저자들은 오스트레일리아 싱크로트론의 MX2 빔라인에서의 지원을 위해 정화 된 미세 결정, Daniel Eriksson 및 Tom Caradoc-Davies의 사진을 제공 한 Chan-Sien Lay과 Monash University의 FlowCore 시설에서 나온 Kathryn Flanagan 및 Andrew Fryga 귀중한 도움.

<ol>
	<li>Tari, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+structural+biology+in+drug+discovery.">The utility of structural biology in drug discovery.</a> Methods Mol Bio (Clifton, N.J). 841, 1-27 (2012).</li><li>Gati, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+crystallography+on+in+vivo+grown+microcrystals+using+synchrotron+radiation.">Serial crystallography on in vivo grown microcrystals using synchrotron radiation.</a> IUCrJ. 1 (2), (2014).</li><li>Redecke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natively+inhibited+Trypanosoma+brucei+cathepsin+B+structure+determined+by+using+an+X-ray+laser.">Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.</a> Science. 339 (2013), 227-230 (2013).</li><li>Shi, D., Nannenga, B. L., Iadanza, M. G., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+electron+crystallography+of+protein+microcrystals.">Three-dimensional electron crystallography of protein microcrystals.</a> eLife. 2, e01345 (2013).</li><li>Evans, G., Axford, D., Waterman, D., Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+microcrystallography.">Macromolecular microcrystallography.</a> Crystallog. Rev. 17 (2), 105-142 (2011).</li><li>Smith, J. L., Fischetti, R. F., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-crystallography+comes+of+age.">Micro-crystallography comes of age.</a> Curr Opin Struct Biol. 22 (5), 602-612 (2012).</li><li>Boudes, M., Garriga, D., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+the+Many+Facets+of+Protein+Microcrystallography.">Reflections on the Many Facets of Protein Microcrystallography.</a> Aust J Chem. 67 (12), 1793-1806 (2014).</li><li>Nelson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cross-beta+spine+of+amyloid-like+fibrils.">Structure of the cross-beta spine of amyloid-like fibrils.</a> Nature. 435 (7043), 773-778 (2005).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+organization+of+cypovirus+polyhedra.">The molecular organization of cypovirus polyhedra.</a> Nature. 446 (7131), 97-101 (2007).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atomic+structure+of+baculovirus+polyhedra+reveals+the+independent+emergence+of+infectious+crystals+in+DNA+and+RNA+viruses.">The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.</a> Proc Natl Acad Sci U S A. 106 (52), 22205-22210 (2009).</li><li>Johansson, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+photosynthetic+reaction+centre+determined+by+serial+femtosecond+crystallography.">Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography.</a> Nat Comm. 4, (2013).</li><li>Li, D., Boland, C., Aragao, D., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+Cryo-cooling+Crystals+of+Membrane+Proteins+Grown+in+Lipidic+Mesophases+for+Structure+Determination+by+Macromolecular+Crystallography.">Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography.</a> J Vis Exp. (67), (2012).</li><li>Roedig, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micro-patterned+silicon+chip+as+sample+holder+for+macromolecular+crystallography+experiments+with+minimal+background+scattering.">A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.</a> Sci Rep. 5, 10451 (2015).</li><li>Koopmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+crystallization+opens+new+routes+in+structural+biology.">In vivo protein crystallization opens new routes in structural biology.</a> Nature Methods. 9 (3), 259-262 (2012).</li><li>Gallat, F. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+crystallography+at+X-ray+free-electron+lasers:+the+next+generation+of+structural+biology.">In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology.</a> Phil Trans R Soc B. 369 (1647), 20130497 (2014).</li><li>Duszenko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+crystallization+in+combination+with+highly+brilliant+radiation+sources+offers+novel+opportunities+for+the+structural+analysis+of+post-translationally+modified+eukaryotic+proteins.">In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.</a> Acta Cryst. F. 71 (8), 929-937 (2015).</li><li>Axford, D., Ji, X., Stuart, D. I., Sutton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+cellulo+structure+determination+of+a+novel+cypovirus+polyhedrin.">In cellulo structure determination of a novel cypovirus polyhedrin.</a> Acta Cryst D. 70 (5), 1435-1441 (2014).</li><li>Sawaya, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystal+structure+obtained+at+2.9+&#197;+resolution+from+injecting+bacterial+cells+into+an+X-ray+free-electron+laser+beam.">Protein crystal structure obtained at 2.9 &#197; resolution from injecting bacterial cells into an X-ray free-electron laser beam.</a> Proc Natl Acad Sci U S A. 111 (35), 12769-12774 (2014).</li><li>Tsutsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Diffraction-Quality+Protein+Crystal+Processed+as+an+Autophagic+Cargo.">A Diffraction-Quality Protein Crystal Processed as an Autophagic Cargo.</a> Mol Cell. 58 (1), 186-193 (2015).</li><li>Boudes, M., Garriga, D., Fryga, A., Caradoc-Davies, T., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pipeline+for+structure+determination+of+in+vivo-grown+crystals+using+in+cellulo+diffraction.">A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.</a> Acta Cryst D. 72, 576-585 (2016).</li><li>Doye, J. P. K., Poon, W. