Predicted models and CCP4.

In late 2020, the results of CASP14, the 14th event in a series of competitions to assess the latest developments in computational protein structure-prediction methodology, revealed the giant leap forward that had been made by Google's Deepmind in tackling the prediction problem. The level of accuracy in their predictions was the first instance of a competitor achieving a global distance test score of better than 90 across all categories of difficulty. This achievement represents both a challenge and an opportunity for the field of experimental structural biology. For structure determination by macromolecular X-ray crystallography, access to highly accurate structure predictions is of great benefit, particularly when it comes to solving the phase problem. Here, details of new utilities and enhanced applications in the CCP4 suite, designed to allow users to exploit predicted models in determining macromolecular structures from X-ray diffraction data, are presented. The focus is mainly on applications that can be used to solve the phase problem through molecular replacement.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

CCP4 Cloud for structure determination and project management in macromolecular crystallography.

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links between experimental observations and the final results deposited in the PDB. In this article, CCP4 Cloud, a new front-end of the CCP4 software suite, is presented which mitigates these effects by providing an online, cloud-based environment for crystallographic computation. CCP4 Cloud was developed for the efficient delivery of computing power, database services and seamless integration with web resources. It provides a rich graphical user interface that allows project sharing and long-term storage for structure-solution projects, and can be linked to data-producing facilities. The system is distributed with the CCP4 software suite version 7.1 and higher, and an online publicly available instance of CCP4 Cloud is provided by CCP4.

Overview of the CCP4 suite and current developments.

The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.

Using Phaser and ensembles to improve the performance of SIMBAD.

The conventional approach to search-model identification in molecular replacement (MR) is to screen a database of known structures using the target sequence. However, this strategy is not always effective, for example when the relationship between sequence and structural similarity fails or when the crystal contents are not those expected. An alternative approach is to identify suitable search models directly from the experimental data. SIMBAD is a sequence-independent MR pipeline that uses either a crystal lattice search or MR functions to directly locate suitable search models from databases. The previous version of SIMBAD used the fast AMoRe rotation-function search. Here, a new version of SIMBAD which makes use of Phaser and its likelihood scoring to improve the sensitivity of the pipeline is presented. It is shown that the additional compute time potentially required by the more sophisticated scoring is counterbalanced by the greater sensitivity, allowing more cases to trigger early-termination criteria, rather than running to completion. Using Phaser solved 17 out of 25 test cases in comparison to the ten solved with AMoRe, and it is shown that use of ensemble search models produces additional performance benefits.

Distributed computing for macromolecular crystallography.

Modern crystallographic computing is characterized by the growing role of automated structure-solution pipelines, which represent complex expert systems utilizing a number of program components, decision makers and databases. They also require considerable computational resources and regular database maintenance, which is increasingly more difficult to provide at the level of individual desktop-based CCP4 setups. On the other hand, there is a significant growth in data processed in the field, which brings up the issue of centralized facilities for keeping both the data collected and structure-solution projects. The paradigm of distributed computing and data management offers a convenient approach to tackling these problems, which has become more attractive in recent years owing to the popularity of mobile devices such as tablets and ultra-portable laptops. In this article, an overview is given of developments by CCP4 aimed at bringing distributed crystallographic computations to a wide crystallographic community.

Recent developments in MrBUMP: better search-model preparation, graphical interaction with search models, and solution improvement and assessment.

Increasing sophistication in molecular-replacement (MR) software and the rapid expansion of the PDB in recent years have allowed the technique to become the dominant method for determining the phases of a target structure in macromolecular X-ray crystallography. In addition, improvements in bioinformatic techniques for finding suitable homologous structures for use as MR search models, combined with developments in refinement and model-building techniques, have pushed the applicability of MR to lower sequence identities and made weak MR solutions more amenable to refinement and improvement. MrBUMP is a CCP4 pipeline which automates all stages of the MR procedure. Its scope covers everything from the sourcing and preparation of suitable search models right through to rebuilding of the positioned search model. Recent improvements to the pipeline include the adoption of more sensitive bioinformatic tools for sourcing search models, enhanced model-preparation techniques including better ensembling of homologues, and the use of phase improvement and model building on the resulting solution. The pipeline has also been deployed as an online service through CCP4 online, which allows its users to exploit large bioinformatic databases and coarse-grained parallelism to speed up the determination of a possible solution. Finally, the molecular-graphics application CCP4mg has been combined with MrBUMP to provide an interactive visual aid to the user during the process of selecting and manipulating search models for use in MR. Here, these developments in MrBUMP are described with a case study to explore how some of the enhancements to the pipeline and to CCP4mg can help to solve a difficult case.

