior. Type ⅢB: intermediatesize crescent fragment and dislocation comprises between one and twothirds of joint. Type ⅢC: a small crescent fragment where dislocation comprises most,but not the entire joint. Different treatment regimens were selected for different types of fractures. Treatment for type Ⅰ sacroiliac joint dislocation: anterior iliac fossa approach pry stripping reset; sacroiliac joint fixed with sacroiliac screw through percutaneous. Treatment for type Ⅱ sacroiliac joint dislocation: posterior sacroiliac joint posterior approach; sacroiliac joint fixed with sacroiliac screw under computer guidance. Treatment for type ⅢA and ⅢB sacroiliac joint dislocation: posterior sacroiliac joint approach; sacroiliac joint fixed with reconstruction plate. Treatment for type ⅢC sacroiliac joint dislocation: sacroiliac joint closed reduction; sacroiliac joint fixed with sacroiliac screw through percutaneous. Treatment for type Ⅳ sacroiliac joint dislocation: posterior approach; sacroiliac joint fixed with spinal pelvic fixation.RESULTS:Results of 24 to 72 months patient follow-up (mean 34.5 months): 100% survival,100% wound healing,and 100% fracture healing. Two cases were identified as type Ⅰ sacroiliac joint dislocation,including one with coexistence of nerve injury. Patients recovered completely 12 months after surgery. Eight cases were identified as type Ⅱ sacroiliac joint dislocation; none had obvious nerve injury during treatments. Twelve cases were identified as type Ⅲ sacroiliac joint dislocation,including one with coexistence of nerve injury. Patients recovered completely 12 months after surgery. Three cases were identified as type Ⅳ sacroiliac joint dislocation with coexistence of nerve injury. Two patients fully recovered 12 months after surgery. One had partial recovery of neurological function.CONCLUSION:The classification and treatment regimen for sacroiliac joint dislocation have achieved better therapeutic effect,which is worth promoting.

Smooth muscle myosin (smM) and heavy meromyosin (smHMM) are activated by regulatory light chain (RLC) phosphorylation but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions involving motor domains and the essential light chain (ELC) [Wendt et al., PNAS 98: 4361 (2001)]. The "free head" can bind to actin, but the actin-binding interface of the "blocked head" is involved in interactions with the free head. We report here a 3-D structure for phosphorylated, active smHMM obtained using electron crystallography of 2-D arrays, and an atomic model obtained by fitting using normal mode flexible fitting. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state, but occur between separate molecules. The interface between heads of phosphorylated smHMM is less extensive and somewhat altered in orientation compared with that of dephosphorylated smHMM. The light chain binding domain of phosphorylated and several dephosphorylated myosin structures show systematic differences. However, the major difference appears to be the relationship between the motor domain and the ELC in a phosphorylated head compared to that of the "blocked head" of dephosphorylated smHMM. We hypothesize that RLC phosphorylation disrupts the inhibited conformation primarily by its effect on the "blocked head" rather than the "free head". Singly phosphorylated smHMM is not compatible with the closed conformation if the "blocked head" is phosphorylated. The implications of this observation for myosin activation at low levels of phosphorylation in smooth muscle will be discussed. Supported by grants from the NIAMS, NHLBI and NSF-MCB.

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-Å density map of the Rubisco–RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.

SPARX (single particle analysis for resolution extension) is a new image processing environment with a particular emphasis on transmission electron microscopy (TEM) structure determination. It includes a graphical user interface that provides a complete graphical programming environment with a novel data/process-flow infrastructure, an extensive library of Python scripts that perform specific TEM-related computational tasks, and a core library of fundamental C++ image processing functions. In addition, SPARX relies on the EMAN2 library and cctbx, the open-source computational crystallography library from PHENIX. The design of the system is such that future inclusion of other image processing libraries is a straightforward task. The SPARX infrastructure intelligently handles retention of intermediate values, even those inside programming structures such as loops and function calls. SPARX and all dependencies are free for academic use and available with complete source.