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+in+vivo.">Protein crystallization in vivo.</a> Curr Opin Colloid Interface Sci. 11 (1), 40-46 (2006).</li><li>Mori, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Bombyx+mori+cytoplasmic+polyhedrosis+virus+polyhedrin+in+insect+cells+by+using+a+baculovirus+expression+vector,+and+its+assembly+into+polyhedra.">Expression of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin in insect cells by using a baculovirus expression vector, and its assembly into polyhedra.</a> J Gen Virol. 74, 99-102 (1993).</li><li>Arevalo, M. T., Wong, T. M., Ross, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+Purification+of+Virus-like+Particles+for+Vaccination.">Expression and Purification of Virus-like Particles for Vaccination.</a> J Vis Exp. (112), (2016).</li><li>Yates, L. A., Gilbert, R. J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Production+and+Purification+of+Recombinant+Murine+Kindlin-3+from+Insect+Cells+for+Biophysical+Studies.">Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies.</a> J Vis Exp. (85), (2014).</li><li>Berger, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MultiBac+Protein+Complex+Production+Platform+at+the+EMBL.">The MultiBac Protein Complex Production Platform at the EMBL.</a> J Vis Exp. (77), (2013).</li><li>Margine, I., Palese, P., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Functional+Recombinant+Hemagglutinin+and+Neuraminidase+Proteins+from+the+Novel+H7N9+Influenza+Virus+Using+the+Baculovirus+Expression+System.">Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System.</a> J Vis Exp. (81), (2013).</li><li>Khurana, A., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+For+Production+of+Recombinant+mCD1d+Protein+in+Insect+Cells.">A Method For Production of Recombinant mCD1d Protein in Insect Cells.</a> J Vis Exp. (10), (2007).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. (16), (2008).</li><li>Baskaran, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+cellulo-derived+structure+of+PAK4+in+complex+with+its+inhibitor+Inka1.">An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1.</a> Nat Comm. 6, 1-11 (2015).</li><li>Armour, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-target+Parallel+Processing+Approach+for+Gene-to-structure+Determination+of+the+Influenza+Polymerase+PB2+Subunit.">Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit.</a> J Vis Exp. (76), (2013).</li><li>Otwinowski, Z., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+X-ray+diffraction+data+collected+in+oscillation+mode.">Processing of X-ray diffraction data collected in oscillation mode.</a> Methods Enzymol. 276, 307-326 (1997).</li><li>Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R., Leslie, A. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iMOSFLM:+a+new+graphical+interface+for+diffraction-image+processing+with+MOSFLM.">iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.</a> Acta Cryst D. 67, 271-281 (2011).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Cryst D. 66, 125-132 (2010).</li><li>French, S., Wilson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+treatment+of+negative+intensity+observations.">On the treatment of negative intensity observations.</a> Acta Cryst.A. 34, 517-525 (1978).</li><li>Winn, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+CCP4+suite+and+current+developments.">Overview of the CCP4 suite and current developments.</a> Acta Cryst D. 67, 235-242 (2011).</li><li>Foadi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustering+procedures+for+the+optimal+selection+of+data+sets+from+multiple+crystals+in+macromolecular+crystallography.">Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.</a> Acta Cryst D. 69 (8), 1617-1632 (2013).</li><li>Rey, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virology:+holed+up+in+a+natural+crystal.">Virology: holed up in a natural crystal.</a> Nature. 446 (7131), 35-37 (2007).</li><li>Sch&#246;nherr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+investigation+of+dynamic+protein+crystallization+in+living+cells.">Real-time investigation of dynamic protein crystallization in living cells.</a> Struct Dyn. 2 (4), 041712 (2015).</li><li>Hasegawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+crystallization+of+human+IgG+in+the+endoplasmic+reticulum+of+engineered+Chinese+hamster+ovary+(CHO)+cells.">In vivo crystallization of human IgG in the endoplasmic reticulum of engineered Chinese hamster ovary (CHO) cells.</a> J Biol Chem. 286 (22), 19917-19931 (2011).</li><li>Ishchenko, A., Cherezov, V., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Delivery+of+Protein+Microcrystals+in+Lipidic+Cubic+Phase+for+Serial+Femtosecond+Crystallography.">Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.</a> J Vis Exp. (115), e54463 (2016).</li><li>Zander, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MeshAndCollect:+an+automated+multi-crystal+data-collection+workflow+for+synchrotron+macromolecular+crystallography+beamlines.">MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines.</a> Acta Cryst. D. 71, 2328-2343 (2015).</li><li>Fan, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calcineurin+crystals+formed+using+the+baculovirus+expression+system.">In vivo calcineurin crystals formed using the baculovirus expression system.</a> Micros Res Tech. 34 (1), 77-86 (1996).</li><li>Nass, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Investigation of protein structure determination using X-ray free-electron lasers. , (2013).</li></ol>

