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基本情報

登録情報
データベース: PDB / ID: 6bbm
タイトルMechanisms of Opening and Closing of the Bacterial Replicative Helicase: The DnaB Helicase and Lambda P Helicase Loader Complex
要素
  • Replication protein P
  • Replicative DNA helicase
キーワードREPLICATION / Helicase Loader / Helicase / DNA replication / ATPase / DNA Replication Initiation / Bacteriophage Lambda
機能・相同性
機能・相同性情報


DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / bidirectional double-stranded viral DNA replication / DNA replication, synthesis of primer / replisome ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / bidirectional double-stranded viral DNA replication / DNA replication, synthesis of primer / replisome / DNA duplex unwinding / response to ionizing radiation / replication fork processing / DNA unwinding involved in DNA replication / DNA replication initiation / DNA helicase activity / helicase activity / DNA replication / DNA helicase / hydrolase activity / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol
類似検索 - 分子機能
Replication P / Replication protein P / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. ...Replication P / Replication protein P / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
類似検索 - ドメイン・相同性
ADENOSINE-5'-DIPHOSPHATE / Replicative DNA helicase / Replication protein P / Replicative DNA helicase
類似検索 - 構成要素
生物種Escherichia coli O111:NM (大腸菌)
Escherichia phage lambda (λファージ)
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
データ登録者Chase, J. / Catalano, A. / Noble, A.J. / Eng, E.T. / Olinares, P.D.B. / Molloy, K. / Pakotiprapha, D. / Samuels, M. / Chain, B. / des Georges, A. / Jeruzalmi, D.
資金援助 米国, 12件
組織認可番号国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM084162 米国
National Science Foundation (NSF, United States)MCB 1818255 米国
National Institutes of Health/National Institute on Minority Health and Health Disparities (NIH/NIMHD)5G12MD007603-30 米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103314 米国
Other governmentPA200A150068 米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM128303 米国
Other privateSF349247 (Simons Foundation) 米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 米国
Other privateF00316 (Agouron Institute) 米国
National Institutes of Health/Office of the DirectorOD019994 米国
Other privateSM-2015-289297 (Silicon Mechanics/Research Cluster Grant program) 米国
引用ジャーナル: Elife / 年: 2018
タイトル: Mechanisms of opening and closing of the bacterial replicative helicase.
著者: Jillian Chase / Andrew Catalano / Alex J Noble / Edward T Eng / Paul Db Olinares / Kelly Molloy / Danaya Pakotiprapha / Martin Samuels / Brian Chait / Amedee des Georges / David Jeruzalmi /
要旨: Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and ...Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.
履歴
登録2017年10月18日登録サイト: RCSB / 処理サイト: RCSB
改定 1.02019年3月6日Provider: repository / タイプ: Initial release
改定 1.12019年3月13日Group: Data collection
カテゴリ: em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
改定 1.22019年8月14日Group: Data collection / Data processing / Experimental preparation
カテゴリ: em_3d_reconstruction / em_ctf_correction ...em_3d_reconstruction / em_ctf_correction / em_experiment / em_image_processing / em_software / em_specimen
Item: _em_experiment.reconstruction_method / _em_software.image_processing_id / _em_specimen.details
改定 1.32019年11月27日Group: Author supporting evidence / カテゴリ: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
改定 1.42019年12月18日Group: Other / カテゴリ: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
改定 1.52024年3月13日Group: Data collection / Database references / Refinement description
カテゴリ: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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構造の表示

ムービー
  • 登録構造単位
  • Jmolによる作画
  • ダウンロード
  • EMマップとの重ね合わせ
  • マップデータ: EMDB-7076
  • UCSF Chimeraによる作画
  • ダウンロード
ムービービューア
構造ビューア分子:
MolmilJmol/JSmol

ダウンロードとリンク

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集合体

登録構造単位
A: Replicative DNA helicase
B: Replicative DNA helicase
C: Replicative DNA helicase
D: Replicative DNA helicase
E: Replicative DNA helicase
F: Replicative DNA helicase
V: Replication protein P
W: Replication protein P
X: Replication protein P
Y: Replication protein P
Z: Replication protein P
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)432,54816
ポリマ－430,41211
非ポリマー2,1365
水00
1


  • 登録構造と同一
  • 登録者が定義した集合体
  • 根拠: mass spectrometry, Native mass spectrometry of DnaB-LambdaP complexes point to a physiological stoichiometry of DnaB6-LambdaP5, which agrees with the data derived from our cryoEM map., mass ...根拠: mass spectrometry, Native mass spectrometry of DnaB-LambdaP complexes point to a physiological stoichiometry of DnaB6-LambdaP5, which agrees with the data derived from our cryoEM map., mass spectrometry, Native mass spectrometry of DnaB-LambdaP in the presence of origin-derived single stranded DNA reveals a single DnaB6-LambdaP5-ssDNA complex, supporting this is the physiologically relevant stoichiometry., cross-linking, Cross-linking mass spectrometry experiments validated that the C-terminus of Lambda P interacts with DnaB. This finding was also supplemented with binding studies.
タイプ名称対称操作数
identity operation1_555x,y,z1

