Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function

BIOMOLECULAR FUNCTION FROM STRUCTURAL SNAPSHOTS

Biological molecules can assume a continuous range of conformations during function. Near equilibrium, the Boltzmann relation connects a particular conformation\u27s free energy to the conformation\u27s occupation probability, thus giving rise to one or more energy landscapes. Biomolecular function proceeds along minimum-energy pathways on such landscapes. Consequently, a comprehensive understanding of biomolecular function often involves the determination of the free-energy landscapes and the identification of functionally relevant minimum-energy conformational paths on these landscapes. Specific techniques are necessary to determine continuous conformational spectra and identify functionally relevant conformational trajectories from a collection of raw single-particle snapshots from, e.g. cryogenic electron microscopy (cryo-EM) or X-ray diffraction. To assess the capability of different algorithms to recover conformational landscapes, we:• Measure, compare, and benchmark the performance of four leading data-analytical approaches to determine the accuracy with which energy landscapes are recovered from simulated cryo-EM data. Our simulated data are derived from projection directions along the great circle, emanating from a known energy landscape. • Demonstrate the ability to recover a biomolecule\u27s energy landscapes and functional pathways of biomolecules extracted from collections of cryo-EM snapshots. Structural biology applications in drug discovery and molecular medicine highlight the importance of the free-energy landscapes of the biomolecules more crucial than ever. Recently several data-driven machine learning algorithms have emerged to extract energy landscapes and functionally relevant continuous conformational pathways from single-particle data (Dashti et al., 2014; Dashti et al., 2020; Mashayekhi,et al., 2022). In a benchmarking study, the performance of several advanced data-analytical algorithms was critically assessed (Dsouza et al., 2023). In this dissertation, we have benchmarked the performance of four leading algorithms in extracting energy landscapes and functional pathways from single-particle cryo-EM snapshots. In addition, we have significantly improved the performance of the ManifoldEM algorithm, which has demonstrated the highest performance. Our contributions can be summarized as follows.: • Expert user supervision is required in one of the main steps of the ManifoldEM framework wherein the algorithm needs to propagate the conformational information through all angular space. We have succeeded in introducing an automated approach, which eliminates the need for user involvement. • The quality of the energy landscapes extracted by ManifoldEM from cryo-EM data has been improved, as the accuracy scores demonstrate this improvement. These measures have substantially enhanced ManifoldEM’s ability to recover the conformational motions of biomolecules by extracting the energy landscape from cryo-EM data.In line with the primary goal of our research, we aimed to extend the automated method across the entire angular sphere rather than a great circle. During this endeavor, we encountered challenges, particularly with some projection directions not following the proposed model. Through methodological adjustments and sampling optimization, we improved the projection direction\u27s conformity to the model. However, a small subset of Projection directions (5 %) remained challenging. We also recommended the use of specific methodologies, namely feature extraction and edge detection algorithms, to enhance the precision in quantifying image differentiation, a crucial component of our automated model. we also suggested that integrating different techniques might potentially resolve challenges associated with certain projection directions. We also applied ManifoldEM to experimental cryo-EM images of the SARS-CoV-2 spike protein in complex with the ACE2 receptor. By introducing several improvements, such as the incorporation of an adaptive mask and cosine curve fitting, we enhanced the framework\u27s output quality. This enhancement can be quantified by observing the removal of the artifact from the energy landscape, especially if the post-enhancement landscape differs from the artifact-affected one. These modifications, specifically aimed at addressing challenges from Nonlinear Laplacian Spectral Analysis (NLSA) (Giannakis et al., 2012), are intended for application in upcoming cryo-EM studies utilizing ManifoldEM. In the closing sections of this dissertation, a summary and a projection of future research directions are provided. While initial automated methods have been explored, there remains room for refinement. We have offered numerous methodological suggestions oriented toward addressing solutions to the challenge of conformational information propagation. Key methodologies discussed include Manifold Alignment, Canonical Correlation Analysis, and Multi-View Diffusion Maps. These recommendations are aimed to inform and guide subsequent developments in the ManifoldEM suite

Imaging and 3D reconstruction of membrane protein complexes by cryo-electron microscopy and single particle analysis