저자는 공개 할 것이 없습니다.

이 프로토콜은 과거에 간과되었을 작은 결정의 분석을 용이하게하기 위해 미정을 분석하는 두 가지 접근법을 제공합니다. 미세 결정 정제를위한 중요한 단계 제시된 프로토콜은 Sf9 세포에서 발현 된 Bombyx mori CPV1 폴리 헤드린을 모델 시스템으로 사용하여 최적화 하였다. 그러나, 생체 내 미결정은 기계적 저항성에 큰 변화를 보인다. 예를 들면, 곤충 세포에서 성장 카 텝신 B의 침상 결정은 강성 및 기계적 스트레스에 매우 내성, 여기 14 설명한 것과 유사한 프로토콜을 사용하여 정제 할 수있다. 한편, 또한 배양 된 곤충 세포에서와 유사한 침상 형태로 성장 반딧불이 루시퍼 결정, 즉시 세포 용해 (38)에 용해. 따라서, 생체 내 추출 및 정제 프로토콜수정은 단계 1.3, 2a.3-4, 2b.4, 3.1.3-4 및 3.2a.5에서 설명한 특정 경우에 시행 착오를 거쳐 조정될 필요가있다. 깨지기 쉬운 결정의 경우 세포 파편으로부터 (부분) 분리는 저속 원심 분리 ( 즉, 200 xg) 또는 반복 된 초음파 처리 대신 튜브 바닥에 자당 쿠션을 추가하여 이루어질 수 있습니다. 일반적으로, 회절 데이터 수집 목적으로 준비된 결정 샘플은 고도로 정제 될 필요가 없다. 따라서 결정이 서서히 냉각되기 전에 급속 정제가 바람직합니다. 생체 외 및 생체 내 미세 결정의 응용 프로그램 두 프로토콜은 생체 내 성장 결정의 분석과 함께 여기에 설명되어 있지만, 정제 된 재조합 단백질의 크리스탈 트레이에서 성장한 미세 결정에이 방법을 쉽게 사용할 수 있습니다. 티에다음의 변경 사항을 고려해야한다. i) 메쉬 지지체는 결정화 방울로부터 미생물을 직접 채취하는데 사용될 수있다. ii) 결정의 양이 제한 요인이 될 수 있기 때문에, 과도한 결정 제거를 피하기 위해 메쉬 지지대에서 초과 된 액체를 닦을 때 특별한주의가 필요하다. iii) 기존의 결정학 (참고 문헌 30 참조)에서와 같이 다른 동결 방지 완충액을 테스트해야합니다. 반면 에 생체 내 성장 결정체의 경우 셀룰로오스 방식이 강력하게 권장됩니다. 세포는 정제 된 결정보다 시각화하고 조작하기가 더 쉽기 때문에이 접근법은 빔 시간 사용 측면에서 더 효율적이며 미세 결정학에 거의 또는 전혀 경험이없는 연구자가 더 쉽게 접근 할 수 있습니다. 그것은 또한 추출 및 pur의 영향을받을 수있는 깨지기 쉬운 결정에 더 적합합니다<html> 화학적 환경의 변화, 주변 단백질의 희석 및 기계적 손상으로 인한 세포로부터의 감염. 게다가, 세포는 결정에 보호 환경을 제공하여 동결 방지제를 추가 할 필요성을 줄여줍니다. cryoprotection 처리 생략 이전 결함의 누적을 방지하기 위해 도시하고, 실온에서 40 데이터 수집 개별 결정의 동형을 개선되었다. 셀룰로오스 프로토콜은 또한 극저온에서 데이터 수집을 허용하면서 동결 방지 솔루션에서 인큐베이션에 대한 요구 사항을 우회합니다. 전반적 cellulo 데이터 컬렉션의 두 가지 장점 갖는다 : 1)이 beamtime (20)의 소정의 기간 동안 더 나은 품질과 더 높은 해상도의 데이터를 생성 할 수있다 단계; 2) 세포 환경에서 결정화 된 단백질을 유지하여 생물학적으로 관련된 리간드를 유지할 가능성을 높입니다. ontent &quot;&gt; 기술의 한계 직렬 미세 결정의 본질적인 복잡성은 데이터 수집 중에 생성되는 부분 데이터 세트의 중요한 수입니다. 결과적으로, 데이터 처리는 점점 더 많은 시간이 소요되는 프로세스가됩니다. 그러나 데이터 처리를위한 기본 워크 플로는 대부분 자동화 할 수 있습니다. 예를 들어, 동형 결정의 최적 선택은 계층 적 클러스터 분석 ( 28 )에 의해 결정될 수 있는데, 예를 들어 BLEND ( 36)에 구현된다. 또한, X 선 자유 전자 레이저 (XFEL) 분석을 위해 개발 된 프로그램은 싱크로트론 빔 라인 2 에서 수집 된 일련의 미세 결정 데이터를 처리하고, 일부 빔 라인에서는 메쉬 및 크리스털 탐지의 자동 래스터 화가 크게 구현되어 일련 번호 데이터 수집 2 , 41 .

