MOLECULAR MODELS OF AVERAGED RIGOR CROSSBRIDGES FROM TOMOGRAMS OF INSECT FLIGHT MUSCLE

Molecular Modeling of Averaged Rigor Crossbridges from Tomograms of Insect Flight Muscle

Real Space Refinement of Acto-Myosin Structures from Sectioned Muscle.

Multivariate Statistical Analysis of Three-Dimensional Cross-Bridge Motifs in Insect Flight Muscle

The Use of Electron Tomography for Structural Analysis of Disordered Protein Arrays.

METHOD--INITIAL MODELS WERE FIT BY HAND USING O. THE FIT WAS THEN REFINED USING REAL SPACE REFINEMENT. REFINEMENT PROTOCOL--RIGID BODY
BEST CORRELATION COEFFICIENT AND FEWEST POOR CONTACTS

3-D MOTIFS WERE IDENTIFIED IN THE TOMOGRAM BY FIRST PRODUCING A CROSS CORRELATION MAP FROM WHICH PEAK COORDINATES WERE DETERMINED FROM THEIR CENTER OF GRAVITY. WE DEFINE A 3-D MOTIF AS ONE, ENTIRE 38.7 NM CROSSBRIDGE REPEAT ALONG ACTIN. THESE MOTIFS USUALLY CONTAIN AT LEAST FOUR MYOSIN HEADS IN TWO PAIRED CROSSBRIDGES (SINGLE CHEVRONS) AND SOMETIMES CONTAIN AS MANY AS SIX MYOSIN HEADS IN FOUR PAIRED CROSSBRIDGES (DOUBLE CHEVRONS). THE REFERENCE FOR THE ANALYSIS WAS SELECTED TO BE CENTERED BETWEEN SUCCESSIVE TROPONIN DENSITIES WHICH COULD BE IDENTIFIED FROM THE IN-PLANE PROJECTION. THE INDIVIDUAL CROSSBRIDGE MOTIFS WERE THEN SUBJECTED TO MULTIVARIATE STATISTICAL ANALYSIS TO IDENTIFY CLUSTERS OF MOTIFS SHOWING SIMILAR CROSSBRIDGE STRUCTURE. THESE CLUSTERS FORMED THE CLASS AVERAGES. THE CHOICE OF STRUCTURE TO BE CLASSIFIED WAS DECIDED BY THE RESOLUTION AND THE LATER PROCESS OF MODEL BUILDING. AVERAGING WAS DONE ACCORDING TO THE HEIRARCHICAL ASCENDENT METHOD. THE RESOLUTION IN EACH OF THE CLASS AVERAGES WAS 7 NM BY THE SPECTRAL SIGNAL TO NOISE RATIO.
DUAL AXIS TILT SERIES ELECTRON TOMOGRAPHY

DORSAL LONGITUDINAL FLIGHT MUSCLES OF THE LARGE WATERBUG, LETHOCERUS MAXIMUS WERE GLYCERINATED IN THE DISSECTED THORAX AND STORED AT -20 DEGREES C. FIXATION INVOLVED SEQUENTIAL TANNIC ACID-GLUTARALDEHYDE (0.2% AND 2%), COLD 1% OSO4 AT PH 6.0, 1% URANYL ACETATE BLOCK STAINING AND ARALDITE 506 EMGEDDING. THIN SECTIONS WERE STAINED WITH A SEQUENCE OF PERMANGANATE AND LEAD. SECTION WERE ~25 NM THICK. MODELS ARE BUILT TO FIT THE THIN, ACTIN CONTAINING FILAMENTS LABELED WITH ENDOGENOUS MYOSIN IN THE RIGOR STATE.
INSECT FLIGHT MUSCLE OF THE LARGE WATER BUG LETHOCERUS MAXIMUS

NO VITRIFICATION. SAMPLES WERE VIEWED AT ROOM TEMPERATURE.