Cryo-electron microscopy (cryo-EM) in combination with single particle image processing and volume reconstruction is a powerful technology to obtain medium-resolution structures of large protein complexes, which are extremely difficult to crystallize and not amenable to NMR studies due to size limitation. Depending on the stability and stiffness as well as on the symmetry of the complex, three-dimensional reconstructions at a resolution of 10-30 ˚ can be achieved. In this range of resolution, we may not be able to answer A chemical questions at the level of atomic interactions, but we can gain detailed insight into the macromolecular architecture of large multi-subunit complexes and their mechanisms of action. In this thesis, several prevalently large membrane protein complexes of great physiological importance were examined by various electron microscopy techniques and single particle image analysis. The core part of my work consists in the imaging of a mammalian V-ATPase, frozen-hydrated in amorphous ice and of the completion of the first volume reconstruction of this type of enzyme, derived from cryo-EM images. This ubiquitous rotary motor is essential in every eukaryotic cell and is of high medical importance due to its implication in various diseases such as osteoporosis, skeletal cancer and kidney disorders. My contribution to the second and third paper concerns the volume reconstruction of two bacterial outer membrane pore complexes from cryo-EM images recorded by my colleague Mohamed Chami. PulD from Klebsiella oxytoca constitutes a massive translocating pore capable of transporting a fully folded cell surface protein PulA through the membrane. It is part of the Type II secretion system, which is common for Gram-negative bacteria. The second volume regards ClyA, a pore-forming heamolytic toxin of virulent Escherichia coli and Salmonella enterica strains that kill target cells by inserting pores into their membranes. To the last two papers, I contributed with cryo-negative stain imaging of the cell division protein DivIVA from Bacillus subtilis and with image processing of the micrographs displaying the siderophore receptor FrpB from Neisseria meningitidis

Advances in image processing for single-particle analysis by electron cryomicroscopy and challenges ahead

Electron cryomicroscopy (cryo-EM) is essential for the study and functional understanding of non-crystalline macromolecules such as proteins. These molecules cannot be imaged using X-ray crystallography or other popular methods. CryoEM has been successfully used to visualize molecules such as ribosomes, viruses, and ion channels, for example. Obtaining structural models of these at various conformational states leads to insight on how these molecules function. Recent advances in imaging technology have given cryo-EM a scientific rebirth. Because of imaging improvements, image processing and analysis of the resultant images have increased the resolution such that molecular structures can be resolved at the atomic level. Cryo-EM is ripe with stimulating image processing challenges. In this article, we will touch on the most essential in order to build an accurate structural three-dimensional model from noisy projection images. Traditional approaches, such as k-means clustering for class averaging, will be provided as background. With this review, however, we will highlight fresh approaches from new and varied angles for each image processing sub-problem, including a 3D reconstruction method for asymmetric molecules using just two projection images and deep learning algorithms for automated particle picking. Keywords: Cryo-electron microscopy, Single Particle Analysis, Image processing algorithms

Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self assembly

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain–heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein–protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis

Structure of the condensin holo complex

Division of one mother cell into two daughter cells resides at the very core of living organisms. To ensure that the cell’s genetic material is equally segregated into both daughter cells, cells undergo a sophisticated succession of highly controlled events. One of these events is the packaging of chromatin fibers into mitotic chromosomes that can be transported by spindle microtubules. The key factor for this chromosome condensation process is the five-subunit condensin complex. Condensin is thought to shape chromosomes by actively extruding large chromatin loops, yet how condensin can create such loops has remained largely unknown. To shed light onto the mechanism of condensin-mediated chromatin condensation, insights into the structure of the complex will be indispensable. While X-ray crystallography proved efficient in providing said information for individual parts of the complex, the architecture of the entire complex remained unknown. The aim of the work described in this PhD thesis was to close this crucial gap in knowledge by elucidating the condensin holo complex structure. I employed cryogenic electron microscopy to first solve the structure of the Saccharomyces cerevisiae condensin holo complex in its apo and nucleotide-bound states. In the absence of nucleotide, condensin adopts a rod-like conformation. The HEAT-repeat subunit Ycs4 stably interacts with the ATPase head domains of closely aligned Smc2 and Smc4 subunits, while the Ycg1 HEAT-repeat subunit is flexibly tethered to the rest of the complex through the Brn1 subunit. Instead of forming a fully stretched rod, the Smc2–Smc4 coiled-coil arm segment contains a kink that results in the hinge folding back onto the coiled coils. In a second apo state, the Smc2 and Smc4 heads are bridged by Ycs4, which splits apart the Smc2–Smc4 coiled coils from the head to the joint regions. Addition of ATP induces a drastic structural rearrangement. The ATPase heads engage, which results in an increase in flexibility and opening of the coiled coils. Furthermore, Ycg1 and Ycs4 swap positions as ATP releases Ycs4 from the Smc4 ATPase head, which in turn provides access for Ycg1 to directly bind the Smc2 ATPase head domain. These data provide a structural framework for the condensin ATPase cycle and suggest that an ATP-driven exchange of the Ycs4 and Ycg1 subunits interconverts DNA binding sites that might form the core of the condensin DNA loop-extruding activity

