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基本情報
登録情報 | データベース: PDB / ID: 6waz | ||||||
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タイトル | +1 extended HIV-1 reverse transcriptase initiation complex core (pre-translocation state) | ||||||
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![]() | VIRAL PROTEIN/RNA / reverse transcriptase / RNA / complex / protein-RNA complex / tRNA / polymerase / VIRAL PROTEIN / VIRAL PROTEIN-RNA complex | ||||||
機能・相同性 | ![]() integrase activity / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Minus-strand DNA synthesis / Plus-strand DNA synthesis / 2-LTR circle formation / Uncoating of the HIV Virion / Vpr-mediated nuclear import of PICs / Early Phase of HIV Life Cycle / Integration of provirus ...integrase activity / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Minus-strand DNA synthesis / Plus-strand DNA synthesis / 2-LTR circle formation / Uncoating of the HIV Virion / Vpr-mediated nuclear import of PICs / Early Phase of HIV Life Cycle / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / Binding and entry of HIV virion / viral life cycle / symbiont-mediated activation of host apoptosis / HIV-1 retropepsin / Assembly Of The HIV Virion / retroviral ribonuclease H / exoribonuclease H / Budding and maturation of HIV virion / exoribonuclease H activity / protein processing / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus / RNA stem-loop binding / RNA-directed DNA polymerase activity / host cell / RNA-DNA hybrid ribonuclease activity / 転移酵素; リンを含む基を移すもの; 核酸を移すもの / symbiont-mediated suppression of host gene expression / peptidase activity / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / 加水分解酵素; エステル加水分解酵素 / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / lipid binding / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / proteolysis / DNA binding / zinc ion binding / identical protein binding / membrane 類似検索 - 分子機能 | ||||||
生物種 | ![]() ![]() ![]() | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.1 Å | ||||||
![]() | Larsen, K.P. / Jackson, L.N. / Kappel, K. / Zhang, J. / Puglisi, E.V. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Distinct Conformational States Underlie Pausing during Initiation of HIV-1 Reverse Transcription. 著者: Kevin P Larsen / Junhong Choi / Lynnette N Jackson / Kalli Kappel / Jingji Zhang / Betty Ha / Dong-Hua Chen / Elisabetta Viani Puglisi / ![]() 要旨: A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have ...A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have identified specific sites along the viral RNA genomic template in which reverse transcriptase (RT) stalls. These stalling points, which occur after the addition of three and five template dNTPs, may serve as checkpoints to regulate the precise timing of HIV-1 reverse transcription following viral entry. Structural studies of reverse transcriptase initiation complexes (RTICs) have revealed unique conformations that may explain the slow rate of incorporation; however, questions remain about the temporal evolution of the complex and features that contribute to strong pausing during initiation. Here we present cryo-electron microscopy and single-molecule characterization of an RTIC after three rounds of dNTP incorporation (+3), the first major pausing point during reverse transcription initiation. Cryo-electron microscopy structures of a +3 extended RTIC reveal conformational heterogeneity within the RTIC core. Three distinct conformations were identified, two of which adopt unique, likely off-pathway, intermediates in the canonical polymerization cycle. Single-molecule Förster resonance energy transfer experiments confirm that the +3 RTIC is more structurally dynamic than earlier-stage RTICs. These alternative conformations were selectively disrupted through structure-guided point mutations to shift single-molecule Förster resonance energy transfer populations back toward the on-pathway conformation. Our results support the hypothesis that conformational heterogeneity within the HIV-1 RTIC during pausing serves as an additional means of regulating HIV-1 replication. | ||||||
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 208.6 KB | 表示 | ![]() |
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PDB形式 | ![]() | 156.7 KB | 表示 | ![]() |
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その他 | ![]() |
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アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 64705.316 Da / 分子数: 1 / 変異: Q258C, E478Q / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 株: isolate BH10 / 遺伝子: gag-pol / 発現宿主: ![]() ![]() 参照: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H |
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#2: タンパク質 | 分子量: 51585.293 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 発現宿主: ![]() ![]() |
#3: RNA鎖 | 分子量: 32623.477 Da / 分子数: 1 / 由来タイプ: 合成 由来: (合成) ![]() ![]() 参照: GenBank: 60651827 |
#4: DNA/RNAハイブリッド | 分子量: 24720.664 Da / 分子数: 1 / 由来タイプ: 合成 / 由来: (合成) ![]() |
-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 |
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分子量 |
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由来(組換発現) |
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緩衝液 | pH: 8 詳細: Full complex was prepared 5-8 hours before freezing. Beta-OG was added just prior to freezing. | |||||||||||||||||||||||||||||||||||
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試料 | 濃度: 5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES / 詳細: Sample was monodisperse. | |||||||||||||||||||||||||||||||||||
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 200 divisions/in. / グリッドのタイプ: Quantifoil R2/1 | |||||||||||||||||||||||||||||||||||
急速凍結 | 装置: LEICA EM GP / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 292 K / 詳細: 3 microliters applied, 2s pre-blot, 1.5s blot. |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD |
撮影 | 電子線照射量: 62.3 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||
3次元再構成 | 解像度: 4.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 394434 / 対称性のタイプ: POINT | |||||||||||||||||||||
原子モデル構築 | 空間: REAL |