Dear Editor,G protein-coupled receptors(GPCRs)play a vital role in regulating almost every aspect of human physiology,making up more than one-third of marketed drug targets(Santos et al.,2017).GPCRs orchestrate their signalling through interactions with three distinct downstream protein families:G proteins,G protein-coupled recep-tor kinases(GRKs),and arrestins(Santos et al.,2017).

Hexadecameric form I Rubisco,which consisting consists of eight large(RbcL)and eight small(RbcS)sub-units,is the most abundant enzyme on earth.Extensive efforts to engineer an improved Rubisco to speed up its catalytic efficiency and ultimately increase agricultural productivity.However,difficulties with cor-rect folding and assembly in foreign hosts or in vitro have hampered the genetic manipulation of hexade-cameric Rubisco.In this study,we reconstituted Synechococcus sp.PCC6301 Rubisco in vitro using the chaperonin system and assembly factors from cyanobacteria and Arabidopsis thaliana(At).Rubisco holo-enzyme was produced in the presence of cyanobacterial Rubisco accumulation factor 1(Raf1)alone or both AtRaf1 and bundle-sheath defective-2(AtBsd2)from Arabidopsis.RbcL released from GroEL is as-sembly capable in the presence of ATP,and AtBsd2 functions downstream of AtRaf1.Cryo-EM structures of RbcL8-AtRaf18,RbcL8-AtRaf14-AtBsd28,and RbcL8 revealed that the interactions between RbcL and AtRaf1 are looser than those between prokaryotic RbcL and Raf1,with AtRaf1 tilting 7° farther away from RbcL.AtBsd2 stabilizes the flexible regions of RbcL,including the N and C termini,the 60s loop,and loop 6.Using these data,combined with previous findings,we propose the possible biogenesis path-ways of prokaryotic and eukaryotic Rubisco.

Dear Editor,Heme is an essential cofactor required across all kingdoms of life utilized in numerous biological processes,including cellular res-piration.CydDC is a prokaryotic ATP-binding cassette(ABC)trans-porter required for heme assembling in respiratory cytochrome bd oxidase(Georgiou et al.,1987;Poole et al.,1989),a promising target for drug discovery(Borisov et al.,2011).CydDC is thought to play a role in maintaining an optimum periplasmic redox poise that is required for the incorporation of heme cofactors(Yamashita et al.,2014).It also suggested that CydDC is involved in heme processing by mediating glutathione/cysteine translo-cation(Cook and Poole,2000;Cook et al.,2002),although a role in heme translocation seems unlikely(Yamashita et al.,2014).To date,CydDC has been shown to be important for disulfide bond formation,motility,respiration,and tolerance to nitric oxide and antibiotics(Poole et al.,2019).

@@

最近,冷冻电镜技术的突破引起结构生物学发生了革命.这一革命导致2017年诺贝尔化学奖授予对冷冻电镜技术发展做出开创性贡献的3位科学家Jacques Dubochet、Joachim Frank和Richard Henderson.本文将综述冷冻电镜的发展历程,导致结构生物学革命的冷冻电镜关键技术,包括电镜、图像记录装置和图像处理算法方面的突破,以及中国科学家应用冷冻电镜取得的重要科学成就,涵盖基因表达/调控、蛋白质合成/降解、膜蛋白、免疫、病毒等相关蛋白复合体.最后,对冷冻电镜的未来发展方向进行展望.

The nucleotide-binding domain-and leucine-rich repeat (LRR)-containing proteins (NLRs) function as intracellular immune receptors to detect the presence of pathogen-or host-derived signals.The mechanisms of how NLRs sense their ligands remain elusive.Here we report the structure of a bacterial flagellin derivative in complex with the NLR proteins NAIP5 and NLRC4 determined by cryo-electron microscopy at 4.28 (A) resolution.The structure revealed that the flagellin derivative forms two parallel helices interacting with multiple domains including BIR1 and LRR of NAIP5.Binding to NAIP5 results in a nearly complete burial of the flagellin derivative,thus stabilizing the active conformation of NAIP5.The extreme C-terminal side of the flagellin is anchored to a sterically constrained binding pocket of NAIP5,which likely acts as a structural determinant for discrimination of different bacterial flagellins by NAIPS,a notion further supported by biochemical data.Taken together,our results shed light on the molecular mechanisms underlying NLR ligand perception.

ATR (ataxia telangiectasia-mutated and Rad3-related) protein kinase and ATRIP (ATR-interacting protein) form a complex and play a critical role in response to replication stress and DNA damage.Here,we determined the cryo-electron microscopy (EM) structure of the human ATR-ATRIP complex at 4.7 (A) resolution and built an atomic model of the C-terminal catalytic core of ATR (residues 1 521-2 644) at 3.9(A) resolution.The complex adopts a hollow "heart" shape,consisting of two ATR monomers in distinct conformations.The EM map for ATRIP reveals 14 HEAT repeats in an extended "S" shape.The conformational flexibility of ATR allows ATRIP to properly lock the N-termini of the two ATR monomers to favor ATR-ATRIP complex formation and functional diversity.The isolated "head-head"and "tail-tail" each adopts a pseudo 2-fold symmetry.The catalytic pockets face outward and substrate access is not restricted by inhibitory elements.Our studies provide a structural basis for understanding the assembly of the ATR-ATRIP complex and a framework for characterizing ATR-mediated DNA repair pathways.

@@

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSinl. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 A resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeat clusters and binds to mTOR through multiple contacts. mSinl is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSinl together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, revealing the mechanism for rapamycin insensitivity of mTORC2. The mTOR dimer in mTORC2 shows more compact conformation than that of mTORC1 (rapamycin sensitive), which might result from the interaction between mTOR and Rictor-mSinl. Structural comparison shows that binding of Rictor and Raptor (mTORC1-specific component) to mTOR is mutually exclusive. Our study provides a basis for understanding the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway.

呼吸作用是生物体最基本最重要的生命活动.在哺乳动物中,呼吸作用(氧化磷酸化)由位于线粒体内膜上的呼吸链复合物完成.一百多年来,科学家们孜孜不倦地对线粒体呼吸链复合物进行研究,想要窥探这一能量大分子机器的全貌,但是一直未能获取该复合物蛋白质结构.我们最新的研究首次纯化出了来源于人类细胞的线粒体呼吸链超超级复合物I2Ⅲ2IV2,通过冷冻电镜技术首次成功解析了它的结构,并且提出呼吸链复合物I、II、III和IV可以一起组成超大型复合物I2Ⅱ2Ⅲ2IV2,这是呼吸链超超级复合物的终极形态.同时,我们所解析的人源呼吸链超级复合物的高分辨率结构,为攻克线粒体缺陷引起的阿尔兹海默综合征、帕金森综合征、多发性硬化、少年脊髓型共济失调以及肌萎缩性脊髓侧索硬化症等多种疾病打下了坚实的基础.