생물 고분자의 고해상도 구조 결정을위한 X 선 결정학의 사용은 지난 20 년간 꾸준히 발전해 왔습니다. 전문가가 아닌 연구자에 의한 X 선 결정학 (X-ray crystallography)의 증가 추세는 생명 과학 분야의 많은 분야에서이 접근법의 민주화를 보여줍니다 1 . 역사적으로 ~ 10 μm 이하의 크기를 가진 결정은 구조 결정을 위해 사용하기에 어렵지 않은 경우 도전적인 것으로 간주되었습니다. 싱크로트론 방사선 소스의 전용 미세 초점 빔라인의 가용성 증가와 미세 결정 조작 도구 개발과 같은 기술적 진보로 인해 X 선 미세 결정의 폭 넓은 사용을 방해하는 이러한 장벽이 제거되었습니다. 직렬 X 선 마이크로 결정학 2 , 3 및 마이크로 전자 회절 4 ha의 발전구조 결정을위한 마이크로 및 나노 결정의 사용이 가능할뿐만 아니라 때로는 큰 결정 5 , 6 , 7 의 사용보다 바람직하다는 것을 보여 주었다. 이러한 발전은 제 8 펩티드 곤충 바이러스 9, 10에 의해 생성 된 천연 결정의 연구에 적용 하였다. 이들은 이제 막 단백질 복합체 크고 11 가장 어려운 시스템을 포함하는 생물학적 고분자의 다양한 사용된다. 이 미세 결정의 분석을 용이하게하기 위해 meso , 특히 멤브레인 단백질 12 와 마이크로 유체 칩 13 에서 분석되었습니다. 이러한 신규 microcrystallography 방법론의 가용성에 사용할 수있는 가능성을 제기in vivo 결정학에 대한 대안을 제공하는 구조 생물학 14 , 15 , 16 의 새로운 경로로서의 생체 내 결정화. 불행하게도, 생체 내 결정체가 생성 될 수있을지라도, 세포로부터의 정제 동안 리간드의 분해 또는 손실, 싱크로트론 빔라인에서의 수정 및 시각화의 어려움 및 지루한 X- 선 회절 실험과 같은 몇 가지 장애물이 남아있다. 대안으로 결정은 정제 단계 17 , 18 , 19 없이 셀 내에서 직접 분석되었습니다. 비교 분석은 cellulo 접근 그러한 정제 된 결정과 높은 해상도 (20)의 수율 데이터의 분석을보다 더 효율적이 될 수 있음을 시사한다. 이 프로토콜은단백질 미세 결정학에 새로운 연구원을 돕는 경향이 있었다. 싱크로트론 빔라인에서 X 선 회절 실험을위한 시료 준비 및 조작에 초점을 맞춘 방법론을 제공합니다. 두 가지 옵션이 고전 microcrystallography 또는 cellulo 분석 ( 그림 1 )에 대한 유동 세포 계측법에 의해 정렬 크리스탈 포함 세포에 대한 격리 된 결정을 사용하여 제안하고 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

