janelia.org/support-team/cryo-electron-microscopy

Workflow@

CryoEM SharedResourceHowardHughesMedicalInstitute

Janelia ResearchCampus

Zhiheng Yu

Twotypesofwork

Collaborationprojectswithcollaborators

WecovertheentirecryoEM workflowfromgridspreparationtomapgeneration.

Routinedatacollectionforusers

Wefocusmainlyoninstrumentoperationanddatacollection.

ToservelabswithcryoEM experiencesandeasyaccesstogridspreparation/screeningequipment.

ToservelabswithlimitedexperienceincryoEM ornoeasyaccesstoequipment.

Receive:ready-to-imagecryo gridsDeliver:largequantityofrawdata

Receive:solution samplesDeliver:3Ddensitymaporfeedbacktoimprovesample

RoutinedatacollectionforusersSpecimenPreparation

DataAcquisition

SpecimenPreparationImage

Processing

Analysis

UserssendcryoEM imagestoqualify fordatacollectiontimeUserssendfrozengridstothefacilityFacilitystaffoftenprovidesuggestions foroptimization ifnecessary

Gridsclipping andloading intothemicroscope.Facilitystafftakecareofaligningandoperatingmicroscope,optimizing imagingcondition/parametersandsettingupautomateddatacollection.Userspickacquisition targets.(canbedone remotelyifnotpresentonsite)Facilitystaffmonitor thedatacollectionprocessanddoqualitycheckregularly.Datamanagementandtransfer

UserstakefullresponsibilityFacilitystaffoftenprovideassistancebyrequest(motioncorrection,CTFestimation)

Users’fullresponsibilityFacilityStaffUsers

Moreondatacollection

AutomatedDataCollection

Gridclipping

andloading4-8grids

persession

MicroscopeAlignment,GIFtuning,cameragainreference

Gridscreening

Atlasacquisition(for fullgridandselectedsquares) Add targets

intoqueue

Setupimagingparameters

(exposure time,mag,framerate,defocusandetc)

Quickmotioncorrectionand

qualityevaluation

Ontheflydatatransfer

Monitor runprogressandtroubleshoot ifnecessary

FacilityStaffUsers

DataTransfertoUsers• rsync viassh directlytoremoteserver.• Yay:allowuserstoprocessdataon-the-fly.• Nay:remotefirewallandsecuritypolicy.Aguestaccountwithpasswordisrequired.

• PortableHardDrives• Yay:easytosetup.• Nay:takestime.

Wecurrentlydon’tgrantaccesstouserstodownloaddatadirectlyfromourserverduetoJanelianetworksecuritypolicy.

DataManagementandStorageRawmoviestackswithsuper-

resolutionmodearegeneratedascompressedTIFfiles,typically

about600MB permovieor1.5TBper3-daysession.

RAIDonK2computer/

FalconcapturePC

Janelianetworkstorage

20TB

Continuouslyupdate

CryoEMFacility

LocalRAID

~300TB

DailybackupbyITDataarekeptfor~2months

JaneliaRAIDoncluster

Rapidread/writedisksfordataprocessing,

i.e.motioncorrection.

10Gnetwork eitheronfiberopticsorcopper

Postdatacollectionfollow-up

• Bookkeeping:parameterssuchastotaldose,exposuretime,magnification,gridtype,issuesencounteredandetc.

• Voluntaryuserfeedbacks.

• Discussionsandsuggestionsfornextsessionifneeded.

• Whendataispublished,usershouldsendalinktothepaperaswellasarepresentativefiguretothefacility.

Collaborationprojects

SpecimenPreparation

DataAcquisition

SpecimenPreparationImage

Processing

Analysis

CollaboratorssendsolutionsamplestothefacilityFacilitystaffscreensamplesbynegativestainorcryo andprovidesuggestions tooptimizeproteinpreparation.Multiple iterationsmightbeneededtillsuitablecryo gridsareobtained.

Facilitystaffcarryoutthedatacollection.Collaboratorsarewelcometojointhedatacollectionandon-the-flyearlyprocessing (helppickparticles).

Facilitystaffleadindataprocessingbutinteractcloselywithcollaborators.

Facilitystaffsendcollaboratorsintermediateresultsandfinalmaps.Collaboratorsanalyzemaps,buildatomicmodels, andinterpretresults.FacilityStaff

Collaborators

Thekeydeterminant:samplepreparation

Highqualityhigh throughput DataCollection

TitanKrios

Providesuggestions foroptimizationandfuturedirection

CryoEMAnalysis

T12Proteinexpression,purification,andcomplexassembly

2DClassAverage

NegativeStainingAnalysis

T12

ImageProcessing/map

generation

ModelBuilding,

analysisandinterpretation

2DClassAverage

Bufferexchangeordilution

ifnecessary

Ifsampleshows

potential

Ifsampleneeds

optimization

FacilityStaffCollaborators

Onetypicalimageprocessingworkflow

Datadecompression

andgaincorrection

IMOD

Motioncorrection,dosefilterandCTFdetermination

cisTEM*(Unblur,sum_movie,CTFFIND4)

Particlepickingandextraction

EMAN2Relion

Reference-free2D

Classification

RelioncisTEM*

3DClassification

cisTEM*/FrealignXRelion

3DRefinement

cisTEM*/FrealignXRelion

Postprocessing

cisTEM*/FrealignXRelion

InitialmodelgenerationEMAN2cisTEM*/FrealignX

*cisTEM fromNikoGrigorieff’s labisacompletepackagefromrawmoviestackto3DclassificationandrefinementwithaGUIinterfaceforsingleparticlecryoEM.Unblur,sum_movie,ctffind4andFrealginX areallpartofcisTEMwhichalsoincludes2Dclassificationandmore.

WorrallLJ,*,HongC*, Vuckovic M,DengW,BergeronJRC, Majewski DD,HuangRK,Spreter T,FinlayBB,YuZ#,Strynadka NCJ#,Nature,Dec2016

Oneexample:T3SSBasalBodyComplex

Acknowledgement

Chuan Hong RickHuang ChrisMorkunas

• ReedGeorgeandHHMI/Janelia management• NikoGrigorieff &Tamir Gonen andlabmembers• MarkPhilipandBuildingFacilityteamatJanelia• PaytonLewis,Haifeng HefromFEIandFredUlmer,DanielRayfromGatan• Allusers

Manyfactorscontributing tothesuccessofasharedfacility:equipment, userbase,management,goodpolicy/proctcol andmoreimportantly, theteammemberswhocarryoutthedaytodayworkandinteractwithusers.

JasondelaCruzalumni