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.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.125 character summary FCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s### Competing Interest StatementThe authors have declared no competing interest.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence–structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.

(Developmental Cell 50, 494–508.e1–e11; August 19, 2019) As a result of an author oversight in the originally published version of this article, the author Susanne Salomon was omitted from the author list. This error has now been corrected in the article online. The authors apologize for the error and any inconvenience that may have resulted. Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 PhosphorylationWrobel et al.Developmental CellAugust 19, 2019In BriefWrobel et al. show that phosphorylation of the mammalian endocytic AP2 adaptor causes a conformational change that allows it to efficiently bind the protein NECAP. This, in turn, recruits membrane-remodeling proteins into clathrin-coated pits, which drive their formation toward final scission from the parent membrane. Full-Text PDF Open Access

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

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.

Aims An overview of the creation, delivery, evaluation and impact of a collaboratively designed health professional degree and master’s module supporting establishment, augmentation and enhancement of transitional services across long term conditions, life threatening illnesses, learning disability and mental health provision. Methods A collaborative engaged multi-disciplinary module team with a combined vision to develop practitioners’ knowledge, skills and abilities concerning effective transition opportunities worked together. We designed an interactive blended-learning module supported by nationally recognised experts in the field, on-line resources and access to the RCPCH Adolescent health module. The module was delivered and evaluated exceptionally well. Students were assessed design and presentation of their proposals to change and develop their services to be more ‘adolescent and transition friendly’. The written assessment was a report to be delivered within the students areas of practice to facilitate the changes intended to realise development of effective transition services accordingly. The module was delivered at level 6 and level 7 to support a multi-disciplinary team approach to embracing transition. Local Education Training Board (LETB) funding supported student attedance. Results Students included Nurses, Doctors, Dietitians, Psychologists and Youth Workers evaluated the module excellently and highlighted opportunities realised within their own practices and specialisms. This demonstrated a change in thinking, renewed energy to face change and tackle potential challenges to providing effective transition. Synergistic opportunities were also realised by managers and commissioners attending the students’ presentations and ‘signing up’ to supporting the fresh approaches and ideas within their services to enable effective transition to be appreciated. Key outcomes for students and practice Developing a clearer vision through action planning, thus developing a wealth of knowledge and resources to stimulate change. Heightened motivation and confidence facilitated renewed energy to initiate practice communication with adult service colleagues. Students had ‘a voice’ rather than hierarchy intimidating their vision for their team. The action plans met patients’ needs for improvement and development to close service gaps. Conclusion Quality transition services can be realised rather than just ‘talked about’. Practitioners are engaged in critical discussions to effect this transformation and are implementing service improvement. Crucially and incrementally managers and commissioners have pledged their support to escalate and influence essential changes in practice.