SIMBAD: a sequence-independent molecular-replacement pipeline.

The conventional approach to finding structurally similar search models for use in molecular replacement (MR) is to use the sequence of the target to search against those of a set of known structures. Sequence similarity often correlates with structure similarity. Given sufficient similarity, a known structure correctly positioned in the target cell by the MR process can provide an approximation to the unknown phases of the target. An alternative approach to identifying homologous structures suitable for MR is to exploit the measured data directly, comparing the lattice parameters or the experimentally derived structure-factor amplitudes with those of known structures. Here, SIMBAD, a new sequence-independent MR pipeline which implements these approaches, is presented. SIMBAD can identify cases of contaminant crystallization and other mishaps such as mistaken identity (swapped crystallization trays), as well as solving unsequenced targets and providing a brute-force approach where sequence-dependent search-model identification may be nontrivial, for example because of conformational diversity among identifiable homologues. The program implements a three-step pipeline to efficiently identify a suitable search model in a database of known structures. The first step performs a lattice-parameter search against the entire Protein Data Bank (PDB), rapidly determining whether or not a homologue exists in the same crystal form. The second step is designed to screen the target data for the presence of a crystallized contaminant, a not uncommon occurrence in macromolecular crystallography. Solving structures with MR in such cases can remain problematic for many years, since the search models, which are assumed to be similar to the structure of interest, are not necessarily related to the structures that have actually crystallized. To cater for this eventuality, SIMBAD rapidly screens the data against a database of known contaminant structures. Where the first two steps fail to yield a solution, a final step in SIMBAD can be invoked to perform a brute-force search of a nonredundant PDB database provided by the MoRDa MR software. Through early-access usage of SIMBAD, this approach has solved novel cases that have otherwise proved difficult to solve.

CCP4i2: the new graphical user interface to the CCP4 program suite.

The CCP4 (Collaborative Computational Project, Number 4) software suite for macromolecular structure determination by X-ray crystallography groups brings together many programs and libraries that, by means of well established conventions, interoperate effectively without adhering to strict design guidelines. Because of this inherent flexibility, users are often presented with diverse, even divergent, choices for solving every type of problem. Recently, CCP4 introduced CCP4i2, a modern graphical interface designed to help structural biologists to navigate the process of structure determination, with an emphasis on pipelining and the streamlined presentation of results. In addition, CCP4i2 provides a framework for writing structure-solution scripts that can be built up incrementally to create increasingly automatic procedures.

Incisional endometriomas after Cesarean section: a case series.

OBJECTIVE: To review a series of women with endometriomas developing in the scar of the skin incision performed for cesarean section. STUDY DESIGN: A total of 37 patients diagnosed with incisional endometrioma at the time of surgical excision from 1975 to 2005 were identified from the comprehensive surgical database, which includes all operative procedures performed at this institution. The medical records of 33 of the 37 patients were available for review. RESULTS: The endometriomas ranged in size from a diameter of 1-12 cm and were initially observed to be present 6 months to 9 years (mean, 3.2) after the surgical procedure. Diagnosis was best made by needle aspiration of chocolate colored fluid from the mass. Medical therapy with a gonadotropin releasing hormone agonist, medroxyprogesterone acetate or combination oral contraceptives had been attempted in 14 patients without a change in lesion size. All patients were cured by surgical excision of the endometrioma. CONCLUSION: The overall incidence of incisional endometriomas following cesarean section during the 30-year period was 0.08%. Optimal treatment is by surgical excision. It is hypothesized that failure to close the parietal and visceral peritoneum with sutures at the time of cesarean section may markedly increase the postoperative occurrence of an endometrioma in the skin incision scar.

Endocervicosis of the bladder: a case report.