액상에 존재하는 개별 나노입자에 대한 3차원 원자구조 분석 방법론

학위논문(박사) -- 서울대학교대학원 : 공과대학 화학생물공학부, 2023. 2. 박정원.Precise three-dimensional (3D) atomic structure determination of individual nanocrystals is a prerequisite for understanding and predicting their physical properties, because the 3D atomic arrangements of materials determine the free energy landscape. We developed a Brownian one-particle reconstruction based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Nanocrystals from the same synthesis batch display what are often presumed to be small but possibly important differences in size, lattice distortions, and defects, which can only be understood by structural characterization with high spatial 3D resolution. The structures of individual colloidal platinum nanocrystals are solved by developing atomic-resolution 3D liquid-cell electron microscopy to reveal critical intrinsic heterogeneity of ligand-protected platinum nanocrystals in solution, including structural degeneracies, lattice parameter deviations, internal defects, and strain. These differences in structure lead to substantial contributions to free energies, consequential enough that they must be considered in any discussion of fundamental nanocrystal properties or applications. We introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo–electron microscopy. Characterization of lattice symmetry is important because the symmetry is strongly correlated with physical properties of nanomaterials. We introduce direct and quantitative analysis of lattice symmetry by using 3D atomic coordinates obtained by liquid-phase TEM. We investigate symmetry of entire unit-cells composing individual platinum nanoparticles, revealing unique structural characteristics of sub-3 nm Pt nanoparticles. We here introduce a 3D atomic structure determination method for multi-element nanoparticle systems. The method, which is based on low-pass filtration and initial 3D model generation customized for different types of multi-element systems, enables reconstruction of high-resolution 3D Coulomb density maps for ordered and disordered multi-element systems and classification of the heteroatom type. Using high-resolution image datasets obtained from TEM simulations of PbSe, CdSe, and FePt nanoparticles that are structurally relaxed with first-principles calculations in the graphene liquid cell, we show that the types and positions of the constituent atoms are precisely determined with root mean square displacement (RMSD) values less than 24 pm. Our study suggests that it is possible to investigate the 3D atomic structures of synthesized multi-element nanoparticles in liquid phase.재료의 3D 원자 배열이 자유 에너지 환경을 결정한다는 점을 고려했을 때, 개별 나노결정의 정확한 3차원(3D) 원자 구조 분석은 물리적 특성을 이해하고 예측하기 위해 필수 불가결하다. 본 연구자는 그래핀 액체 세포 투과 전자 현미경을 사용하여 콜로이드 나노입자의 앙상블 이미징을 기반으로 하는 "브라운 단일 입자 재구성"을 개발했다. 동일한 합성 배치의 나노입자는 크기, 격자 왜곡 및 결함 등에서 종종 작지만 중요한 것으로 추정되는 것으로 간주되는 구조적 차이점이 있으며, 이는 3D 고해상도 구조 분석에 의해서만 이해할 수 있다. 구조적 퇴화, 격자 매개변수 편차, 내부 결함 및 변형을 포함한 개별 콜로이드 백금 나노입자의 구조적 특성은 원자 분해능 3D 액체 세포 전자 현미경을 개발하여 풀어낼 수 있다. 