EMDB-28841: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter ...

基本情報

登録情報

データベース: EMDB / ID: EMD-28841

• タイトル: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Classification and subtomogram averaging - Class 5
• マップデータ: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Classification and subtomogram averaging - Class 5

試料

• 細胞器官・細胞要素: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Class 5
  • Other: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain

• キーワード: Extracellular matrix protein adhesin A Bacterial Adhesin Glycosylated protein Trimeric Autotransporter / CELL ADHESION
• 生物種: Aggregatibacter actinomycetemcomitans (バクテリア)
• 手法: サブトモグラム平均法 / ネガティブ染色法 / 解像度: 14.5 Å
• データ登録者: Ruiz T / Radermacher M / Mintz KP / Tang-Siegel GG

資金援助

米国, 3件

Organization	Grant number	国
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)	DE024554	米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	GM078202	米国
National Science Foundation (NSF, United States)	DBI 1660908	米国

• 引用: ジャーナル: J Bacteriol / 年: 2022; タイトル: Serotype-Specific Sugars Impact Structure but Not Functions of the Trimeric Autotransporter Adhesin EmaA of Aggregatibacter actinomycetemcomitans.; 著者: Gaoyan G Tang-Siegel / Michael Radermacher / Keith P Mintz / Teresa Ruiz / ; 要旨: The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors, including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with -polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202-kDa protein was expressed in a non-serotype b, mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by two-dimensional (2D) electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were posttranslationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII, and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes, however, did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. The human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for colonization by this pathobiont . This adhesin is modified with sugars associated with the -polysaccharide (O-PS), and the modification is mediated using the enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the posttranslational modification of this adhesin is exchangeable without compromising functional activities.

履歴

登録	2022年11月9日	-
ヘッダ(付随情報) 公開	2022年11月23日	-
マップ公開	2022年11月23日	-
更新	2024年1月17日	-
現状	2024年1月17日	処理サイト: RCSB / 状態: 公開

マップ

• ファイル: ダウンロード / ファイル: emd_28841.map.gz / 形式: CCP4 / 大きさ: 25.8 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• 注釈: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Classification and subtomogram averaging - Class 5
• ボクセルのサイズ: X=Y=Z: 3.08 Å

密度

表面レベル	登録者による: 0.00117
最小 - 最大	-0.0008794135 - 0.0055044703
平均 (標準偏差)	0.0000042745205 (±0.00009586966)

• 対称性: 空間群: 1

詳細

EMDB XML:

マップ形状	Axis order X Y Z Origin 0 0 0 サイズ 189 189 189 Spacing 189 189 189
セル	A=B=C: 582.12 Å α=β=γ: 90.0 °

添付データ

ハーフマップ: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter...

• ファイル: emd_28841_half_map_1.map
• 注釈: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Half-Map-Odd - Class 5

ハーフマップ: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter...

• ファイル: emd_28841_half_map_2.map
• 注釈: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Half-Map-Even - Class 5

試料の構成要素

全体 : EmaA (extracellular matrix protein adhesin A) of Aggregatibacter ...

• 全体: 名称: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Class 5

要素

• 細胞器官・細胞要素: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Class 5
  • Other: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain

超分子 #1: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter ...

• 超分子: 名称: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain expressed in a serotype a strain - Class 5; タイプ: organelle_or_cellular_component / ID: 1 / 親要素: 0 / 含まれる分子: all
• 由来(天然): 生物種: Aggregatibacter actinomycetemcomitans (バクテリア)

分子 #1: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter ...

• 分子: 名称: EmaA (extracellular matrix protein adhesin A) of Aggregatibacter actinomycetemcomitans serotype b strain; タイプ: other / ID: 1 / 分類: other
• 由来(天然): 生物種: Aggregatibacter actinomycetemcomitans (バクテリア)

配列

文字列:

MNKVFKVIWC KTSQTWIAVS ELSKAFSLST TTDIPKKTKI FIAAAPLLFL SFNTNAYIAI GSVENNSVK SEGAEASPNK RKGSQALNYY NPGSKSYDDK DKPSNPERRY SNGEAYGIAI G KNTDVRDS SKDSNGIALG DYSKATGGLA MALGSFSRAE KNGGIAIGIA SRSSGINSLA MM RQSAATG DYSTAIGSVA WAAGQSSFAL GASATAKGNQ SIAIGSLEQK ISPNGSGVPI TKY NGLDNT QTNGNRSMAL GTAAKTNGDD SFAIGYKAHT GEFKVEHDNY LKENVTSPDL SKKA DKAIA VGTSALAQKE SAIAFGYQAN ASGINAISLG ANAKASQDNV VAIGKDATAT ESGSM AIGQ GAKSTFKNSL ALGTGTIVNS VDGGQSKFTA QNYDANNGVV AVANAGKERR IINVAG GRN DTDAVNVAQL KFVNDNLAKS IAGAGYNGYE TDGHTYKAPV FSIKNTNYHD VKTAVEA AQ TNYVSVNSTN TAADSNYDNK GAKAVGSIAL GEKATTGRAA MNSIAIGLNS NVSGQNTV A LGANITATTN GSVILGNSST TEGSHPVSNV SSATVNGYTY SGFTGTVKES GHFVSIGSK GNERQIKNVA AGNVAANSTD AVNGSQLFAV ASRVEQGWQI TSGVENGGTQ NGAASTATIK PSNQVKLLA GKNLAVKQNG TNFTFSTQEN VTFTNVTTQD LTATGNTTVK NFSVQNGGTI N MGNNRITG VAEGTQDDDA VNFKQLKSLL GGSASTEIVE KKAAQAGDEN LADISVANGK NA GDMGAKY EVSVSKKAVQ SAAKEAVKVT GSAPINVNKT DVNGVDTYAV TFNGTEAAKS IPL TYKANG SGDKTVMLDK GLNFTNGMMT TASVANDGVM KYDVNLSTIK VEDGKAAVAG TPGT NGANG TDGKDGVATV KNVVEALNNA AWTITASKSD GEVVSNASNS VKNGDTVTYD AGKNI KITQ RDKKFSFATK DNVEFTSVTT GNTKLTGNGV EITNGPKLTQ SGVDAGGKKI TNVADG VIA ANSKDAVNGG QLFAETAKAK TTVEKGDDNI QITSETATDG HINYKVALNP SLTVGPR TN GHPITIDGNN GYITGLTNTS WTGAPTTGRA ATEDQLSIVD KKFDNKVSLG GDNGSTTE K SLSHNGGIKF NIKGGDSQKY VTTSGSGDDV TVDLAQTTKN KIDNAADKDL ANITDNGKK VITALGAVVK AADSTITVTD ETDNTTGQKT YKIKANIPTP EKTAMAPGNN TTIEGDGSAA NPFKVNLKD DLALGQKDAN GVTGKDSSIK VNGKDGSGVA INGKDGSIAL NGKDGANPVT I KTAQGPAG VNETNPKDRL MVNNDAVATL KDGLKFAGDN STEVITKTLN QKLEIVGGAD KN KLSDNNI GVNANNGKLE VKLAKELNEL TSAQFKNGDN TTVINGNGIT ITPKDPTKAV SLT DKGLNN GGNQIVNIDS GLKQADGSTV ALKDASGDTL KNAANIGDLQ KSINDITDAS KNGG FGLSD DNGATAKANL GETVKVKGDG SVITKVVTDN GKPTLQVGLS NDITVGDDAQ AGTIS VKGE NGKDGVSING KEASVTFAKD GQPGMSIAAT RSADGKDALT LKGKDGKDGI SFQEDG RIT QVADGVNDKD AVNKSQLDRS IAQAKSGVSA GKNITVTPQK NADGSTTYTV ETQKDVE FS TVKTGDTTLD SNGVNINGGP SVTKDGIHAN DKKITGVKDG EISAHSKEAV NGSQLHQT N QNVTNLANNV DKGLNFQGDN QEVTVNRKLG DQLNIRGGAD PKKLTQNNIG VTADKNGTM TVQLAKEVNL GADGSLTVGN TTVNNDGVTI KDGPSMTSHG INAGGKRIAN VAKGKAPTDA VNMSQLQDV GSAINNRIDN IDKRVKKMDK RRKAGTASAL ATAGLMQPHR DGQSALVAAV G QYQSETAV AVGYSRISDN GKYGVKVSFS TNSQGEVGGT AGAGYFW

実験情報

構造解析

• 手法: ネガティブ染色法
• 解析: サブトモグラム平均法
• 試料の集合状態: cell

試料調製

緩衝液

pH: 7.4
構成要素:

濃度	名称	式
10.0 mM	Sodium Phosphate
150.0 mM	Sodium Chloride	NaCl

• 染色: タイプ: NEGATIVE / 材質: NanoW; 詳細: Electron microscopy grids were prepared as previously described. Briefly, a 5 ul aliquot of bacterial suspension was placed on either 300 or 200 mesh carbon-coated grids, and deep stained with NanoW (Nanoprobes, Yaphank, NY). For 3D electron tomography, the grids were pretreated with Poly-L-lysine (1000-5000 Da. Sigma, St. Louis, MO) and colloidal gold (SPI, West Chester, PA) to be used as fiducial markers.
• グリッド: モデル: Homemade / 材質: COPPER / メッシュ: 200 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: CONTINUOUS / 前処理 - タイプ: PLASMA CLEANING / 前処理 - 時間: 20 sec. / 前処理 - 雰囲気: OTHER