microcrystallography of protein crystals and in cellulo diffraction

많은 싱크로트론 설비에서 고품질의 미세 초점 빔라인이 출현함에 따라 도전을 대표하는 가장 큰 차원에서 10μm보다 작은 결정의 일상적인 분석이 가능 해졌다. 우리 는 생체 내에서 성장한 결정에 특별한 초점을 맞춘 X 선 결정학에 의한 단백질 미결정의 구조 결정을위한 두 가지 대안 적 작업 흐름을 제시합니다. 미세 결정은 초음파 처리에 의해 세포로부터 추출되고 차등 원심 분리에 의해 정제되거나 결정 함유 세포의 유동 세포 계측법에 의한 세포 분류 후에 셀룰로오스로 분석된다. 선택적으로, 정제 된 결정 또는 결정 - 함유 세포를 실험적 단계적 조정을 위해 중질 원자 용액에 침지시킨다. 이 샘플들은 액체 질소에서의 마이크로 메쉬지지 및 플래시 냉각에 대한 적용에 의해 유사한 방식으로 회절 실험을 위해 준비됩니다. 격리 된 미세 결정과 수정 결정의 연속 회절 실험을 간략하게 기술하고 비교한다.microfocus 싱크로트론 빔라인을 사용하여 셀을 포함하는 셀을 조정하여 위상 조정, 모델 구축 및 미세 조정에 적합한 데이터 세트를 생성합니다. 이러한 워크 플로우는 곤충 세포에 재조합 배큘로 바이러스 (baculovirus)가 감염되어 생성 된 Bombyx mori cypovirus 1 (BmCPV1) 폴리 헤드린의 결정체를 예로들 수 있습니다. 이 사례 연구 에서 셀룰로오스 분석은 정제 된 결정의 분석보다 효율적이며 표현에서부터 정제까지 ~ 8 일 내에 구조를 산출합니다.