이러한 구조의 차이는 자유 에너지에 상당한 기여를 하므로 결과적으로 기본적인 나노입자 특성 또는 응용에 대한 논의에서 고려되어야 한다. 본 논문에서는 성공적인 원자 해상도 3D 재구성에 필요한 계산 방법론을 소개한다. 그 방법론에는 다음과 같은 알고리즘이 포함된다. (1) 시계열 이미지에서 개별 나노입 자를 추적하는 알고리즘, (2) 그래핀 액체 셀의 배경 노이즈를 제거하는 알고리즘, (3) 저해상도 이미지를 검출 및 제거하는 알고리즘, (4) 극저온 전자현미경을 이용한 바이오 입자의 재구성에 쓰이는 알고리즘과는 다른 나노입자만을 위해서 고안된 2차원/3차원 정렬 알고리즘. 격자 대칭성은 나노 물질의 물리적 특성과 강한 상관관계가 있기 때문에, 격자 대칭성 분석은 중요하다. 본 논문에서는 액상 투과 전자 현미경을 통해서 얻은 3차원 원자 좌표를 이용하여 격자 대칭을 직접적, 정량적으로 분석할 수 있는 방법론을 소개하고자 한다. 개별 백금 나노입자를 구성하는 전체 unit cell의 대칭성을 조사함으로써, 3 나노미터 이하의 백금 나노입자가 갖는 독특한 구조적 특징을 밝혀내였다. 본 논문에서는 다원소 나노입자 시스템을 위한 3차원 원자 구조 분석법을 소개하고자 한다. 제시된 low-pass filtering과 initial 3D modeling 방법은 다양한 유형의 다원소 시스템에 맞춰져 있으며, 이를 통해 ordered multi-element system과 disordered multi-element system에서 원자의 위치를 파악하고 원소의 종류를 구분할 수 있다. First-principles calculation을 통해 얻은 PbSe, CdSe, FePt 나노입자 구조로부터 그래핀 액체 셀 안에서의 TEM 시뮬레이션 이미지를 얻고, 이를 활용하여 구성 원자의 유형과 위치를 24 피코미터 미만의 오차로 정확도 높게 판별할 수 있음을 확인하였다. 우리의 연구는 액상에서 합성된 다원소 나노입자의 3차원 원자 구조를 조사하는 것이 가능함을 시사한다.Chapter 1. Introdution 1 1.1. Atomic structure property relationships in nanoparticles 1 1.2. Toward atomic structure characterization 2 1.3. Direct observation of 3D atomic structures of individual nanoparticles: Electron tomography and Brownian one-particle reconstruction 3 1.4. Purpose of Research 4 Chapter 2. 3D atomic structures of individual ligand-protected Pt nanoparticles in solution 7 2.1. Introduction 7 2.2. 3D reconstruction from electron microscopy images of Pt nanoparticles in liquid 8 2.2.1. Synthesis of Pt nanoparticles 8 2.2.2. Preparation of graphene liquid cells 9 2.2.3. Acquisition of TEM images 9 2.2.4. 3D reconstruction 10 2.2.5. Atomic position assignment 11 2.2.6. Validation 11 2.2.7. Atomic structure analysis 13 2.3. Atomic structural characteristics of Pt nanoparticles in liquid 16 2.2.1. Effect of surface ligands on the 3D atomic structures of Pt nanoparticles 16 2.3.2. Structural heterogeneity of Pt nanoparticles 18 2.3.3. Strain analysis of individual Pt nanoparticles from the 3D atomic maps 19 2.4. Conclusion 21 Chapter 3. SINGLE: Computational methods for atomic-resolution 3D reconstruction 57 3.1. Introduction 57 3.2. Results 58 3.2.1. Overview of 3D SINGLE 58 3.2.2. The SINGLE workflow 58 3.3. Conclusion 66 Chapter 4. 3-Dimensional scanning of unit cell symmetries in individual nanoparticles by using Brownian one-particle reconstruction 75 4.1. Introduction 75 4.2. Results 77 4.2.1. Quantitative symmetry analysis from 3D atomic coordinates 77 4.2.2. Direction of symmetry breakage 79 4.2.3. Structural heterogeneity 80 4.2.4. Relationship between symmetry and surface interactions 80 4.3. Conclusion 84 Chapter 5. Method for 3D atomic structure determination of multi-element nanoparticles with graphene liquid-cell TEM 102 5.1. Introduction 102 5.2. Results 104 5.2.1. Overview of multi-element nanoparticle 3D reconstruction 104 5.2.2. Principles for multi-element nanoparticle reconstruction 105 5.2.3. Demonstration using simulated TEM images 106 5.3. Conclusion 111 Bibliography 136 국 문 초 록 144박