- EMDB-8728: CryoEM Structure of the Zinc Transporter YiiP from helical crystals -
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基本情報
登録情報
データベース: EMDB / ID: EMD-8728
タイトル
CryoEM Structure of the Zinc Transporter YiiP from helical crystals
マップデータ
primary map
試料
複合体: YiiP dimer in an inward-facing conformation
タンパク質・ペプチド: Cadmium and zinc efflux pump FieF
リガンド: ZINC ION
キーワード
zinc antiporter / membrane protein / cation diffusion facilitator / metal transport / helical crystals / Structural Genomics / PSI-Biology / Transcontinental EM Initiative for Membrane Protein Structure / TEMIMPS
機能・相同性
機能・相同性情報
zinc efflux active transmembrane transporter activity / cadmium ion transmembrane transporter activity / ferrous iron transmembrane transporter activity / intracellular zinc ion homeostasis / metal ion binding / plasma membrane 類似検索 - 分子機能
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
U54 GM94598
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM095747
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2018 タイトル: Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP. 著者: Maria Luisa Lopez-Redondo / Nicolas Coudray / Zhening Zhang / John Alexopoulos / David L Stokes / 要旨: YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn across bacterial membranes. Previous work defined the atomic structure of ...YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.
想定した対称性 - らせんパラメータ - Δz: 28.2 Å 想定した対称性 - らせんパラメータ - ΔΦ: -17.0 ° 想定した対称性 - らせんパラメータ - 軸対称性: D5 (2回x5回 2面回転対称) 解像度のタイプ: BY AUTHOR / 解像度: 4.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 2.0) 詳細: Reconstruction was achieved using the IHRSR method implemented in RELION 2.0. 使用した粒子像数: 72333
Segment selection
選択した数: 141904 ソフトウェア:
名称
詳細
SPARX/EMAN2 (ver. EMAN2.1)
sxhelixboxer.py
RELION (ver. 2.0)
2D Classissification
詳細: 2293 filaments selected with SPARX/EMAN2, then windowed into 141,904 segments (450x450 pixel overlapping segments)
初期モデル
モデルのタイプ: OTHER 詳細: A map was obtained previously with images acquired on a JEOL2100 microscope. This map was used as a startup model (That map was obtained as follow: we started with a noisy cylinder. Then, a ...詳細: A map was obtained previously with images acquired on a JEOL2100 microscope. This map was used as a startup model (That map was obtained as follow: we started with a noisy cylinder. Then, a first reconstruction was obtained with Helicon (from SPARX) and refined with Frealix).
最終 角度割当
タイプ: NOT APPLICABLE / ソフトウェア - 名称: RELION (ver. 2.0)
chain_id: B, residue_range: 7-288, source_name: PDB, initial_model_type: experimental model
詳細
The residues were manually adjusted in COOT relying on the large side-chain residues, and the whole system was further optimized using the real_space_refine algorithm in Phenix to ensure proper fit. The conformation was subjected to 13 rounds of COOT/Phenix refinements.
精密化
空間: REAL / プロトコル: FLEXIBLE FIT 当てはまり具合の基準: cross-correlation coefficient
得られたモデル
PDB-5vrf: CryoEM Structure of the Zinc Transporter YiiP from helical crystals