생체 내 미세 결정을 이용한 구조 결정을위한 두 가지 대체 방법의 개요가 제시됩니다 ( 그림 1 ). Polyhedra는 초음파 처리 및 원심 분리로 쉽게 정제 할 수 있습니다. 밀도 때문에, 그들은 pipetting ( 그림 3a 와 3b )에 의해 제거 할 수있는 파편 층 밑에 튜브의 바닥에 레이어를 형성합니다. 그런 다음 샘플을 여러 차례의 초음파 처리하고 펠렛의 흰색 및 백악면으로 판단 할 때 충분한 순도에 도달 할 때까지 세척합니다 ( 그림 3c ). in cellulo approach의 경우, 감염되지 않은 세포의 유동 세포 계측 프로파일을 결정을 함유하는 세포의 프로파일과 비교하고, 결정 함유 세포를 멀리 떨어 뜨려 분류 할 세포의 수를 결정하는데 사용합니다다른 세포 ( 그림 4 ). 이 예에서 다면체를 포함하는 세포는 감염되지 않은 세포보다 더 높은 측면 산란을 보입니다. 산란 패턴의 다른 차이가 다른 결정 유형에서 관찰 될 수 있으며 이에 따라 게이팅이 수정되어야합니다. 정제 된 미세 결정 또는 결정을 함유하는 세포를 마이크로 메쉬에 피펫 팅합니다 ( 그림 5 ). trypan blue로 염색 된 세포는 싱크로트론 빔라인의 대부분에서 사용 가능한 카메라를 사용하여 쉽게 시각화 될 수 있으며 ( 그림 5b ), 정제 된 크리스털은 크기가 작기 때문에 X 선 빔을 식별하고 정렬하기가 힘들다 ( 그림 5c ). 데이터를 수집 할 때, 중심 정렬 절차를 최소화하고 동일한 셀 또는 결정을 두 번 노출 시키거나 누락시키지 않도록 그리드 패턴으로 진행하는 것이 가장 좋습니다 ( 그림 5a ). 데이터 collec회절 한계의 차이, 방사선 손상의 영향 및 동형의 부재 가능성을 고려하여주의 깊게 처리해야합니다. <img alt="그림 1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> 그림 1 : In Vivo Microcrystals를 이용한 구조 결정을위한 두 가지 방법의 개요. 미세 결정은 재조합 발현 시스템을 사용하여 목적 단백질의 과발현에 의해 세포 배양에서 생산된다. 특정 경우, 미정 제는 또한 특정 조건에서 세포에 의해 자연적으로 생성 될 수있다. 상단 행은 세포가 용해되고 차동 원심 분리에 의해 결정이 정제되는 고전적 접근법을 보여줍니다. 정제 된 미세 결정을 마이크로 메쉬에 피펫 팅합니다. 과량의 액체를 제거한 후에, 동결 방지제 용액을 첨가하고, 과량의 액체를 다시 블로 팅하고 메쉬를 플래시 냉각시켰다. X 선 촬영시<html> 분수 실험에서 결정은 X 선 광선과 조심스럽게 정렬되며 데이터 수집의 속도 제한 단계 일 수 있습니다. 하단 행 은 셀룰로오스 방식으로 나타납니다. 세포는 유동 세포 계측법에 의해 분류되어 결정 함유 세포를 특이 적으로 선택한다. 세포는 시각화를 촉진하고 마이크로 메쉬로 퍼지기 위해 트리 판 블루로 염색됩니다. 이 경우, 동결 방지제의 첨가는 선택적이다. X 선 회절 실험의 경우, 센터링 프로세스를 용이하게하는 싱크로트론 빔라인에서 사용 가능한 일반적인 카메라를 사용하여 셀을 쉽게 시각화 할 수 있습니다. 스웨덴 웁살라 GE Healthcare AB로부터의 자동화 된 액체 크로마토 그래피 시스템 이미지 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> = &quot;xfig&quot;&gt; 그림 2 : In Vivo Microcrystals의 예. 생체 내 결정의 선택된 예 : ( a ) 사이 폰 바이러스 다면체를 가진 Sf9 세포; ( b ) 엔도 포 톡신 스페 로이드를 갖는 LD652 세포; ( c ) Bryillus thuringiensis 와 Cry3A 결정 (화살표); ( d ) Trypanosoma brucei 카 텝신 B 결정을 갖는 Sf9 세포; ( e ) 칼시 뉴린 결정을 갖는 Sf21 세포 (화살표); ( f ) PKA를 갖는 COS7 세포 단층 : Inka1 결정체; ( g ) IgG 결정을 갖는 CHO 세포. 허락을 받아 14 , 18 , 29 , 37 , 39 , 42 에서 복제되었습니다. 눈금 막대 = 10 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">larg를 보려면 여기를 클릭하십시오.</a>이 그림의 어 버전입니다. <img alt="1 번 테이블" src="/files/ftp_upload/55793/55793table1.jpg" /> 표 1 : 재조합 단백질을 발현하는 배양 세포에서 성장한 생체 내 결정의 요약. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793table1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

단백질 미세 결정을 사용하는 X 선 결정학을위한 프로토콜이 제시됩니다. 정제 후 또는 cellulo에서 미세 -grown 생체 분석 두 예가 비교된다.

단백질 결정의 미세 결정학 및<em&gt; 셀룰로에서</em&gt; 회절

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

microcrystallography-of-protein-crystals-and-in-cellulo-diffraction

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K 조회수.  Monash University.  단백질 미세 결정을 사용하는 X 선 결정학을위한 프로토콜이 제시됩니다. 정제 후 또는 cellulo에서 미세 -grown 생체 분석 두 예가 비교된다. 

비디오: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

공동 면역 침전에 의한 핵 및 세포질 분수에서 단백질 - 단백질 상호 작용의 시각화 및<em&gt; 현장에서</em&gt; 근접 내고 분석

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

연구

생화학

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

시간 해결 전기 분무 이온화 수소 - 중수소 교환 질량 분석 단백질 구조와 역학 연구를위한

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

사용 하 여 다른 크기와 성장 단백질 결정 자동화 현장에서 동적 산란과 함께 결정 화

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 1: 원스텝 단백질 단백질과 단백질 대사 산물 단지의 병렬 분석에 대 한 친 화력 정화

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

측정 및 해석의 전체 산소 소비 속도 비행 머리 세그먼트

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

경쟁 적정 형광 현미경 검사 법을 사용 하 여 녹음 방송 요인 DNA 바인딩 선호도의 높은 감도 측정

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

단백질 폴딩, 전송, 및 방사성 펄스 체이스 하 여 살아있는 세포의 저하의 분석

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

완전히 자치 특성화 및 생물학 고분자의 결정에서 데이터 수집

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

전사에서 단백질-단백질 상호 작용을 연구하기 위한 생체층 간섭측정기(BLI) 적용

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.