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要素

#1: タンパク質
Replicative DNA helicase


分子量: 52450.945 Da / 分子数: 6 / 由来タイプ: 組換発現 / 由来: (組換発現) Escherichia coli O111:NM (大腸菌) / 遺伝子: DQW49_15020 / プラスミド: pET24a / 細胞株 (発現宿主): BL21(DE3) / 発現宿主: Escherichia coli BL21(DE3) (大腸菌)
参照: UniProt: A0A365Q7M1, UniProt: P0ACB0*PLUS, DNA helicase
#2: タンパク質
Replication protein P


分子量: 23141.221 Da / 分子数: 5 / 由来タイプ: 組換発現
由来: (組換発現) Escherichia phage lambda (λファージ)
プラスミド: pET24a / 細胞株 (発現宿主): BL21(DE3) / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 参照: UniProt: P03689
#3: 化合物
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / ADP


分子量: 427.201 Da / 分子数: 5 / 由来タイプ: 合成 / 式: C10H15N5O10P2 / コメント: ADP, エネルギー貯蔵分子*YM
配列の詳細The complete sequence of Bacteriophage Lambda P is ...The complete sequence of Bacteriophage Lambda P is MKNIAAQMVNFDREQMRRIANNMPEQYDEKPQVQQVAQIINGVFSQLLATFPASLANRDQ NEVNEIRRQWVLAFRENGITTMEQVNAGMRVARRQNRPFLPSPGQFVAWCREEASVTAGL PNVSELVDMVYEYCRKRGLYPDAESYPWKSNAHYWLVTNLYQNMRANALTDAELRRKAAD ELVHMTARINRGEAIPEPVKQLPVMGGRPLNRAQALAKIAEIKAKFGLKGASV

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実験情報

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実験

実験手法: 電子顕微鏡法
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
ID名称タイプ詳細Entity IDParent-ID由来
1DnaB helicase - Lambda P helicase loader DNA replication complexCOMPLEXpET24a containing full-length DnaB was co-expressed with pCDFDuet containing full-length LambdaP in BL21(DE3) cells. The resolution of the LambdaP portion of our EM map did not permit the unambiguous assignment of the amino acid sequence to the structure. As such, the model for LambdaP was built as a poly alanine model. Additionally, only half of LambdaP was observed in our maps due to the intrinsic flexibility of the amino and carboxy terminal domains of LambdaP. Subsequent experiments determined that the observed portion of LambdaP in our maps corresponds to the C-terminal domain.#1-#20RECOMBINANT
2E coli DnaB helicaseCOMPLEXE coli DnaB helicase is observed as an open-spiral hexamer, in which one of the interfaces is breached. Five ADP molecules are observed at the five intact ATP binding sites. Additionally, clear density is observed for five of six linkers permitting unambiguous assignment of NTD to parent CTD domain.#11RECOMBINANT
3Lambda P helicase loaderCOMPLEXFive lambda P molecules were observed bound to the five intact DnaB subunit interfaces. Unambiguous assignment of side chain density for lambda P was not possible due to the resolution of this region of the EM map. Instead, a polyalanine model was built for each lambda P molecule. Additionally, density for approximately half of the expected 233 residues of lambda P was observed owing to flexibility between domains. Subsequent experiments confirmed that the observed region of Lambda P is the C-terminal domain, which interacts with DnaB.#21RECOMBINANT
分子量
IDEntity assembly-ID値 (°)実験値
110.4465 MDaYES
21NO
31NO
由来(天然)
IDEntity assembly-ID生物種Ncbi tax-ID
21Escherichia coli (大腸菌)562
32Escherichia coli (大腸菌)562
43Enterobacteria phage lambda (λファージ)10710
由来(組換発現)
IDEntity assembly-ID生物種Ncbi tax-ID細胞プラスミド
21Escherichia coli (大腸菌)562BL21(DE3)pET24a and pCDFDuet
32Escherichia coli (大腸菌)562BL21(DE3)pET24a
43Escherichia coli (大腸菌)562BL21(DE3)pCDFDuet
緩衝液pH: 7.5
詳細: Concentrated BP sample (18mg/mL) was diluted with freshly prepared buffer to desired concentration (~1.5 micromolar) for grid preparation.
緩衝液成分
ID濃度名称式Buffer-ID
120 mMSodium HEPESNa-HEPES1
2450 mMSodium ChlorideNaCl1
32 mMDithiothreitolDTT1
40.5 mMMagnesium ChlorideMgCl21
50.5 mMAdenosine triphosphateATP1
試料濃度: 0.48 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
詳細: The sample was monodisperse. Side views were more electron weak than top or bottom views creating challenges for particle picking. This issue was overcome with cryo-electron tomography ...詳細: The sample was monodisperse. Side views were more electron weak than top or bottom views creating challenges for particle picking. This issue was overcome with cryo-electron tomography techniques used for 1) initial model generation and 2) template generation for particle picking.
試料支持詳細: The grid was coated with 50 nm of evaporated gold prior to use. All remaining carbon was removed by plasma cleaning for 5 minutes in a Gatan Solarus plasma cleaner.
グリッドの材料: GOLD / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R0.6/1
急速凍結装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277.15 K
詳細: 3uL of sample was adhered to a fresh plasma cleaned grid and allowed to adsorb for 30 seconds, blotted for 3 seconds with a blot force of 4 and plunge frozen into liquid nitrogen-cooled ethane.