Despite the breadth of studies on complexes of the mitochondrial electron transport chain, molecular details of the tendency of these complexes to assemble into supermolecular respirasomes are unclear. Previously we established that in mild detergent extracts of mitochondria from Saccharomyces cerevisiae lacking cardiolipin (crd1 null strains), cytochrome bc1 complex (Complex III) and cytochrome c oxidase (Complex IV) do not form a tetrameric (III2IV2) supermolecular complex as observed in extracts from wild type mitochondria (J. Biol. Chem. 277: 43553–56). This result correlates with the cytochrome c pool-like kinetic behavior in electron transfer between Complexes III and IV in intact mitochondria lacking cardiolipin as compared to their respirasome-like behavior in wild type mitochondria (J. Biol. Chem. 280: 29403–08). In this study classical biochemical techniques were applied to purify the supercomplex (III2IV2) in the active state. Negative stain electron microscopy was used to investigate the structural organization of the supercomplex. Image processing was performed combining tomographic and single particle analysis. These studies revealed the organization of the supercomplex to be a central dimer of III flanked at opposite sides with monomers of IV (IV-III2-IV), suggesting an overall symmetry of the C2 type. The first two authors contributed equally. Supported by NIH grants GM56389 and GM60635.

A112 Pancreatic cancer is a lethal disease because it is difficult to detect and treat. However, pancreatic cancer progresses over many years, which offers the opportunity for prevention. Over 90% of pancreatic tumors have a point mutation in K-Ras at codon, which leads to activation of the Ras, epidermal growth factor receptor (EGFR), and vascular endothelial cell growth factor receptor (VEGFR) signaling pathways. Pancreatic cancer is an epigenetic disease with profound alterations in gene methylation. In contrast, histone deacetylase inhibitors (HDACi) can restore epigenetic alterations in gene functioning. This study investigated whether the combination of the EGFR/ VEGFR inhibitor ZD6474 and the HDACi, Trichostatin A (TSA) was more protective in human pancreatic cancer cells than higher doses of these agents alone. PK-9 and BxPC-3 cells were treated with IC50 concentrations of ZD6474 and TSA for 72 hours for measurement of cell survival and apoptosis. Treating the pancreatic cancer cells with ZD6474 in combination with TSA decreased survival and induced apoptosis in a synergistic manner. The combination was associated with decreased activation of EGFR, ERK1/2, and Akt survival signaling. The synergistic apoptotic effect was associated with an increased in cleaved poly (ADP-ribose)polymerase (PARP). These findings with HDACi combined with agents that target EGFR may represent a novel strategy for preventing pancreatic cancer in a high-risk population.

The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs; comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs; on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

Template matching together with the comprehensive theory of image formation in electron microscope provides an optimal (in Bayesian sense) tool for solving one of the outstanding problems in single particle analysis, i.e., automatic selection of particle views from noisy micrograph fields. The method is based on the assumption that the reference three-dimensional structure is known and that the relevant parameters of the model of the image formation process can be estimated. In the first stage of the procedure, a set of possible particle views is generated using the available reference structure. The template images are constructed as linear combinations of available particle views using a clustering technique. Next, the micrograph noise characteristic is established using an automated contrast transfer function (CTF) estimation procedure. Finally, the CTF parameters calculated are used to construct a matched filter and correlation functions corresponding to the available template images are calculated. In order to alleviate the problem of the biased caused by varying image formation conditions, a decision making strategy based on the predicted distribution of correlation coefficients is proposed. It is demonstrated that due to the inclusion of CTF considerations, the template matching method performed very well in a broad range of microscopy conditions.

The current theory of image formation in electron microscopy has been semi-quantitatively successful in describing data. The theory involves parameters due to the transfer function of the microscope (defocus, spherical aberration constant, and amplitude constant ratio) as well as parameters used to describe the background and attenuation of the signal. We present empirical evidence that at least one of the features of this model has not been well characterized. Namely the spectrum of the noise background is not accurately described by a Gaussian and associated "B-factor;" this becomes apparent when one studies high-quality far-from focus data. In order to have both our analysis and conclusions free from any innate bias, we have approached the questions by developing an automated fitting algorithm. The most important features of this routine, not currently found in the literature, are (i). a process for determining the cutoff for those frequencies below which observations and the currently adopted model are not in accord, (ii). a method for determining the resolution at which no more signal is expected to exist, and (iii). a parameter-with units of spatial frequency-that characterizes which frequencies mainly contribute to the signal. Whereas no general relation is seen to exist between either of these two quantities and the defocus, a simple empirical relationship approximately relates all three.