電子顕微鏡法

• 顕微鏡: FEI TECNAI 12
• 撮影: フィルム・検出器のモデル: TVIPS TEMCAM-F216 (2k x 2k); 実像数: 80 / 平均電子線量: 3.0 e/Ų; 詳細: Data were collected using a Tecnai 12 electron microscope (FEI, Hillsboro, OR) equipped with a LaB6 cathode (Kimball Physics, Wilton, NH), operated in point-mode, a 2048 by 2048 pixel CCD camera with a pixel size of 14 um, (TVIPS, Gauting, Germany) and a dual axis tilt tomography holder (Fischione, Export, PA). All images were recorded on the CCD camera at an acceleration voltage of 100 kV and a nominal magnification of 42,000, which corresponds to 0.308 nm pixel size on the specimen scale. Tomographic tilt series were acquired at least within a +/-64 degree angular range in 2 degree angular intervals. Data were collected under low dose exposure conditions (0.10 e-/nm2 for 2D imaging and 0.03 e-/nm2 per image for the tomographic tilt series data) as previously described.
• 電子線: 加速電圧: 100 kV / 電子線源: LAB6
• 電子光学系: 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 最大 デフォーカス（公称値）: 1.6 µm / 最小 デフォーカス（公称値）: 1.3 µm / 倍率（公称値）: 42000
• 試料ステージ: 試料ホルダーモデル: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER; ホルダー冷却材: NITROGEN

画像解析

• 最終 再構成: 使用したクラス数: 8 / 想定した対称性 - 点群: C₁ (非対称) / アルゴリズム: FOURIER SPACE / 解像度のタイプ: BY AUTHOR / 解像度: 14.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: EMIRA (ver. 1.1.4); 詳細: Final 3D structures were calculated by de novo reconstruction from 2D Radon transforms of the combined subprojections of all subvolumes in each group. Class 5 (of 8 classes) was reconstructed using 17 subtomograms. In addition, to assess variability within groups, average subvolumes and variances were calculated from the reconstituted subvolumes after processing with PPCA-EM, which results in reconstituted subvolumes with the missing data filled in. The resolution for the average subvolume of each class was calculated using Fourier shell correlation methods. The subprojections of each subvolume belonging to a class were divided into two groups (odd and even) and combined to calculate two subvolumes per class. The Fourier shell correlation was calculated using Spider and the 0.143 nm resolution criterion was used to determine resolution.; 使用したサブトモグラム数: 151
• 抽出: トモグラム数: 84 / 使用した粒子像数: 151 / 手法: Manual / ソフトウェア - 名称: IMOD; 詳細: Tomographic tilt series were processed using IMOD. Projections were initially aligned by cross correlation and further refined using fiducial markers located outside of the bacterial surface. EmaA functional domains were orientationally selected from the tomograms by marking two points along their long axis with the first point located at the tip of the adhesin.
• 最終 3次元分類: クラス数: 8 / 平均メンバー数/クラス: 18 / ソフトウェア - 名称: EMIRA (ver. 1.1.4) / ソフトウェア - 詳細: PPCA-EM; 詳細: Subvolumes were visualized in Chimera and further aligned to a reference subvolume of the wild-type EmaA using the "fit in map" command. Angles and shifts were applied to the subprojections and aligned subvolumes were reconstructed. The aligned subvolumes were grouped using Probabilistic Principle Component Analysis with Expectation Maximization (PPCA-EM), an algorithm to analyze 3D volumes with missing data, followed by clustering using Diday's method of moving centers as implemented in EMIRA. Outliers (with sigma of 5) were identified and excluded from this stage of the analysis. A representative of each group was selected, and all representatives were aligned to each other. The members of each group were subsequently realigned to the aligned representative subvolume using the "fit in map" command in Chimera. Angles and shifts were applied to the subprojections and new aligned subvolumes were reconstructed. The newly aligned subvolumes were grouped anew using PPCA-EM followed by non-linear mapping and Diday's method of moving centers for clustering.
• 最終 角度割当: タイプ: OTHER / ソフトウェア - 名称: EMIRA (ver. 1.1.4); 詳細: For each selected molecule, a tilt series of subprojections was extracted, subprojection angles were recalculated and subvolumes were reconstructed with the molecules long-axis oriented approximately parallel to the Y-axis using algorithms implemented in EMIRA. The 3D reconstruction algorithms based on Radon transforms, as implemented in EMIRA, contain an occupancy index that keeps track of the number of 2D transforms averaged into it and is used to determine the location of the missing data