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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
顕微鏡モデル: FEI TITAN KRIOS
詳細: Preliminary grid screening was performed prior to Krios data collections. All microscope alignments were completed by the New York Structural Biology SEMC team.
電子銃電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM
電子レンズモード: BRIGHT FIELD / 倍率（公称値）: 22500 X / 最大 デフォーカス（公称値）: -3 nm / 最小 デフォーカス（公称値）: -1 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: COMA FREE
試料ホルダ凍結剤: NITROGEN
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
最高温度: 70 K / 最低温度: 70 K
撮影平均露光時間: 10 sec. / 電子線照射量: 8 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)
撮影したグリッド数: 3 / 実像数: 2426
詳細: Single particle movies were recorded at a pixel size of 1.07 angstroms/pixel. Three 24-hour sessions produced 2,426 micrograph movies. In addition, five tilt series were collected from the ...詳細: Single particle movies were recorded at a pixel size of 1.07 angstroms/pixel. Three 24-hour sessions produced 2,426 micrograph movies. In addition, five tilt series were collected from the same grids bi-directionally over a tilt range of -45 degrees to +45 degrees in 3 degree increments at a dose of 2.57 to 3.3 electrons per angstrom squared (total accumulated dose of 90 electrons per angstrom squared). Tilt series were collected at a pixel size of 1.76 angstroms and at defocus values of -2.8um, -6.1um and -9.3um.
電子光学装置球面収差補正装置: The Krios this data was collected on has a Cs of 2.7.
画像スキャン横: 3838 / 縦: 3710 / 動画フレーム数/画像: 50 / 利用したフレーム数/画像: 1-50

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解析

ソフトウェア名称: PHENIX / バージョン: 1.14_3260: / 分類: 精密化
EMソフトウェア
ID名称バージョンカテゴリ詳細
1Leginon0画像取得Leginon was used for automatic hole targeting during single particle movie collection.
2Appion0画像取得Single particle micrograph movies were aligned on-the-fly using MotionCorr in Appion.
3Appion0画像取得Tilt angle
5GctfCTF補正Gctf was used to estimate CTF for summed micrographs.
8MOLREPモデルフィッティングMolrep was used to unambiguously assign six DnaB CTD and six DnaB NTD homology models into our BP map.
9CootモデルフィッティングCoot was used to manually build 5 lambda P protomers and five DnaB NTD-CTD linkers
11RELIONseries alignment2D and 3D classifications and auto-refinement were performed in Relion.
12Gautomatchseries alignmentTemplate-based particle picking was performed with Gautomatch.
13EMAN2series alignmentEMAN2 was used to project tomography-derived initial model for templates used in auto picking procedures.
14RELION３次元再構成Auto refine and post processing was performed in Relion.
16TOMO3Dseries alignmentTomo3D was used to reconstruct tomograms from aligned images.
17Dynamoseries alignmentDynamo was used to pick and align particles from tomograms.
18EMAN2series alignmentEMAN2 was used to generate 2D projections of sub-tomogram average.
19Dynamo３次元再構成Dynamo was used to generate sub-tomogram average, later used as initial model.
20PHENIXモデル精密化Phenix real_space_refine was used to assess model validity.
画像処理詳細: Images collected were CTF corrected and selected based on CTF estimates of less than 10 angstrom at a confidence cutoff of 0.8 for subsequent processing.
CTF補正詳細: CTF correction was performed before data processing on summed micrographs using Gctf.
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: 90883
詳細: Relion was used to independently refine half sets using 0.143 gold-standard to a resolution of 4.1A. A total of 90,883 particles went into this reconstruction.
原子モデル構築プロトコル: FLEXIBLE FIT / 空間: REAL
詳細: The initial fitting was done with the 2R5U and 3BH0 models onto which the E. coli amino sequence had been built. The linker segments that connected these segments were built by hand. PHENIX ...詳細: The initial fitting was done with the 2R5U and 3BH0 models onto which the E. coli amino sequence had been built. The linker segments that connected these segments were built by hand. PHENIX real_space_refine was used to refine the complete model for the B6P5 entity.
原子モデル構築

3D fitting-ID: 1 / Source name: PDB / タイプ: experimental model

IDPDB-IDAccession codeInitial refinement model-IDPdb chain residue range
12R5U

2r5u

2R5U11-173
23BH0

3bh0

3BH02203-441

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