BACKGROUND: Endocervicosis of the bladder is a rare, benign lesion characterized by mucinous endocervical epithelium within the detrusor muscle of the bladder. CASE: A 48-year-old woman presented with a history of dysuria for the past week and pelvic pain for the past 6 months. Her history was significant for 2 prior cesarean sections. Transvaginal pelvic ultrasound revealed a mass protruding into the bladder. Cystoscopy and magnetic resonance imaging of the pelvis confirmed this finding. A total abdominal hysterectomy ing. A total abdominal hysterectomy and partial cystectomy were performed. Pathology of the bladder wall revealed endocervicosis. The patient remained symptom free at 6 months. CONCLUSION: Endocervicosis of the bladder wall is a rare lesion, and the diagnosis is difficult to make both clinically and pathologically. The diagnosis should be considered in any woman presenting with pelvic pain, dysuria and frequency. Gynecologists should be aware of this condition for proper diagnostic and surgical management.

Successful extraction of cardiac-extending intravenous leiomyomatosis through gonadal vein.

OBJECTIVE: To report a conservative surgical management of cardiac-extending intravenous (IV) leiomyomatosis. DESIGN: Case report. SETTING: Tertiary care center. PATIENT(S): A 40-year-old nulligravid with incidentally identified IV leiomyomatosis arising from the right gonadal vein and extending into the right atrium. INTERVENTION(S): First, intraoperative transesophageal echocardiogram was performed that demonstrated the IV leiomyomatosis stalk to be 1.1 cm in diameter without an enlarged tip or adherence to the vessel lumen. Next, the 20-week-size uterus was gently pulled caudally under live visualization of the IV leiomyomatosis tip with transesophageal echocardiogram. As the uterus was pulled caudally, the IV leiomyomatosis tip obviously protruded from the right atrium and down into inferior vena cava. Lastly, the gonadal vein was incised longitudinally and the stalk of the tumor was grasped and extracted through the incision. MAIN OUTCOME MEASURE(S): One-step abdominal surgery for complete tumor resection without sternotomy or cardiac bypass surgery. RESULT(S): To our knowledge, this is the first reported case of a cardiac-extending IV leiomyomatosis successfully extracted through the gonadal vein. CONCLUSION(S): In a selected case with logistic step-by-step approach, conservative surgical treatment via gonadal vein extraction could be a feasible option in the management of cardiac-extending IV leiomyomatosis. Systematic literature review highlights important clinical characteristics and management options for IV leiomyomatosis.

Polypoid cystitis resulting from ovarian abscess.

We present the case of a 41-year-old woman who presented with gross hematuria, urinary urgency and frequency, and intermittent abdominal pain after vaginal hysterectomy 2 years prior. The presence of an adnexal mass was suspected by pelvic examination and confirmed by transvaginal pelvic ultrasonography. Cystoscopy with biopsy was consistent with an inflammatory bladder polyp, which was initially discovered by pelvic ultrasonography and noted to be contiguous with the adnexal mass. The patient underwent exploratory laparotomy, lysis of adhesions, left salpingoophorectomy, and transvesical resection of the bladder mass. Histology was consistent with ovarian abscess and chronic sinus tract formation that was contiguous with an inflammatory bladder polyp.

Urethral and paraurethral leiomyomas in the female patient.

Urethral leiomyomas in women arise from the smooth muscle of the urethra and are rare, benign urethral tumors seen primarily in women. We present three cases of urethral leiomyomas identified over a 30-year period at our institution. A 45-year old woman presented with a 1 year history of frequency, nocturia, and hesitancy and was found to have both a 2-cm proximal urethral and a 3-cm posterior bladder leiomyoma. She developed stress urinary incontinence postoperatively and was treated with a Burch colposuspension. A 33-year old woman with hematuria was found to have both a 3-cm urethral and a 3-cm paraurethral leiomyoma at the bladder neck. A 21-year old without urinary complaints was found to have a 3-cm leiomyoma at the urethral meatus. Urethral leiomyomas must be differentiated from paraurethral leiomyomas, which are often asymptomatic and may be removed without disrupting the urethral mucosa or smooth muscle. The removal of urethral myomas may be complicated by the development stress urinary incontinence or urethral stricture.

Bladder injury during transobturator sling.

The new minimally invasive transobturator sling for surgical treatment of female genuine stress urinary incontinence is designed to reproduce the natural suspension of the urethral fascia while eliminating the need for retropubic needle passage. We report 3 cases of bladder perforation during the transobturator sling procedure. All injuries were identified intraoperatively by cystoscopy, and successful reinsertion of the mesh was accomplished. Transurethral bladder drainage with a Foley catheter was maintained for 5 to 7 days postoperatively. All 3 patients recovered uneventfully. Routine intraoperative cystoscopy is, therefore, recommended for the identification of bladder injuries during the transobturator sling procedure.