SKELETAL MUSCLE MYOSIN II REGULATORY LIGHT CHAIN

SKELETAL MUSCLE MYOSIN II ESSENTIAL LIGHT CHAIN

DAEMAAFGEAAPYLR(MLY)SEKERIEAQN(MLY)PFDA(MLY)SSVFVVHPKQSFV(MLY)GTIQS(MLY)EGG(MLY)
VTV(MLY)TEGGETLTVKEDQVFSMNPP(MLY)YD(MLY)IEDMAMMTHLHEPAVLYNL(MLY)ERYAAWMIYTYSGLFC
VTVNPY(MLY)WLPVYNP(MLY)VVLAYRGKKRQEAPPHIFSISDNAYQFMLTDRENQSILITGESGAGKTVNT(MLY)R
VIQYFATIAASGEKKKEEQSGKMQGTLEDQIISANPLLEAFGNA(MLY)TVRNDNSSRFG(MLY)FIRIHFGATGKLASA
DIETYLLE(MLY)SRVTFQLPAERSYHIFYQIMSN(MLY)(MLY)PELIDMLLITTNPYDYHYVSEGEITVPSIDDQEEL
MATDSAIDILGFSADE(MLY)TAIY(MLY)LTGAVMHYGNLKF(MLY)Q(MLY)QREEQAEPDGTEVAD(MLY)AAYLMG
LNSAELLKALCYPRVGVGNEAVT(MLY)GETVSEVHNSVGALA(MLY)AVYE(MLY)MFLWMVIRINQQLDTKQPRQYFI
GVLDIAGFEIFDFNSFEQLCINFTNE(MLY)LQQFFNHHMFVLEQEEY(MLY)(MLY)EGIEWEFIDFGMDLAACIELIE
(MLY)PMGIFSILEEECMFPKATDTSF(MLY)N(MLY)LYDEHLGKSNNFQKPKPAKGKAEAHFSLVHYAGTVDYNISGW
LE(MLY)N(MLY)DPLNETVIGLYQ(MLY)SSV(MLY)TLALLFATYGGEAEGGGGKKGGKKKGSSFQTVSALFRENLN
(MLY)LMANLRSTHPHFVRCIIPNET(MLY)TPGAMEHELVLHQLRCNGVLEGIRICRKGFPSRVLYADFKQRYRVLNAS
AIPEGQFMDSKKASEKLLGGGDVDHTQYAFGHT(MLY)VFF(MLY)AGLLGLLEEMRDD(MLY)LAEIITATQARCRGFL
MRVEYRAMVERRESIFCIQYNVRSFMNV(MLY)HWPWM(MLY)LFF(MLY)I(MLY)PLLK

DAEMAAFGEAAPYLRKSEKERIEAQNKPFDAKSSVFVVHPKQSFVKGTIQSKEGGKVTVKTEGGETLTVKEDQVFSMNPP
KYDKIEDMAMMTHLHEPAVLYNLKERYAAWMIYTYSGLFCVTVNPYKWLPVYNPKVVLAYRGKKRQEAPPHIFSISDNAY
QFMLTDRENQSILITGESGAGKTVNTKRVIQYFATIAASGEKKKEEQSGKMQGTLEDQIISANPLLEAFGNAKTVRNDNS
SRFGKFIRIHFGATGKLASADIETYLLEKSRVTFQLPAERSYHIFYQIMSNKKPELIDMLLITTNPYDYHYVSEGEITVP
SIDDQEELMATDSAIDILGFSADEKTAIYKLTGAVMHYGNLKFKQKQREEQAEPDGTEVADKAAYLMGLNSAELLKALCY
PRVGVGNEAVTKGETVSEVHNSVGALAKAVYEKMFLWMVIRINQQLDTKQPRQYFIGVLDIAGFEIFDFNSFEQLCINFT
NEKLQQFFNHHMFVLEQEEYKKEGIEWEFIDFGMDLAACIELIEKPMGIFSILEEECMFPKATDTSFKNKLYDEHLGKSN
NFQKPKPAKGKAEAHFSLVHYAGTVDYNISGWLEKNKDPLNETVIGLYQKSSVKTLALLFATYGGEAEGGGGKKGGKKKG
SSFQTVSALFRENLNKLMANLRSTHPHFVRCIIPNETKTPGAMEHELVLHQLRCNGVLEGIRICRKGFPSRVLYADFKQR
YRVLNASAIPEGQFMDSKKASEKLLGGGDVDHTQYAFGHTKVFFKAGLLGLLEEMRDDKLAEIITATQARCRGFLMRVEY
RAMVERRESIFCIQYNVRSFMNVKHWPWMKLFFKIKPLLK

FDETEIEDFKEAFTVIDQNADGIIDKDDLRETFAAMGRLNVKNEELDAMIKEASGPINFTVFLTMFGEKLKGADPEDVIM
GAFKVLDPDGKGSIKKSFLEELLTTGGGRFTPEEIKNMWAAFPPDVAGNVDYKNICYVITHGEDA

SKAAADDFKEAFLLFDRTGDAKITASQVGDIARALGQNPTNAEINKILGNPSKEEMNAAAITFEEFLPMLQAAANNKDQG
TFEDFVEGLRVFDKEGNGTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCINYEAFVKHIMSV

DEDETTALVCDNGSGLVKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIITNWD
DMEKIWHHTFYNELRVAPEEHPTLLTEAPLNPKANREKMTQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVT
HNVPIYEGYALPHAIMRLDLAGRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALDFENEMATAASSSSLEKSY
ELPDGQVITIGNERFRCPETLFQPSFIGMESAGIHETTYNSIMKCDIDIRKDLYANNVMSGGTTMYPGIADRMQKEITAL
APSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWITKQEYDEAGPSIVHRKCF

ORGANISM FROM WHICH THE MYOSIN FOR THE CRYSTAL STRUCTURE THAT PROVIDED 2MYS WAS OBTAINED

ORGANISM FROM WHICH THE ACTIN THAT PROVIDED THE CRYSTAL STRUCTURE FOR 1ATN WAS OBTAINED

VOLUME DATA INTO WHICH THESE MODELS WERE FIT. INCLUDES THE RAW TOMOGRAM AND THE CLASS AVERAGES.

The first model of the 11 models was presented in this entry.

SEQUENCE
Since the models used to fit the data are not from the same
source, the DBREF and the SEQADV remarks were suppressed.